antimicrobial activity of shilajit aliya hayat1*, fatima sher ali1 1department of microbiology, jinnah university for women, karachi – 74600. abstract shilajit is a pale-brown to blackish-brown exudation of variable consistency, exuding from layers of rocks in the mountainous ranges of this world, especially himalayan and hindu kush ranges of indian subcontinent. the aim of study was to determine the antimicrobial activity of shilajit against different microorganisms. the diluted sample (80%) was used to find the activity which was assessed by agar well diffusion method. the microbial inhibition was more effective. among all species, candida albicans was found to be highly susceptible while acinetobater was highly resistant. other bacterial species were also susceptible to this material. shilajit not only helps to overcome physiological activities of human, but can also protect the body by inhibiting the pathogenic organism of respiratory and gastrointestinal tract. this indicates that shilajit contain antimicrobial compounds to control pathogens. keywords: shilajit, agar well diffusion, susceptible introduction shilajit is also known as salajit, shilajatu, mumie or mummiyo (cornejo et al., 2011) means "winner of rock" (mukherjee, 1992) or "sweat of the rock" (tirtha, 1998). it has a unique composition as a phytocomplex. it is very rich in fulvic acid, elagic acid, some fatty acids, resins, latex, gums, albumins, triterpenes, sterols, aromatic carboxylic acids, 3,4benzocumarins, amino acids, polyphenols and phenolic lipids (chopra et al., 1958).there are four different varieties of shilajit which have been described in charka samhita, namily savrana, rajat, tamra and lauha shilajit (agarwal et al., 2007). it is a compact mass of vegetable organic matter composed of a dark-red gummy matrix interspersed with vegetable fibers, sand and earthy matter (chopra et al., 1958). traditionally, shilajit was believed to be a general restorative tonic, meaning it helps fight a range of illness and is also taken to maintain good health. variety of benefits was reported that is against common colds, ulcers, cancers and act as antiviral agent. it has great affinity for human viruses. literature about the antimicrobial activity of shilajit is lacking. therefore, present study was conducted to identify the antimicrobial potential of shilajit (black shilajit/lauha shilajit) against gram positive, gram negative and yeast species. it was found that this black rock has distinct antifungal activity than any other bacteria. materials and methods shilajit sample was purchase from a local market of gilgit, pakistan. the processed sample was diluted in sterile distilled water to make 80% solution. the sample was filtered through whattman filter paper and filtrate was stored under refrigeration condition until use. twenty five pure cultures of bacterial and fungal species were collected from culture bank of department of microbiology, jinnah university for women. among them 13 were gram negative, 8 were gram + and 4 were candida species. microbial susceptibility was determined by agar well diffusion method (betoni et al, 2006) using 5mm borer. it was*corresponding author: aliyaap@yahoo.com 10 vol 4 (2), july 2013; 10-12 carried out for e. coli, pseudomonas florescense, klebsiella oxytoca, citrobacter, salmonella typhi, salmonella para typhi a, acinetobacter, shigella dysenteriae, staphylococcus aureus, staphylococcus saprophyticus, streptococcus pneumoniae, bacillus subtilis and candida albicans. overnight cultures (37ºc) in brain heart infusion (bhi) were adjusted to 0.5 macfarland’s standard and inoculated on mha. results and discussion shilajit is an oriental medicine, from steep rocks of different formations found throughout the whole world (phillips, 1997). it is said to be very effective against chronic bronchitis and asthma, digestive troubles, nervous disease, leprosy, parasitic diseases of skin etc (mittal et al., 2009).it is also believed to slow down the process of aging(sharma et al., 1978). most research papers described its physiological properties and activity in humans and animals. antiviral and antifungal activities of shilajit have been reported. studies showed that it has effective anti-inflammatory (acharya et al., 1988) and immunomodulatory (ghosal s, 1990) effects. it was also found that this compound has the property of reducing viral load (ghosal s, 1990). in this study it was found that shilajit gave intermediate zone of inhibition with gram positive and negative bacteria. only acinetobacter spp. was resistant to its action. among gram positive (+) organism; streptococcus pneumonia and b.subtilis showed greater zone of inhibition (20mm). while s.aureus showed a zone of (17mm) and s. saprophyticus showed a zone of (15mm). among gram negative (-) organism, shigella dysenteriae and salmonella paratyphi a showed greater zone of (17mm) in diameter. e.coli showed a zone of (16mm), p.florescense and klebsiella oxytoca showed a zone of (16mm), salmonella typhi showed a zone of (14mm) and citrobacter showed a zone of (13mm). greater zone of inhibition (50mm) was found in candida albicans as compare to bacteria. the literature reported mostly antifungal activity of shilajit. inhibitory effects of shilajit were reported by shalini & rachana srivastava, (2009) against phytopathogenic fungi. conclusion it is concluded that this rock material not only helps to overcome physiological activities of human but simultaneously protects the body by inhibiting various p a t h o g e n i c o rg a n i s m s o f r e s p i r a t o r y a n d gastrointestinal tract. references acharya sb, fortan mh, goel rk, tripathi sk and das pk. (1988). pharmacological actions of shilajit. indian journal of experimental biology, 26: 775777. agarwal sp, khanna r,karmarkar,anwer mk,khar rk. (2007). shilajit: a review. phytother res., 21(5):401-405. 11 vol 4 (2), july 2013; 10-12 zone of inhibition (mm) table i. zone of inhibition of different isolates. alberto cornejo, josé m. jiménez, leonardo caballero, francisco melo, ricardo b. maccioni (2011) fulvic acid inhibits aggregation and promotes disassembly of tau fibrils associated with alzheimer’s disease journal of alzheimer’s disease 27:143–153. doi 10.3233/jad-2011110623. betoni, jec, mantovani rpp, barbosa ln, di stasi lc, fernandes junior a. (2006). synergism between plant extract and antimicrobial drugs used on staphylococcus aureus diseases. mem. inst. oswaldo cruz., 101: 387-390. chopra, rn, chopra i c, handa k l & kapur l d. (1958). chopra’s indigenous drug of india. 2nd ed. b.k. dhur of academic publishers, calcutta india. mittal p.kaushik d. gupta v. bansal p, khokra s. (2009). therapeutic potential of “shilajit rasayana” a review, international journal of pharmaceutical and clinical research; 1(2): 47-49. mukherjee, biswapati. (1992). traditional medicine, proceeding of an international seminar. pp. 398319. hotel taj bengal, calcutta india. oxford & ibh publishing, new delhi. paul p. (1997). unearthing the evidence. chemistry in britain, pp.32-34. ghosal s. (1990). chemistry of shilajit, an immunomodulatory ayurvedic rasayan:, pur and applied chemistry, 62(7):1285-1288. sharma rk, dash b, sambita tc. (2000). chowkhamba sanskrit series office, varanasi-1,. vol iii chap 1:3 pg 50-54.varanasi, india. srivastava sr. (2009). antifungal activity screening and hplc analysis of crude extract from tectona grandis, shilajit, valeriana wallachi, electrical journal of environment, agricultural and food chemistry, 8(4): 218-229. tritha, swami sada shiva. (1998). the ayurvedic encyclopedia. ayurveda holistic centre press. bayville, ny. 12 vol 4 (2), july 2013; 10-12 introduction the manufacture of injectable pharmaceuticals presents challenges from many perspectives, especially for products that must be processed aseptically and cannot be terminally sterilized. food and drug administration (fda) inspections for good manufacturing practice (gmp) compliance in injectable drug product manufacturing facilities commonly find problems in the following areas: issues with environmental control problems with equipment, especially older equipment media fill failures and/or issues with media fills truly simulating actual practices · lack of proper training of operators · documentation failures besides the need to be in gmp compliance, manufacturing requires competency and resiliency in such matters as: inventory management, scheduling of facilities, equipment and personnel, management of changes in schedule, management of shutdowns for maintenance and repairs, management of change control, management of changes in product demand and departmental growth. it takes well-organized and competent management and highly trained and competent operators and manufacturing support personnel to deal with all these challenges successfully. this article will focus on a few of the challenges involved in the manufacturing of injectable drug products the process, equipment, people and environment. process unit operations in sterile product processing include: c o m p o u n d i n g ( o r f o r m u l a t i o n ) , m i x i n g , homogenization (dispersed systems), filtration, filling, stoppering, sealing and terminal sterilization (although a significant majority of small volume injectable products cannot be terminally sterilized for stability reasons). all segments of the manufacturing process present many challenges, especially as the process is scaled-up from bench to full production. the pathway of a process is usually initially defined in a lab setting at small-scale by scientists who are frankly not highly experienced in the world of manufacturing. some process methods are simple activities, such as weighing and mixing, while others are more complex and require special procedures, equipment, temperature control, inert atmospheres, protection from light and explosion proofing. special criteria must be considered during the transfer of a process from the development lab to the manufacturing environment. ideally, processes should be designed to be transferable on multiple projects in order to maintain a key principle in quality manufacturing. that principle is consistency for the manufacturing of sterile injectable products, consistency is critical. it is well known that media fills are only a snapshot in time, and merely demonstrate that under the 'correct conditions', aseptic filling of solutions is possible for that particular filling line. more importantly, however, is the fact that we base the entire principle of media fill validation on the ability, and expectation, to consistently perform each operation in the same quality manner for each and every batch. these 'correct conditions' include the current challenges in the manufacturing of injectable drug products mohammad shakil siddiqui1*, ghulam sarwar1 1department of pharmacy, jinnah university for women, karachi-74600, pakistan. * corresponding author: mshakil@hotmail.com 47 vol 4 (1), january 2013; 47-49 proper environment, equipment, components, personnel and process methods. equipment many challenges posed by manufacturing process methods can be solved by the proper choice of equipment. however, applying proper equipment always has a price. once the initial cost of the equipment is considered, then come installation and validation issues. it is imperative to consider space and utilities required before getting started. once in place, the company budget must be prepared to support a preventative maintenance programme, including a sufficient spare parts inventory. there is nothing more frustrating than loss of production due to equipment failure and worse, not having the means to repair or replace the parts necessary to resume manufacturing. during annual budget planning sessions, it is important to balance the push for cost reduction and optimization with robust processes and capable equipment-support programmes. once the right equipment has been sourced for the process, proper identification and control for all product contact parts is vital to quality manufacturing and preventing the risk of cross contamination. elements of this challenge include the handling, cleaning, testing, release, storage, issuing and return of each product contact part. furthermore, the importance of each of these areas is easily confirmed by the fact that they are frequently primary auditing targets. people some may believe that people are the source of most, if not all, problems encountered in manufacturing, particularly aseptic manufacturing. in fact, the development of isolators and automation is driven primarily by the desire (and possibly need) to remove people from direct contact with sterile products during manufacturing. the root cause, however, may be better correlated to how the people are developed. training and experience are the primary solutions to minimizing or eliminating operator errors. training a strong training programme is essential in the manufacture of pharmaceutical products. training must be well organized to effectively maintain the compliance of all operators, who must be trained on the most current versions of all applicable standard operating procedures (sops). a solid change control process for procedures is required to properly maintain compliance with operator training. training curricula are helpful tools that outline all sop training and qualifications required for each position. these curricula are a valuable reference for auditing, and an easy way for operators and management to measure their development over time. experience most manufacturing operations are sop-driven and are not conducive to classroom training for new operators. job shadowing, with repetitive sop review and hands-on demonstration, is the most direct method of transferring knowledge to new operators. for this reason, it pays in large dividends to develop experienced operators into effective trainers by investing in courses on effective communication and training techniques. experienced operators are not automatically the best trainers, but most can become quite capable with proper guidance. it might seem obvious, but new operators should always be paired with the most experienced operators/trainers. furthermore, they should remain paired with the same trainer for as long as possible. it takes some time for a trainer to assess the trainee's strengths, weaknesses and rate of learning, then adopt the best pattern to use in each training opportunity. for example, some trainees may require more verbal repetition, hands-on practice, demonstrations, or sop review. consequently, successful trainer/trainee teams will more efficiently and effectively work their way through all procedures on a training curriculum. a vol 4 (1), january 2013; 47-49 48 vol 4 (1), january 2013; 47-49 strong training programme is also imperative for a fast growth rate in personnel, which can introduce a large number of new operators into the manufacturing operations at the same time. any operation saturated with inexperience will likely incur an increase in errors. again, this can be managed by pairing the new with the experienced. environment the impeccable quality of manufacturing environments for pharmaceutical processing is vitally important. we routinely measure the suitability of these environments with a variety of testing methods. the air is evaluated for direction of flow, velocity, pressure differentials, control of temperature, humidity, non-viable and viable particle content. room and equipment surfaces are evaluated for general debris, microbial contamination and chemical contamination. as people and equipment are part of the environment during the process, monitoring must be performed under dynamic conditions. the operators are also monitored for proper gowning and handling techniques using media contact plates in the search for microbial contamination at multiple bodily sites, such as chest, forearms, hands and fingertips. to meet the challenging specifications for environmental monitoring, the manufacturing operations must include proven methods for proper room clearance, cleaning and sanitization. additionally, the gowning and cleanroom behaviour techniques must be both well trained and well practiced. batch deviations at some point, the challenges of manufacturing injectable products will eventually lead to some sort of batch deviation or variance. batch deviations are typically due to a planned change or loss of control in at least one of the 'correct conditions' as discussed in this article. some common root causes for deviations include: documentation errors, equipment/software problems, product/material defects, performance errors and incorrect process methods. it should be noted here that the above challenges with respect to people and environment have generated great interest and progress in the area of isolation technology. successful application and validation of isolators enables the manufacture of sterile products to be distinctly separated from the direct intervention of personnel and ideally provides a safer environment for sterility assurance. however, beyond the scope of this brief article, there are many obstacles in the validation and routine use of isolators in aseptic manufacturing. conclusion we hear the common 'buzzwords' such as cost reduction, optimization, efficiency and capacity utilization. in our efforts to meet these goals it is important that we do not swing the pendulum too far from some other key manufacturing principles, such as training, practice, validation, preventative maintenance and availability of spare parts. it is important to find a balance among all these needs to meet the many challenges of manufacturing i n j e c t a b l e p h a r m a c e u t i c a l d o s a g e f o r m s . references reminngtons practice of pharmacy vol.1 ich guidance for industry q2b validation of analytical procedures fda code of federal regulations 49 oral health status: the level of oral microbial flora in healthy girls sughra hasan1* amna shafiq2, aziz fatima2, sayyada ghufrana nadeem2 1department of environmental sciences, jinnah university for women, karachi-74600, pakistan. 2department of microbiology, jinnah university for women, karachi-74600, pakistan. abstract the normal oral flora comprises a diverse group of micro-organisms and then 300 species inhabit the oral cavity of which about 50 are found routinely and account for the majority of the cultivable strains, these factors, together with the fact that the indigenous microbiota plays an important role in health and disease of a human and animals.it contribute to the development of the immune system and provide resistance to colonization by allochthonous or pathogenic micro-organisms. the aim of our study is to determine the most frequent micro-organisms as normal flora in healthy individuals. a total of 30 healthy students, all belongs to jinnah university for women of same age group were studied. throat swabs were plated with human-blood agar, chocolate agar and apply bacitracin disk on both plates, sda agar were incubating for 24 hours for isolation of candida species. organisms were cultured on blood agar, sda agar, mannitol salt agar to observe the colonies. different spot test like catalase and coagulase test were performed for identification of staphylococcus species. identification was also done by microscopic examination by gram stain. catalase-positive, gram-positive rods were found to be as most frequent micro-organisms as normal flora of healthy students and were only micro-organisms detected 46.6% and total staphylococcus species were (30%); staphylococcus aureus ( 13%) and other staphylococcus species (17 %) where as streptococcus species (catalase -negative gram positive cocci) isolated from oral specimen of healthy individuals. key words: microbiota, oral flora, staphylococcus sp., throat swab. introduction the microbial flora of the oral cavity are rich and extremely diverse. this reflects the abundant nutrients and moisture and hospitable temperature, and the availability of surface on which bacterial populations can develop. the presence of a myriad of microorganisms is a natural part of proper oral health. however, a imbalance in the microbial flora can lead to the production of acidic compounds by some micro-organisms that can damage the teeth and gums. damage to the teeth is referred to a dental caries. it has been estimated that at least 35% of denate u.s adult s aged 30 to 90 years have peridontitis (yamamoto et al., 1994). in addition specific oral b a c t e r i a l s p e c i e s h a v e b e e n i m p l i c a t e d severalsystemic disease, such as bacterial endocarditis (michalek et al., 1990) aspiration pneumonia (childers et al., 1989), osteomylitis in children (farnaud et al., 2003) preterm low birth weight (corthesy et al., 1999) and cardiovascular disease (tenovuo et al., 1994). surprisingly, little known about the microflora of the healthy oral cavity. these include the tougue , epithelial cells lining the roof of the mouth and the cheeks , the hard enamel of the teeth. in particular , the microbial communities that exist on the surface of the teeth are known as dental plaque. the adherent communities also represent a biofilm. oral biofilm develop over it time into*corresponding author: sughra_888@hotmail.com 13 vol 4 (2), july 2013; 13-15 exceedingly complex communities. hundreds of species of bacteria have been identified in such biofilm. examples of some bacteria that are typically present as primary colonizers include streptococcus, actinomycetes, neisseria and veillonella. example of secondary colonizers include fusobacterium n u c l e a t u m , p r e v o t e l l a i n t e r m e d i a a n d capnocytophaga species. with further time, another group of bacteria can become associated with the adherent community. examples of these bacteria include campylobacter rectus, eikenella corroden, actinobacillus actinomycetem comitans, and the oral spirocheates of the genus treponema. the mouth is the largest natural opening in the human body, and is a major component in the mucosal barrier system. it has its own immune barriers, which we call the oral immune system. the primary function of the immune system of the mouth is to protect the teeth, jaws, gingivae and the rest of the oral mucosa against infection (yamamoto et al., 1994). the flow of saliva has a mechanical effect, flushing microorganism from mucosal and tooth surfaces. saliva also contains important antimicrobial agents (tenovuo et al., 1994). the intact stratified squamous epithelium supported by the lamina propria presents a mechanical barrier to oral microorganism. the continous shedding by exfoliation of epithelial squamous limits microbial colonization of the surface. intra-epithelial dendritic langerhans cells are peripheral antigen-presenting cells which can process antigen in their mhc-class ii abundant intracellular compartments. they migrate to the regional lymph nodes to present antigenic peptides complexed to mhc-class ii molecules to prime naïve helper t cells. the oral epithelium also forms part of an intercommunicating network of the immune system, in which signals are regularly exchanged in dynamic interactions (yamamoto et al., al 1994) as the nonspecific defense factors include mucins, nonimmune salivary glycoproteins, lactoferrin, lysozyme, peroxidase, histatins, and cystatins. siga is considered the first line of defense against pathogens which colonize and invade surfaces bathed by secretions (mcnabb et al., 1981). siga antibodies may play an important role in the homeostasis of oral resident microbiota and in the prevention against caries and periodontal diseases (michalek et al., 1990). the normal oral flora comprises a diverse group of micro-organism, including bacteria, fungi, protozoa and possibly even viruses (marsh et al., 1999). more than 300 species inhabit the oral cavity of which about 30 are found routinely and account for the majority of the cultivable strains, these factors, together with the fact that the oral cavity has a wide range of sites with different environment condition, make the study of oral microbiology complex and difficult. the indigenous microbiota plays an important role in health and disease of humans and animals. it contributes to the development of the immune system and provides resistance to colonization by allochthonous or pathogenic microorganisms (crabbe´et al., 1968). materials and methods specimen: a sterile cotton swab specimen, vigorously rub on both tonsillar surface and the posterior pharynx. remove swab from mouth and insert tip down into wrapper. requirements: petri plates, sterile cotton swabs, test tubes, pipettes. media and reagents: catalase reagent, blood base, mannitol salt agar, bacitracin disk, gram staining reagents. procedure: throat swabs were plated with humanblood agar, and sda agar. place bacitracin disk on blood agar plates. both plates were incubated at 37 ºc for 24 hours. gram staining was performed to observe the gram reaction and morphology of microorganism. the organisms were streaked in order to check haemolysis. catalase test and coagulase test were also performed. results catalase-positive, gram-positive rods were found to be as most frequent micro-organisms as normal 14 vol 4 (2), july 2013; 13-15 15 vol 4 (2), july 2013; 13-15 flora of healthy students and were only microorganisms detected 46.6% and total gram positive cocci were found 53% in which total staphylococcus species were 30%; staphylococcus aureus 13% while other staphylococcus species were 17 % where as catalase-negative gram-positive cocci were also isolated from oral specimen of healthy individuals (table i). discussion and conclusion there is a distinctive bacterial flora in the healthy oral cavity which is different from that of oral disease for example, many species specifically associated with periodontal disease, such as porphyromonas gingivalis, tanneralla forsythia, and treponema denticola, were not detected in any sites tested. in addition, the bacterial flora commonly thought to be involved in dental caries and deep dental cavities, represented by streptococcus mutans, lactobacillus species, bifidobacterium species and atopobium species, were not detected in supra and sub gingival plaques from clinically healthy teeth. the bacterial species associated with sore throat such as lancifield group a ß-haemolytic streptococcus pyogenes were also not detected. it is necessary to first define the bacterial flora of the healthy oral cavity before we can determine the role of oral bacteria n disease. references childers nk, bruce mg and mcghee jr. 1989. molecular mechanism of immunoglobulin a defense. annu. rev. microbiol., 43:503–536. c o r t h e s y b , s p e r t i n i f. 1 9 9 9 . s e c r e t o r y immunoglobulin a: from mucosal protection to vaccine development. boil chem, 380:1251. crabbe pa, bazin h, eyssen h and heremans jf. 1968. the normal microbial flora as a major stimulus for proliferation of plasma cells synthesizing iga in t h e g u t . i n t . a r c h . a l l e rg y 3 4 : 3 6 2 – 3 7 5 . farnaud s, evans rw. 2003. lactoferrin--a multifunctional protein with antimicrobial properties. mol. immunol., 40(7): 395–405. marsh p, martim mv. 1999. oral microbiology, 4th edn. oxford,wright. mcnabb p c and tomasi tb. 1981. host defense mechanisms at mucosal surfaces. annu. rev. microbiol., 35:477–496. michalek sm and childers nk. 1990. development and outlook for a caries vaccine. crit. rev. oral biol. med., 1:37–54. tenovuo j, lagerlof f . saliva in: thylstrup a, fejerskov o,editor textbook of clinical cardiology. c o p e n h a g e n : m u n k s g a a r d , 1 9 9 4 : 3 8 4 1 . yamamoto t, osaki t, ueta e. cytokine production by keratinocytes and mononuclear infiltrates in oral lichen planus. j oral pathol med 1994;23:30915. 4 5 14 7 13 17 47 23 microorganisms no. of individuals percentage (%) staphylococcus aureus other staphylococcus species catalase positive, gram-positive rods catalase-negative gram-positive cocci candida 0 0 table i. frequency rate of isolated micro-organisms from oral cavity. keratitis causing micro-organisms isolated from ophthalmic contact lens solutions mohniyal abbasi1, aliya hayat1 1department of microbiology, jinnah university for women, karachi. abstract this study aimed to determine the disinfecting potential of some contact lens solution used by the students of jinnah university for women. we investigated the possible contaminating rate and identified microbial contamination. 25 samples were collected in this study. were used multipurpose disinfecting solutions with protein remover of different companies. we have isolated 43 strains among which 20 were gram positive and 23 were gram negative. s.aureus, pseudomonas serratia was most common organisms isolated i.e. 26%, 16% & 14% respectively. all isolated strains were resistant to antibiotic used in this study except bacillus which gives intermediate-resistant to streptomycin. keywords: contact lens, contamination, disinfection, microorganisms introduction besides having optical, protective and therapeutic significance, contact lenses are widely used as cosmetic aids by the new generation (kumar, 1985). the contact lens industry has grown rapidly over the past four decades due to the widespread demands of the population for a convenient alternative to spectacle wear for the correction of myopia. unfortunately, many people who wear contact lenses are not aware of the potential risks associated with them, and consumer education about lens care has not been adequate (levey et al., 1996). most contact lens-related infections are often associated with inadequate contact lens hygiene, and therefore, contact lens care products should be able to sufficiently minimize the amount of pathogens that are responsible for these infections (hildebrandt et al., 2012). the widespread use of contact lenses has resulted in an increased incidence of microbial keratitis (wilson et al., 1981; weismann et al., 1984; alfonso et al., 1986; moore et al., 1987). there are approximately 125 million contact lens wearers globally (wu et al., 2010). corneal ulcers are a major cause of vision loss worldwide (sharma et al., 2007). the rate of progression of microbial keratitis is dependent on the virulence of the offending pathogen and host factors (ahn et al., 2011; aao, 2008). pseudomonas aeruginosa, one of the most common pathogens in clmk, is highly destructive and difficult to neutralize. another highly common pathogen in clmki.e. staphylococcus, may account for 45% of all bacterial keratitis (ahn et al, 2011; giraldez et al., 2010). enterobacter, serratia and klebsiella species which are usually of faecal origin can be transferred to the disinfectants by the wearers during the process of immersion or removal of the lenses from the solutions. in addition, some of the organisms like serratia and pseudomonas species are resistant to some disinfecting solutions (willcox et al., 2010). there is no doubt that antimicrobial performance of contact lens disinfection systems is an important factor in reducing contamination (zanetti et al., 1995). the objective of this study was to isolate the micro*corresponding author: aliyaap@yahoo.com 05 vol 5 (1), january 2014; 05-08 organisms which contaminate the ophthalmic disinfecting solutions and caused microbial keratitis, conjunctivitis and vision loss. materials and methods ophthalmic contact lenses solutions were obtained from 25 students of jinnah university for women who were the regular user of contact lenes. the sample was collected in sterile tubes and stored in room temperature. the tubes were opened under aseptic conditions. 50µl from each sample solutions inoculated in tryptic soy broth for enrichment and incubated at 24hrs at 37 °c. from enrichment broth, mannitol salt agar and macconkey agar were inoculated and incubated at 37°c for 24-48 hours. bacterial isolates were characterized by gram’s staining and following standard biochemical test such as imvic, catalase, tsi. antimicrobial susceptibility test was determined by kirby-bauer method. all strains were tested with lincomycin (l), streptomycin (s), cephalexin (cfx), oxacilin (ox), methicillin (met), penicillin (p), cefaclor (cfc). results and discussion in-use ocular solutions samples from 25 students commonly yielded gram-negative bacteria and gram positive bacteria. the age of students ranged from 22 to 25 years. results showed that a total of 43 strains were isolated including 20 gram positive and 23 gram negative bacteria. the bacterial species were enterobacter, pseudomonas, serratia, staphylococcus aureus, klebsiella, bacillus., e.coli, proteus, salmonella, and citrobacter and s. epidermidis. from contact lens solutions, s. aureus was the most common microorganisms found in this study. all 25 contact lens wearers (100%) were contaminated contact lenses solutions. figure 1 showed the percentage of isolated microorganisms. it was reported previously that p. aeruginosa was the most common contaminant of contact lenses solutions. in our study asymptomatic subjects were analyzed during the study, which showed that s. aureus was the highest number of all the isolate (26%) than pseudomonas spp. it has been implicated in several lens wearer complications including keratitis and corneal ulcers. the results of the present study are reflective of the observation that p. aeruginosa and s. aureus are the dominant bacteria that cause ocular infections among contact lens wearers. these finding are in confirmation with the earlier reports. in this study, gram+ bacillus sp. rate were 16%. few cases of bacillus keratitis among contact lens wearers were reported earlier (pinna et al., 2001). bacillus spores survived multiple lens disinfection treatments. key words: lincomycin (l), streptomycin (s), cephalexin (cfx), oxacilin (ox), methicillin (met), penicillin (p), cefaclor (cfc), resistant (r), intermediate(1.6cm). 06 vol 5 (1), january 2014; 05-08 figure 2: resistivity pattern of isolated organisms s.aureus figure 1: distributions of bacterial strains isolated from lens solutions s. aureus s. epidermidis e.coli the mic test results of pseudomonas show that resistivity against lincomycin, streptomycin, cephalexin, oxacilin, methicillin, penicillin, and cefaclor antibiotics. except bacillus spp, showed intermediate against streptomycin, all organisms were resistant and show no inhibitions (fig.2). a m i n o g l y c o s i d e s a n d f l u o r o q u i n o l o n e s (ciprofloxacin) are the choice of drugs than ß-lactams and given to those which show resistant with ßlactams conclusion in conclusion, our findings suggest that most contact lens disinfecting solutions may be ineffective if contact lens care systems become contaminated with micro-organisms. the most common bacteria that contaminate contact lenses and its accessories were s. aureus and p. aeruginosa. the results suggested that p. aeruginosa isolated from different infectious samples may have different characteristics. we found that all strains of p. aeruginosa were resistant to the antibiotic ß-lactam while three strains (serratia, s.aureus, and bacillus) were also resistant; except bacillus they were show intermediate to streptomycin so these antibiotics cannot be used in the treatment of infections caused by these resistant strains of p. aeruginosa and s. aureus. contact lens disinfecting solutions with same formulation but manufactured by different companies, possess different disinfecting potential. references aao. [2008]. american academy of ophthalmology cornea/external disease panel. preferred practice patterns guidelines. bacterial keratitis. san francisco, ca: american academy of ophthalmology. available at: www.aao.org/ppp. ahn m, yoon kc, ryu sk, cho nc, you ic. [2011]. clinical aspects and prognosis of mixed microbial (bacterial and fungal) keratitis. cornea; 30:409-13. alfonso e, mandelbaum s, fox mj, et al. [1986]. ulcerative keratitis associated with contact lens wear. am j ophthalmol. 101:429-433. giraldez mj, resua cg, lira m, et-al. [2010]. contact lens hydrophobicity and roughness effects on bacterial a d h e s i o n . o p t o m vi s s c i ; 8 7 : e 4 2 6 3 1 . hildebrandt c, wagner d, ohlmann t and kramer a. [2012]. bmc infectious diseases: 12:241. kumar d. [1985]. a textbook on contact lens practice. 1st edition. new delhi, india: cbs publishers and distributors. levey sb, cohen ej. [1996]. methods of disinfecting contact lenses to avoid corneal disorders, volume 06. pages 245251. moore mb, mc culley jp, newton c,et al .[1987]. acanthamoeba keratitis: a growing problem in soft and hard contact lens wearers. ophthalmology. 94:1654¬1661. pinna a, sechi la, zanetti s, usai d, delogu g, cappuccinelli p, carta f. [2001]. bacillus cereus keratitis associated with contact lens wear. ophthalmology 108: 1830-1834. sharma s, taneja m, gupta r, et-al. [2007]. comparison of clinical and microbiological profiles in smear-positive and smear-negative cases of suspected microbial keratitis. indian j ophthalmol; 55:21-5. weissman ba, mondino bj, pettit th, et al. [1984]. corneal ulcers associated with extended wear soft contact lenses. am j ophthalmol. 97:476-481. willcox mdp, carnt n, diec j, naduvilath t, evans v, stapleton f, et al. [2010]. contact lens case contamination during daily wear of silicon hydrogels. optom vis sci;87:456–64. wilson la, schilitzer rl, ahearn dg. [1981]. pseudomonas corneal ulcers associated with soft 07 vol 5 (1), january 2014; 05-08 contact lens wear. am j ophthalmol. 92:546-554. wu yt, zhu h, harmis ny, iskandar sy, willcox m, stapleton f. [2010]. profile and frequency of microbial contamination of contact lens cases. optom vis sci.; 87:153-158. zanetti s, fiori pl, pinna a, usai s, carta f, fadda g. [1995]. susceptibility of acanthamoeba castellanii to contact lens disinfecting solutions. antimicrob agents chemother 39: 1596-1598. 08 vol 5 (1), january 2014; 05-08 call for papers publication is free of cost rads journal of biological research and applied sciences welcomes the submission of research papers, review articles and short communications in the following subject fields: t biochemistry t biotechnology t botany t chemistry t environmental science t medical & health sciences t microbiology t molecular biology t pharmacy t physics t zoology t clinical research note:all manuscripts should be sent by e-mail to the editor with your mobile mumber. e-mail address : rads@juw.edu.pk or deanresearch@juw.edu.pk our aim is to provide a platform for budding scientists researchers, research scholars, academicians etc. to present their research findings in the field of biological and applied sciences. citrus fruit peels in combating various foodborne pathogens vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 64 r a d s j . b i o l . r e s . a p p l . s c i 64 op en ac ces s f u l l l e n g t h a r t i c l e citrus fruit peels in combating various foodborne pathogens aziz fatima*, maham ghouri and toba sajid jinnah university for women, karachi, pakistan a b s t r a c t foodborne diseases are the world's main problem in a society nowadays; many phytochemicals are of great interest to scientists. herbal medicines can be used to treat many infectious diseases. in our study, we focus on screening of antibacterial activity of the lemon (citrus limon) and orange (citrus sinesis) peels against various pathogens. bioactive compounds have been found in variety of citrus fruits that have favorable impact on human health. the major component; flavonoids have been found in the peels of citrus fruits in large amount. citrus peel has a rich amount of flavonoid and other poly methylated flavonoids (exceptional to some plants). these antibacterial compounds exhibit antibacterial activity and have wide application in both food and pharmaceuticals industries. the citrus peel extracts show effective antimicrobial activity. the antagonistic activity of selected citrus peel extracts was determined by agar well diffusion; against test organisms i.e. escherichia coli, salmonella typhi and bacillus subtilis isolated from food products. effectiveness of peel extracts are measured in terms of inhibitory zones in millimeter. the methanol extract of lemon peel displayed zone of inhibition in between 5mm to 8mm and methanol extract of orange peel displayed zone of inhibition about 10mm and 11mm. it was observed that lemon peel extract has maximum antibacterial activity against bacillus subtilis (8mm) and minimum antibacterial activity against salmonella typhi (5mm) whereas orange peel extract has maximum antibacterial activity against bacillus subtilis (11mm) and minimum antibacterial activity against salmonella typhi (10mm). both citrus peels extract showed considerable antibacterial activity against all tested foodborne pathogens. our main focus was the use of herbal treatments against various infections which overcome the emergence of upcoming superbugs. keywords citrus peel, antimicrobial activity, foodborne pathogens, flavonoids, agar well diffusion. *address of correspondence azizfatima1988@gmail.com article info. received: april 06, 2018 accepted: october 02, 2018 cite this article: fatima a, ghouri m, sajid t. citrus fruit peels in combating various foodborne pathogens. rads j. biol. res. appl. sci. 2018; 9(2): 64-66. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n the resistance of microbes has been increased due to extensive use of antimicrobial drugs. these organisms have various gene pools that carry such genes which have ability to acquire resistance from adjacent organisms1. in previous studies, plants and other natural sources are considered as vital agents’ natural compounds that have been used to cure many healthrelated issues2. the extracts of such medicinal plants can contribute a lot at industrial level especially in pharmaceuticals, due to their antibacterial properties3. among these plants, the products of citrus fruits have shown effective inhibitory activity against pathogenic bacteria. lemon and orange are important in relation to medicine, belongs to the family rutaceae (citrus family). o r i g i n a l a r t i c l e citrus fruit peels in combating various foodborne pathogens vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 65 r a d s j . b i o l . r e s . a p p l . s c i 65 these are the compounds which have potent bioactive component that have provided beneficial effect on human health such as vitamins, flavonoid, minerals, and alkaloids. flavonoids extracted from citrus peels have shown high spectrum of bactericidal, fungicidal activity along with, antidiabetic, anticancer and antiviral activities4,6. these bioactive components can act as free radical scavengers and are also capable to control different enzymatic reactions and inhibit cell progression. extracts of citrus peels have also shown the activity against phytopathogens5,7. the research study was design off to evaluate the inhibitory effect of peel extract of two citrus fruits namely lemon (citrus limon) and orange (citrus sinesis) peels against food borne pathogens. m a t e r i a l a n d m e t h o d extract preparation bacterial isolates microbes were isolated from different food products and identified using standard protocols. cultures were stored and maintained for screening antibacterial activity. the fruits were properly washed with distilled water for about 2 or 3 times. fruit peels were then separated, shredded into small pieces, dried in shaded area and then a fine powder were made. twenty-five grams of peel powder was then added into separate flask, mixed with 100 ml of methanol and kept for 24 hours. flask content was then filtered by using sterile what man filter paper as prescribed and allowed to dry by evaporation. condensed peel extract was then used for screening the bactericidal potential. collection of citrus fruits the ripe citrus fruits (orange and lemon) were collected from supermarket of karachi. agar well diffusion assay 24 hours old prepared nutrient broth inoculated with test organisms was matched to 0.5 mcfarland tube. twentyfive ml of mha was poured in labeled petri plates, test organisms were aseptically streaked on surface of sterile muller-hinton agar surface, followed by formation of agar wells of 10 mm diameter using a sterile borer. an amount of 0.5 ml of extract was then poured in respective wells. tetracycline disc was used as positive control. plates were then kept incubated for 1 day at 37ºc. zone of inhibition were then measured around the agar well. r e s u l t s & d i s c u s s i o n s antimicrobial activity of orange peel and lemon peel was assessed against the above-mentioned foodborne pathogens using agar well diffusion technique. the results are presented in fig. 1-4. fig. 1, 2 and 3: represent the inhibitory effects on e. coli, s. typhi and b. subtilis respectively. fig. 4: shows graphs of orange and lemon peels against test organisms. according to our findings, orange peel showed excellent inhibition against all the test organisms showing the highest zone of inhibition against b. subtilis, s. typhi and e. coli. orange peel thus showed a broad spectrum activity against all the selected foodborne isolates screened in this research study. on the other hand, lemon peel showed inhibition against all the test organisms with a maximum zone of inhibition against b. subtilis and minimum zone of inhibition against s. typhi whereas control i.e. tetracycline inhibits s. typhi, b. subtilis and e. coli shows resistance to tetracycline. we then compared result of this with the previous studies. demonstration with antibacterial activity of commonly available fruitpeels8 showed similar results as revealed in present study i.e. citrus peel extract of orange fruit showed with an inhibitory zone size of 11-12mm against bacillus subtilus. whereas s. typhi and e. coli were found resistant against both peel extract than the current study in which we found zone of inhibition 5-10 mm in lemon and orange peel respectively in case of s. typhi and 6-10 mm zone of inhibition in lemon and orange peel respectively in e. coli. it has been experimentally shown that in our study peel extracts of citrus fruit inhibit microbial growth. similar to citrus fruit peels in combating various foodborne pathogens vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 66 r a d s j . b i o l . r e s . a p p l . s c i 66 the study conducted in 2009. they evaluated bactericidal potential of peel oil using citrus fruits. it was found in the study that citrus plant including, citrus sinesis and citrus limon exhibit antibacterial activity against their test organisms or bacterial isolates. the resistance patterns of gram-negative bacteria against plants are not surprising. commonly the group of gramnegative bacteria have shown high resistance in comparison to gram-positive bacteria. this resistance pattern is due to permeability hence provided by their cell wall chemistry or by the membrane accumulation mechanism1. in this study, the ratio of salmonella was slightly inhibited by the activity of orange extracts. such results are very interesting and can be related to previous studies in which concentrated extracts produce more bacterial inhibition. the bioactive compounds of citrus peels can kill pathogenic bacteria using different mechanism and have high therapeutic value as an antibacterial agent against a number of multi-drug resistant bacterial populations. the extent of variation in the sensitive pattern of the gram-negative bacteria and gram-positive bacteria is because of their cell wall composition or its chemistry. gram-positive bacterial cell wall is the permeable due to presence of peptidoglycan as their outer cell layer on the other side gram-negative bacteria have high lipid content10. result plotted from the above research study provides the effective scientific basis for the use of methanol extract of citrus sinesis and citrus limon as antibacterial agent. this citrus fruit peel extract against the foodborne pathogen reveals them as better inhibitory agents than synthetic compounds. however, a number of studies have been required to screen the active constituent of these fruit peels with their pharmacology benefits. studies on citrus fruit peels are necessary to determine their active constituent activity11. the inhibitory effect of these extracts can be enhanced by the purification of the bioactive compounds and also measurable doses design for the administration. statistical analysis all the data obtained from secondary screening was analyzed by one-way anova. the level of significance was determined using spss version 15 and the results having a p value <0.05 were defined to be significant. r e f e r e n c e s 1. abu-shanab b, adwan g, abu-safiya d, jarrar n adwan k. antibacterial activities of some plant extracts utilized in popular medicine in palestine. turk j biol. 2004; 28: 99-102. 2. gislene gf, locatelli nj, paulo cf. giuliana ls. antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. braz j microbial. 2000; 31: 247-56 3. kirbaslar gf, tavman a, dulger b, turker g. antimicrobial activity of turkish citrus peel oil. pak j bot 2009; 41(6): 3207-12. 4. ortuno aa, baidez p, gomez mc, arcas i, porras ag, del rio j.a. citrus paradisi and citrus sinensis flavonoid: their influence on the defense mechanism against penicillium digitatum. food chem 2006; 98(2): 351-8. 5. seenivasan p, manickkam j, savarimuthu i. in vitro antibacterial activity of some plant essential oils. bmc complem altern. m2006 ;6: 39. 6. burt sa. essential oils: their antibacterial properties and potential applications in foods: a review. inter. j food microbiol 2004; 94: 223-53. 7. sohn hy, son kh, know cs, kang ss. antimicrobial and cytotoxic activity of 18 prenylated flavonoids isolated from medicinal plants: morus alba l., morusmongolica schneider, broussonetia papyrifera (l.) vent, sophora flavescens ait and echino sophora koreensis nakai. phytomed. 2004; 11: 666672. 8. jannatun t. demonstration of anti-bacterial activity of commonly available fruit extractsin dhaka, bangladesh. amer j microbiol research 2014; 68-73. 9. ghouri m, afshan n, javed s, fatima a, sadat a, chohan a, nadeem s.g. physiochemical evaluation and liability of dromedary camels milk in combating various pathogens. afri j microbiol res 2016; (17): 39-45. 10. malanovic n, lohner k. gram-positive bacterial cell envelopes: the impact on the activity of antimicrobial peptides. biochimica et biophysica acta (bba)biomembranes. 2016; 936-46. 11. gyawali r, salam a. natural products as antimicrobial agents. food cont 2014; 412-29. detection of du antigen in rh negative blood group individuals naheed afshan1*, sarah tariq1 1department of microbiology, jinnah university for women, karachi -74600, pakistan abstract du is the weak expression of d antigen. the cells which are not immediately agglutinated by anti-d sera cannot be easily classified as d negative because some of these agglutinate after addition of antiglobulin sera. this weak reactivity is termed as du. du positive cells are likely to elicit an immune response in d negative individuals and the du cells could be destroyed if the recepient is already immunized. therefore, du positive donor is treated as d positive and recipient is treated as negative. this report is based on du antigen and the testing of du antigen. in this report we discussed about the detection of du antigen using 2 different protocols that show how to test the presence and absence of du antigen in rh negative blood group individuals. in this study we included 100 blood group dnegative individuals .the result showed there were 3% that have du antigen in their blood. keywords: agglutination, blood group, du antigen, rh antigen, serum introduction the rh system includes many antigens but the major one is d, alternatively reffered as rho. the term rh positive is used to denote red cells that carry the d (rh) antigen or its variant du. red cells that have neither d nor du on their membranes are termed rh negative. with the exception of a and b, the most important of all blood group antigens is undoubtedly d. this is because the consequence of the presence can be severe and rh haemolytic disease of the newborn can be tragic: transfusion reaction due to rh antibodies can be heartbreaking experience. however, unlike the situation in the abo system, an rh negative person does not usually have antid in his or her serum. rh antigens are confined to red cells and are not found in body fluids or natural substances; therefore, exposure to red cells is the only way a person can become immunised to rh. also contributing to the importance of the rh system is the fact that the d antigen is one of the most effective blood group immunogens. as stated above, no natural substances chemically similar to the d antigen have been found; therefore when an rh negative person is found to have anti-d, that individual has invariably been exposed to rh positive cells. the two most likely ways for rh positive red cells to reach the circulation of an rh negative individual are: (1) transfusion of red cells from an rh positive donor to an rh negative recipient. except in rare circumstances, this is contrary to good transfusion practice; therefore it is usually the result of clerical or technical error, (2) passage of red cells from an rh positive foetus through the placenta to the rh negative mother. this almost always occurs to some extent at delivery and occasionally late in pregnancy. du antigen: in transfusion medicine, after the abo blood groups, the d antigen is the most significant. a high pro¬portion of people whose red blood cells (rbcs) lack d will make anti-d if exposed to the d antigen by pregnancy or transfusion. accordingly, all d– patients, especially girls and women who may become pregnant, should be transfused with d– rbcs. the d antigen is in the rh blood group sys¬tem, which with 49 distinct antigens is the most polymorphic blood group system. this document reviews fundamental in¬formation for the d antigen.*corresponding author: naheedafshan07@hotmail.com 16 vol 4 (2), july 2013; 16-19 du is the phenotypic term used to denote a weakened expression of the d antigen. du originally defined as those red cells reacting with anti-d only when a more sensitive indirect antiglobulin test was used. du phenotype can arise from three different genetic situations. a) a person may inherit a gene coding for weakened quantitative expression of d antigen. b) one gene may interact with another to modify and weaken the expression of the d antigen. c) a gene may not code for the total material that makes up the antigen.the frequency of du antigen is relatively low less than 1%. du is a poor immunogen, however, accelerated destruction of du red cells can result if transfused to a person already making anti-d. hence du donor units are currently labelled as rah positive. du recipients are labelled as rah negative. newborn of rah negative mother are tested for d & du and rh ig is recommended for mothers of d positive or du positive infants in order to prevent potential immunisation. the terms du variant or partial du are recommended when there is both a qualitative and quantitative difference noted in the d antigen. du testing: not all red cells can be classified as rh positive or negative by direct agglutination tests. the cells of a few persons react weakly with antid or requires a longer reaction time than most rh positive cells. an even smaller number of persons have red cells that are not agglutinated by not not all red cells can be classified as rh positive or negative by direct agglutination tests. the cells of a few persons react weakly with anti-d or requires a longer reaction time than most rh positive cells. antiglobulin serum. an even smaller number of persons have red cells that are not agglutinated by antiglobulin serum. these cells are called du. cells of the du phenotype may fall anywhere within this spectrum of reactivity with anti-d. because du is a form of d, red cells of the du phenotype can stimulate the production of anti-d in rh negative recipients and, more importantly, react with anti-d in vivo. it is for these reasons that donor blood must be shown to be negative not only in the test for d but also in the test for du. in general, testing the red cells of recipients for du is considered unnecessary. the recipient's welfare is not compromised if he or she is of the du phenotype but is typed as d negative and receives rh negative red cells. in such circumstances rh negative donor blood may be used unnecessarily. it is important that the du status of the d negative pregnant woman be established early in pregnancy. if the mother is found to be rh positive, du varient, she is not a canditate for rh immunoglobulin prophylaxiseither antepartum or postpartumwhereas the rh negative (d and du negative) mother is a canditate. the reason for performing the du test early in pregnancy is to avoid mis-interpreting the cause of a positive fetal cell screening test at the time of delivery. in addition to prenatal patients, newborn babies are also tested for du if they type as d negative. again, this relates to the need for rh immune globulin: the d negative, du negative baby cannot immunize its mother; for this reason she does not need rh immune globulin protection. however, the mother should receive rh immune globulin if the baby is of the du phenotype. du red cells fall into a wide spectrum of reactivity when tested with anti-d reagents. how each cell is detected depends on the type of anti-d that is used and the kind of test that is performed. to test for du, red cells are incubated at 37oc with an igg anti-d and an antiglobulin test is performed. if serum suspended cells are used, some blood samples at the upper end of the du spectrum will be agglutinated weakly by most anti-d reagents prior to the antiglobulin test, either at room temperature or at 37oc. when the same red cells are suspended in saline, direct agglutination may not be observed, or it may be seen with one reagent and not another. regardless of whether the red cells are agglutinated directly by anti-d or they absorb anti-d and it is detected in the antiglobulin phase of the test, they are rh positive, provided both controls for d typing and the du test are negative. 17 vol 4 (2), july 2013; 16-19 materials & methods blood sample: any blood group sample with edta which is rh-negative. reagents: anti-d antisera, 3-5% red cell suspension, normal saline, coombs reagent, 37 c incubator, albumin. procedures: i) without albumin: prepare a washed, 3-5% suspension of rbcs. add 50ul anti d in a tube containing 50ul 3-5% red cell suspension. incubate the tube at 37 c for 40-45 minutes. after 40-45 min, suspend the tube & examine agglutination, if agglutination occurs it means rh is positive & if no agglutination present it confirmed that rh is negative. centrifuge the suspension at 3500 rpm for 15 seconds. after centrifugation, washed the suspension 3 times with normal saline. after 3rd time washing, tapping should be done so that all the remaining saline should be removed from the cell suspension. add 1-2 drops of coombs reagent in the tube containing washed red cells. after addition of coombs reagent, centrifuge the tube at 3500 rpm for 15 seconds. immediately resuspend the tube and examine for agglutination using electron microscope. confirm all negative results by adding one drop coombs control cells to all tubes showing no agglutination and centrifuge1530 seconds at 3500 rpm. gently resuspend & examine for agglutination. agglutination should be present in this step or the test is invalid. ii) with albumin: prepare a washed, 3-5% suspension of rbcs. add 50ul anti d & 1-2 drops albumin in a tube containing 50ul 3-5% red cell suspension. incubate the tube at 370c for 40-45 minutes. after 40-45 min, suspend the tube & examine agglutination, if agglutination occurs it means rh is positive & if no agglutination present it confirmed that rh is negative. centrifuge the suspension at 3500 rpm for 15 seconds. after centrifugation, washed the suspension 3 times with normal saline. after 3rd time washing, tapping should be done so that all the remaining saline should be removed from the cell suspension. add 1-2 drops of coombs reagent in the tube containing washed red cells. after addition of coombs reagent, centrifuge the tube at 3500 rpm for 15 seconds. immediately resuspend the tube and examine for agglutination using electron microscope. confirm all negative results by adding one drop coombs control cells to all tubes showing no agglutination and centrifuge1530 seconds at 3500 rpm. gently resuspend & examine for agglutination. agglutination should be present in this step or the test is invalid. observation 18 vol 4 (2), july 2013; 16-19 table ii. results showing presence and absence of du antigen. table i. how to read the result. results & discussion 100 blood samples taken from different individuals having different blood groups but all were rh negative individuals. from 100 samples, 3 were du positive. as described earlier, people whose rbcs have a weak d phenotype (quantitative d variant) do not make anti-d, whereas people whose rbcs have a partial d phenotype (qualitative d variant with or without weakening of the d antigen) can make alloanti-d. this presents a different problem depending on whether the person is a donor or a patient. for donors, detection of weak and partial d antigens would eliminate the possibility of immunization should such blood be transfused to a true d-negative patient. however, historical data show that weakly expressed d antigens are most unlikely to be immunogenic. clinical complications result from rbc destruction due to the interaction of an alloantibody with rbcs carrying the corresponding antigen. the d antigen is highly immunogenic and induces an immune response in 80% of d-negative persons when transfused with 200 ml of d-positive blood. for this reason, in most countries d typing is performed routinely on every blood donor and transfusion recipient so that d-negative patients receive dnegative rbc products. consequently, clinical complications due to mismatched transfusions are infrequent. in contrast, despite the use of i m m u n o s u p p r e s s i v e t h e r a p y w i t h a n t i d immunoglobulin prophylaxis, d alloimmunization in pregnancy still occurs. patients with acute or chronic myeloid leukemia, myeloid metaplasia, polycythemia, or myelofibrosis occasionally have 2 populations of rbcs of different rh type. in some cases, a loss of rh antigens is a s s o c i a t e d w i t h c h r o m o s o m e a b e r r a t i o n s . conclusion from the result, it is concluded that there were rare cases in peoples that have du antigen in their blood. we cannot avoid to detect du antigen before transfusion because if donor is rh negative and recipient is also rh negative but du antigen is present in the donor blood so if we can’t test du antigen mismatched in transfusion occur which will results in mild to life threatening complications or death. references reid me and lomas-francis c. 2004. blood group antigen factsbook. 2nd ed. san diego: academic press. reid me and mohandas n. 2004. red blood cell blood group antigens: structure and function. semin hematol, 41: 93-117. reid me and yazdanbakhsh k. 1998. molecular insights into blood groups and impli¬cations for blood transfusions. curr opin hematol, 5:93-102. tayyab m, malik ar and khan as. 2000. du phenotype a review. jamc,12(3): 41-44. westhoff cm. 2007. the structure and function of the rh antigen complex. semin hematol, 44:42-50. 19 vol 4 (2), july 2013; 16-19 prevalence study of stress throughout pregnancy vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 18 r a d s j . b i o l . r e s . a p p l . s c i . 18 op en ac ces s f u l l l e n g t h a r t i c l e prevalence study of stress throughout pregnancy zehra ashraf*, nimra waheed* faculty of pharmacy, jinnah university for women, karachi, pakistan a b s t r a c t background: stress in pregnancy refers to a particular type of stress-related with the interaction with people or any other cause like workload, society etc. today, however, we are increasingly exposed to prolonged stress which gives rise to problems such as sleep problems, headache, fatigue, memory disturbances and so on. stress in pregnancy can have a major impact on the mother as well as the developing fetus. various symptoms and issues faced by the mother can indirectly have an adverse effect on the baby’s development. aim of study: the purpose of this study is to understand and identify the effect of stress on women during pregnancy. method: the research performed included 100 women which give us a general overview of how women of our society whether of high or low socioeconomic status cope with their stress or even understand the term "stress" in the first place. this study was carried out in various hospitals (maternity wards) to get an insight into our research. various aspects were identified in order to draw out clear conclusions. result and discussion: 83% of women agreed on the fact of suffering from stress during pregnancy, with the other 16% replying "no" and the remaining 1% had no clue. considering the relationship of socioeconomic factors are mostly affecting in pregnancy which is agreed by 66% of the female population while 18% disagree with this factor. conclusion: our overall approach throws light on understanding and detecting these aspects during pregnancy. to help provide better alternatives and have a stress-free pregnancy. keywords pregnancy, problems, behavior, health, stress. *address of correspondence dr.zehra.ashraf@gmail.com, nimra_w@hotmail.com article info. received: april 20, 2018 accepted: february 13, 2019 cite this article: ashraf z, waheed n. prevalence study of stress throughout pregnancy. rads j. biol. res. appl. sci. 2019; 10(1): 18-22. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n stress is a feeling of strain and pressure. small amounts of stress may be desired, beneficial, and even healthy1-3. stress in pregnancy is defined as the imbalance that a pregnant woman feels when she cannot cope with demands which are expressed both behaviorally and physiologically4-6. stress is the result of a cognitive appraisal (your mental interpretation) of what is at stake and what can be done about it7-9. stress during pregnancy is associated with multiple complications like insomnia anorexia etc. however interventions to prevent stress during pregnancy have been almost evaluated but up to some extent10-12. the importance of conducting such research helps us to better understand the possible outcomes on the health of the mother as well as the baby and to minimize the adverse effects caused by such forms of stress. it is essential to understand the sensitivity of the matter, each woman experiences and reacts differently to the kind of situation they are encountered with which further leads to the development of sometimes known and otherwise unconscious stress. we sought to identify the factors associated with stress and to find new interventions to prevent and overcome such problems during the course of pregnancy. our goal to conduct such research on this topic was for the o r i g i n a l a r t i c l e prevalence study of stress throughout pregnancy vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 19 r a d s j . b i o l . r e s . a p p l . s c i . 19 betterment for our women and to spread awareness and highlight the problems which can be resolved within our society, to be able to acknowledge why being stress free during pregnancy can have an astonishing impact not only for the mother and the baby but for all the members directly or indirectly related to them. m e t h o d o l o g y place of study karachi is one of the largest cities in the world. it is known as cities of lights and also the 2nd largest populous city of the world, a cosmopolitan city of pakistan. propose of study cross-sectional analyses of data were conducted into various hospitals, parental care of karachi from the 1st of october to 31st of december 2016. the questionnaire was m.c.qs based asking the women about how they are experiencing stress during pregnancy. we also provide some awareness to these women who didn't even aware of suffering from stress and diseases that cause by psycho-social stress. we also asked about their lifestyles that how it is affecting their health and what type of symptoms they observed other than pregnancyrelated complications. we collected the data after the survey and discussed our findings in this article which is represented by graphs for better understanding. r e s u l t upon calibrating the results from our survey we concluded that about 83% of women were aware of the term stress whereas the other 16% don't know, shown in figure 1. figure 1. aware with the term “stress” in pregnancy. further questions about whether they experienced stress during pregnancy lead to the observation that 77% suffered through stress whereas 23% of women were stress-free, presented in figure 2. figure 2. illustrates the proportion of women facing various forms of stress during their pregnancy. moreover, we emphasized on the varied sorts of stresses in which 41% female experience pregnancy-related stress, 26% are dealing with discomfort, 14% occurrence of stress due to negative life experience, 8% of the women are scared of gender discrimination issues, 2% have long-lasting stress throughout the pregnancy while 9% occurrence of stress due to other issues (figure 3). figure 3. points out to the varied sorts of stress that women face. figure 4 represents the 21.5% of women who preferred to take some sort of medication while 78.5% of female does not prefer to take any medication to relieve their stress. prevalence study of stress throughout pregnancy vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 20 r a d s j . b i o l . r e s . a p p l . s c i . 20 figure 4. taking medications for relieving stress during pregnancy. most of the vivid dreams occur in 3rd trimester that is 23.7% while 11.8% in the 2nd trimester and 8.6% in the 1st trimester and 55.6% of the women does not have any vivid dream in whole pregnancy period (figure 5). figure 5. occurrence of vivid dreams in pregnancy. socioeconomic factors are mostly affecting in pregnancy which is agreed by 66% of female population while 18% disagree this factor that is shown in figure 6. 46.2% of housewife faced psychosocial stress during pregnancy while a higher rate of stress that is 53.7% was experienced by working women, this is illustrated in figure 7. figure 6. socioeconomic factors affecting on pregnancy. figure 7. percentage of women who agree upon the fact that working women experience more psychosocial stress than women who are home makers. many women experience a lot of symptoms which are 22.6% insomnia, 21.5% vomiting, 18.3% anxiety, 13.9% nausea, 7.5 headache, and 16.1% are other symptoms during pregnancy, presented in table 1. alternative ways of stress relief during pregnancy are represented in table 2. 41.9% think that their stress can be reduced by talking with friends or other partners and 9.7% of women believed that exercise and healthy food can reduce their stress. physiological changes that have been observed during pregnancy are preeclampsia (6%), diabetes (9%), edema (39%) and others (28%), these are shown in table 3. prevalence study of stress throughout pregnancy vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 21 r a d s j . b i o l . r e s . a p p l . s c i . 21 table 1. symptoms of stress during pregnancy. symptoms of stress percentage % anxiety 18.3% nausea 13.9% vomiting 21.5% insomnia 22.6% headache 7.5% others 16.1% table 2. demonstrates the various other ways women prefer to relieve their stress rather than choosing to take medicines. how do you cope up with your stress usually, if you are not taking medication percentage % 1: eating healthy food 9.7 2: having plenty of sleep 20.4 3: exercise (yoga or massage) 9.7 4: talk to someone (friend or partner) 41.9 5: if other 9.7 table 3. physiological changes observed during pregnancy. type of physiological changes observed during pregnancy percentage of these changes observed preeclampsia 6% diabetes 9% edema 39% others (obesity, stomach problems, severe pain, etc.) 28% d i s c u s s i o n the survey was aimed to study stress in women that lead to specific pregnancy outcomes. the survey shows that 83% of women aware of the term stress, while 77% of women felt stress during pregnancy which includes different sort of stress. most of the women took medications to cope with stress while some relay on diet and some took enough of sleep. the survey also elaborates that working women experience more stress than housewives, and the most common symptom due stress was vomiting and insomnia and occurrence of vivid dream especially in the 3rd trimester. the stress leads to certain physiological changes in the body like preeclampsia, edema. while considering the relationship between socioeconomic status and stress it was found that 61% of women felt stress during pregnancy. (but this study is still in progress for the treatment of stress). c o n c l u s i o n evaluating the results from this study it can be concluded that women of our society face various forms of stress which can indirectly be the reason/cause of complication during pregnancy and may also have adverse effects on the fetus during/even after birth. according to our study stress-free pregnancy is not possible because 83% women face stress whereas the remaining 16% have no idea about stress but they have a responsibility to other questions of which they experience during their pregnancies. this depicts that they were facing stress while the remaining 1% didn't experience stress during their pregnancies which is very less ratio comparatively to others. stress can be controlled by following alternative ways such as eating healthy foods, taking enough sleep, exercise like yoga and massage, talk to someone close to them etc. if stress is migrated it can cause harmful effects like preeclampsia, diabetes, edema, obesity severe pain etc. to avoid such situations proper guidance and counseling sessions must be provided to the conceived mother for better physical and mental wellbeing essential for both the mother and her child. r e f e r e n c e s 1. woods sm, melville jl, guo y, fan my, gavin a. psychosocial stress during pregnancy. american journal of obstetrics and gynecology. 2010;202(1):61e1. 2. ruiz r, fullerton j. the measurement of stress in pregnancy. nursing & health sciences. 1999; 1(1):1925. prevalence study of stress throughout pregnancy vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 22 r a d s j . b i o l . r e s . a p p l . s c i . 22 3. halbreich u. prevalence of mood symptoms and depressions during pregnancy: implications for clinical practice and research. cns spectrums. 2004; 9(03):177-84. 4. curry ma. the interrelationships between abuse, substance use, and stress during pregnancy. journal of obstetric, gynecologic, & neonatal nursing. 1998; 27(6):692-9. 5. nuckolls kb, cassel j, kaplan bh. psychosocial assets, life crisis and the prognosis of pregnancy. american journal of epidemiology. 1972;95(5):43141. 6. hobfoll se. conservation of resources: a new attempt at conceptualizing stress. american psychologist. 1989; 44(3):513. 7. côté-arsenault d. threat appraisal, coping, and emotions across pregnancy subsequent to perinatal loss. nursing research. 2007; 56(2):108-16. 8. colman ad. psychological state during the first pregnancy. american journal of orthopsychiatry. 1969; 39(5):788. 9. alder j, fink n, urech c, hösli i, bitzer j. identification of antenatal depression in obstetric care. archives of gynecology and obstetrics. 2011;284(6):1403-9. 10. okun ml, roberts jm, marsland al, hall m. how disturbed sleep may be a risk factor for adverse pregnancy outcomes a hypothesis. obstetrical & gynecological survey. 2009;64(4):273. 11. sangsawang b, sangsawang n. stress urinary incontinence in pregnant women: a review of prevalence, pathophysiology, and treatment. international urogynecology journal. 2013; 24(6):90112. 12. lupien sj, mcewen bs, gunnar mr. effects of stress throughout the lifespan on the brain, and cognition. nature reviews neuroscience. 2009; 10(6):434. probiotics as human health promoters vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 102 r a d s j . b i o l . r e s . a p p l . s c i 102 op en ac ces s f u l l l e n g t h a r t i c l e probiotics as human health promoters shiekh ajaz rasool*, fehmida mirza, hera waheed and muhammad munir department of microbiology, jinnah university for women, karachi, pakistan a b s t r a c t probiotics (pro-life live entities) provide the health and well being with multitude of beneficial effects on humans and animals (and relief against varied disorders). probiotics may manage lactose intolerance, elevate immune profile, prevent colorectal cancers, reduce cholesterol and triglyceride profile, lowering blood pressure and inflammatory process. they also prevent osteoporosis, allergic reactions and help suppress h. pylori infections and other pathological manifestations. microbial metabolites (even in the absence of live entities) may exert (analogous) effects on signal pathways and barrier functions. such substances are referred as ‘postbiotics’ (the plain metabolic byproduct of probiotics, bioactive manifestations in the host). generally, postbiotics include secondary metabolites such as bacteriocins, organic acids, ethanol, acetaldehyde, reactive oxygen species (ros). such metabolites are inhibitory against pathogenic strains of different broad spectrum drug resistant microbial groups (mdr, xdr etc). postbiotics are safe, apathogenic which may resist hydrolysis by enzymes of mammalian origin. it has been described that micro-rna profile of human milk may exert the inhibitory effects of probiotics. our research group has been investigating the merits of mammalian milk as a viable source of probiotics that secrete bioactive peptides against mdr/biofilm producing strains (ref. streptococcus thermophilus and enterococcus faecalis, a git probiont). these peptides are in the range of 10-16kda molecular mass (sensitive to proteolytic enzymes as well). genes coding for these peptides are plasmid associated. mode of action of these peptides is bacteriostatic. molecular identification of these probiotic strains is being followed. this, on the whole marks an emphasis on biological operation of novel strains of probiotic and their applications in medico-clinical areas to improve the human health and wellness. keywords probiotics, postbiotics, bioactivity, mdr, biofilm. *address of correspondence drajazrasool@gmail.com article info. received: april 02, 2018 accepted: september 24, 2018 cite this article: rasool sa, mirza f, waheed h, munir m. probiotics as human health promoters. rads j. biol. res. appl. sci. 2018; 9(2): 102-105. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n probiotics constitute live microorganisms that are laced with medical and health advantages. human gut is a unique reservoir for such microbes. despite the fact that microorganisms are frequently considered as destructive "germs", numerous microorganisms enable our body to work legitimately. for instance, microbes that are typically present in our digestive tract (git) help in digestion of food, repulse infection causing microorganisms, and deliver vitamins. substantial quantities of microorganisms live on and in our bodies. actually, microorganisms in the human body out number human cells by 10:1. probiotics play a vital role in digestive tract and maintain healthy balance between normal flora of gut and in turn healthy digestive tract filters out toxins, chemicals, pathogens and waste products 1. the probiotics help boost immune system. they protect body against pathogens (fig. 1). r e v i e w a r t i c l e probiotics as human health promoters vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 103 r a d s j . b i o l . r e s . a p p l . s c i 103 otherwise, allergic manifestations, autoimmunity processes (e.g. colon ulcer, ibd) and infection based diarrheas, dermatitis and vagino-urinary clinical indications may ensue 2,3. fig. 1: mode of action of probiotics. in 1908 a russian noble laureate, ellie metchnikoff, revealed the useful effects of probiotics on human wellbeing. he proposed that natives of bulgaria feel comfortable and enjoy longevity as they consume probiotic processed milk products (courtesy lab strains). the term probiotic was first coined by parker as microorganisms and metabolites that establish git microbiome equilibrium. the updated statement regarding probiotics was referred by havenaar and huisint veld as live single or consortia of bacterial strains that (after application) do benefit the host by boosting the merited characteristics of resident microbiome (fig. 2). some of the common probiotic bacterial genera include lactobacillus (lab), bifidobacterium, enterococcus, streptococcus, bacillus, pediococcus and saccharomyces 4,5. postbiotics are non-viable, non-toxic and non-pathogenic bacterial secondary metabolites of probiotics which exhibit bioactivity within the host. they constitute bacteriocins, ethanol, hydrogen peroxide, acetaldehyde, diacetyl and organic acids. researchers have revealed that these metabolic byproducts carry broad spectrum bioactivity against pathogenic microorganisms and hence, may be used as an alternative to antibiotics. bacteriocins constitute the most appealing research topic of recent era. antibiotic resistance has lead to loss of efficacy of conventional antimicrobials. in that case bacteriocins can be used to target mdr, edr, xdr strains and biofilm formers. probiotics possess important aspects that can fulfill our daily nutritional requirements and help our body combat different clinical conditions. a few prominent characteristics regarding probiotics include strategies that may be applied to encounter cancers, pathogenicity, obesity, allergies and diabetes etc 2,6 (fig. 3). fig. 2: beneficial effects of ‘biotics’. defering the cancer activity cancers are referred as the syndromes that affect people all over the world. radiotherapy, chemotherapy and surgical interventions are used to treat different kinds of tumors and cancers, but they leave behind the side effects which outnumber the positive aspects. the natural products and sources with anti cancerous activity are of major value in recent era. probiotics have attracted interest by medical researchers to develop effective drugs with anti-cancerous property and minimal side effects. l. acidophilus and l. rhamnosus are known for their antitumor activity. moreover, l. casai and l. acidophilus have also shown anti-cancer property to encounter cancers of colon and rectum in vitro 2,7 (fig. 3). encountering pathogenesis apathogenesis is considered as an important function of the biotics. tejero-sarinena et al. revealed the pathogen inhibition by producing short chain fatty acids e.g. acids of butyrate, propionate, acetate and lactate. they assist in maintaining ph of the gut thereby, creating favorable environment for normal flora to flourish 8. mohseni et al. concluded that lactobacillus acidophilus, due to the production of the antimicrobial compounds, effects on bacteria and yeast pathogens of vagina. obviously, it also helps in prevention and treatment of uro-genital infections 2,9. anti-obesity activity obesity or excessive weight gain is a well known side effect of modern life style and unhealthy food intake. it is probiotics as human health promoters vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 104 r a d s j . b i o l . r e s . a p p l . s c i 104 the cause of many other underlying medical conditions like hypertension, hypercholesteremia and diabetes. probiotics possess physiological functions that facilitate weight loss by lipolytic and thermogenic response by regulating sympathetic nervous system. l. gasseri with its metabolites inhibits the enhancement in adipocyte tissues which principally produces leptin and adiponectin. generally, strains of lab and bifidobacteria have been recorded for hypocholestremic state 10. anti-inflammatory activity inflammatory chronic disorders like ibs/ibd could be rescued by probiotic–postbiotic supplements. malfunction of short chain fatty acids may result in ibd onset. scfas are involved in regulating homeostasis and also defer allergic reactions 11. for this purpose, lab, bifidobacteria and members of family enterobacteriaceae carry due importance (e.g. e. coli) 6. anti-diabetic activity diabetes is a non-communicable chronic condition associated with absence or insufficient production of insulin. there is no definitive cure for this condition and is managed by multiple medications (e.g. the use of synbiotics in curing the diabetic condition). larsen et al. proposed that by increasing the number of probiotics, metabolic diseases like diabetes can be managed. firmicutes dominate the gut microenvironment. research shows that patients suffering from metabolic disorders have reduced numbers of these species (fig. 3). in this aspect, probiotics could help in regulation of gut flora and managing the chronic conditions 12,13. fig. 3: multiple role of the ‘biotics’. antagonism by probiotic lactic acid bacteria and others lactobacillus reuterii produces a small molecular mass metabolite “reuterin” (a class i bacteriocin with broad spectrum bioactivity) 14. e. feacalis also synthesizes a class i bacteriocin against mdr strains. a class ii protein antibiotic is produced by l. plantarum. l. lactis produces “nisin”, an fda approved food preservative/additive 15. bacteriocins such as lugdunin, streptococcin are also known for manifestation of bioactivity against mdr strains16. micro-rnas are in abundance in milk of the mammals. it can mediate the inhibitory effects of probiotics. it helps in boosting the immunity process and gastrointestinal tract of the newborn, also prevents atopic dermatitis by probiotic intake in perinatal period 17,18 (fig. 4). fig. 4: valuable components of human milk. we have been focusing on the significance of mammalian milk as a rich source of probiotics that possess peptides with bioactive potential against mdr and biofilm producer strains. the isolated producer strains include: streptococcus thermophilus from goat (capra aegagrus hircus) milk and enterococcus fecalis (a gut probiont). these secondary metabolites are peptides (10kda streptococcin and enterococcin of 16kda. mass). genes that code for these bioactive peptides are plasmid borne. mode of action is bacteriostatic. experiments on the molecular identification of these probiotic strains are underway. these studies are in accordance with the emphasis on natural activities of novel strains of probiotics and their application in the field of biomedicoclinical sciences for enhancing the human wellbeing 5,6. probiotics as human health promoters vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 105 r a d s j . b i o l . r e s . a p p l . s c i 105 r e f e r e n c e s 1. patel rm, denning pw. therapeutic use of prebiotics, probiotics, and postbiotics to prevent necrotizing enterocolitis: what is the current evidence? clinics perinatol. 2013; 40(1), 11-25. 2. kerry rg, patra jk, gouda s, park y, shin hs, das g. benefaction of probiotics for human health: a review. jfda. 2018; 1-13. 3. bermudez-brito m, plaza-díaz j, muñoz-quezada s, gómez-llorente c, and gil a probiotic mechanisms of action. ann nutr metab. 2012; 61:160-174. 4. parker ga. assessment strategy and the evolution of fighting behaviour. j theoret biol. 1974; 47:223-243. 5. havenaar r, huis jh. probiotics: a general view. in the lactic acid bacteria. springer, boston, ma. 1992; volume 1: 151-170. 6. gowri rs, meenambigai p, prabhavathi p, rajeswari pr, yesudoss la. probiotics and its effects on human health; a review. int j curr microbiol appl sci. 2016; 5: 384-392. 7. awaisheh ss, obeidat mm, al-tamimi, hj, assaf, am, el-qudah jm, al-khazaleh jm. in vitro cytotoxic activity of probiotic bacterial cell extracts against caco-2 and hrt-18 colorectal cancer cells milk sci int. 2016; 69: 2731. 8. tajero-sarinena s, barlow j, costabile a, gibson gr, rowland i. antipathogenic property of probiotics against salmonella typhimurium and clostridium difficile in anaerobic batch culture system: is it due to synergies in probiotic mixtures or the specificity of single strain? anaerobe. 2013; 24:60-5. 9. mohseni ah, bahmani s. and yaghoubch s. antipathogenic activity of probiotic lactobacillus acidophilus against urogenital infections. conference paper, islamic azad university, tehran, science and research branch. res gate. 2014; 304019626. 10. kang jh, yun si, park mh, park jh, jeong sy, park ho. anti-obesity effect of lactobacillus gesseri bnr17 in high-sucrose diet induced obese mice. plos one. 2013; 8:1-8. 11. currò d, ianiro g, pecere s, bibbò s and cammarota g. probiotics, fibre and herbal medicinal products for functional and inflammatory bowel disorders. british j pharmacol. 2017; 174(11):1426-1449. 12. larsen n, vogensen fk, van-den-berg fw, nielsen ds, andraesen as, pedersen bk. gut microbiota in human adults with type 2 diabetes differs from non-diabetic adults. plos one. 2010; 5:1-10. 13. membrez m, blancher f, jaquet m, bibiloni r, cani pd. gut microbiota modulation with norfloxacin and ampicillin enhances glucose tolerance in mice. faseb j. 2018; 22: 2416-2426. 14. talarico tl, casas ia, chung tc, dobrogosz wj. production and isolation of reuterin, a growth inhibitor produced by lactobacillus reuteri. antimicrobe agents chemotherap. 1988; 32(12):1854-1858. 15. amenu d. probiotic properties of lactic acid bacteria from human milk. j med microb diagn. 2014; s3-005. 16. rasool sa, ajaz m. probiotics: the live therapies. (muhammadi advertizing, karachi, pakistan). isbn no: 978-969-23188-0-8. 2017; 1-209. 17. simpson mr, brede g, johansen j, johnsen r, storrø o, sætrom p, oien t. human breast milk mirna, maternal probiotic supplementation and atopic dermatitis in offspring. plos one. 2015; 10(12): e0143496. 18. parigi, sm, eldh m, larssen p, gabrielsson s, villablanca, ej. breast milk and solid food shaping intestinal immunity. front immunol. 2015; 6:415. to study the efficacy of bio-sand filter for commercial use vol 8 (2), july 2017 issn (print): 2305 – 8722 issn (online): 2521 – 8573 46 r a d s j . b i o l . r e s . a p p l . s c i 46 op e n ac c e ss f u l l l e n g t h a r t i c l e to study the efficacy of bio-sand filter for commercial use maria aslam department of biochemistry, jinnah university for women a b s t r a c t water is essential for life. without water, not only, no one can survive but it also affects health, education and well being of a person. industrialization, climate change and other environmental factors contributes in contamination of water. access to safe potable water is basic human right for drinking, and other purposes. filtration through biosand filter is among one of several methods for the purification of contaminated water. biosand filter is easy to construct, use and maintain. it can be made from local materials and is also low in cost. the purpose of this study was to investigate the effectiveness of the biosand filter. in this study 30 samples were run to detect the effectiveness of biosand filter. results suggest that biosand filter effectively reduced microbial load up to 98%. it is also effective in removing suspended particles, turbidity and other impurities from water and improves the taste and quality of water for drinking, sanitation and other purposes. keywords water contamination, biosand filter, microbial reductions, house hold drinking water treatment. address of correspondence provide email address article info. received: april 3, 2017 accepted: may 29, 2017 cite this article: aslam m. to study the efficacy of bio-sand filter for commercial use. rads j. biol. res. appl. sci 8(2):46-50. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n today widespread problem which the world facing is poor and deprived access to clean drinking water. since access to sufficient water and sanitation services influences everybody’s health, education, life expectancy, well-being and social development (1). safe potable water accessibility has become a human right. poor water quality and insufficiency has becoming a serious problem worldwide, especially in the rural areas. this situation has been brought about by industrialization and climate change resulting in safe drinking water being a scarce resource. approximately one billion people have no approach to safe drinking water (2, 3). 2.6 billion people lack basic sanitation. safe drinking water and basic sanitation are necessary to health and they are being taken for granted (4). water should be free from contamination (5). for this purpose household water treatment and safe storage is as an effective way to provide clean water to the people in rural areas (6). the household water treatment process is focused on removing pathogens from drinking water (7)] by biosand filtration unit (bsf), designed for household use (8). it is small and does not require continuous delivery of untreated water. a bsf consists of a concrete or plastic container, approximately 0.9 meters tall and 0.3 meters wide. it is filled with specially selected sand and gravel. as water is passed through the filter, physical straining eliminates pathogens, iron, turbidity etc from water. a shallow layer of water is present at the top of sand and forms biofilm (9, 10). it takes 10-20 days for biofilm to develop which consists of bacteria, fungi, protozoa, and o r i g i n a l a r t i c l e to study the efficacy of bio-sand filter for commercial use vol 8 (2), july 2017 issn (print): 2305 – 8722 issn (online): 2521 – 8573 47 r a d s j . b i o l . r e s . a p p l . s c i 47 aquatic insect larvae (11).the diffuser plate contains a number of small holes that lower the flow of the influent water and prevent it from disturbing the bio-layer (12). the influent water displaces the water in the filter, and is collected by a drain system present at the bottom of the filter that is removed through a vertical tube. the outlet of the tube is present at a few centimeters above the surface of the sand (13). the biofilm contributes to the removal of pathogens by predation and competition for food (10). the biosand filter can be constructed locally, it is easy to use and maintain, and it is low cost. water produced by the filters is free of discoloration, odor, undesirable taste and this filtered water can be used for drinking, food preparation and other purposes. these filters are effective in the elimination of viruses, protozoa and helminthes up to 99.9%, and 85% of turbidity (14). m a t e r i a l s a n d m e t h o d s sample size: the sample size of the project is 30. sample collection: the water sample was collected from different areas of karachi including sabzimandi, gulshan, nazimabad #3, eissa nagri, lalukhait, north karachi, buffer zone, north nazimabad, gulistan-e-johar, dastageer, shadman, nazimabad #2, fb area, and shahra-e-faisal, nazimabad # 4, nazimabad # 7, clifton, garden east, karimabad, m.a jinnah road, malir, orangi town, saddar, tower, azizabad, lasbaila, al-noor and golimar respectively. the samples were checked for microbial load before and after filtration through biosand filter. construction of biosand filter: pet bottle is taken and upper part of the bottle was cut with the help of hot and sterile cutter. holes were made with the help of sterile needle in the separated part of the bottle and also in the cap which was already fixed to the bottle. another hole is made at the lower part of the bottle using the needle. stick straw from outside at the lower part hole of the bottle with the help of squash tape. wash the pebbles and gravels 3-4 times with clean water and place them up to 1 inch. wash the sand 4-5 times with water and let it dry. place the sand on to the gravels in the bottle up to 2.5 inch. place the upper separated part of the bottle in an upside down position in the bottle. standard plate count: all the samples were tested by spread plate method before and after filtration. a small volume of serially diluted bacterial suspension is spread evenly over the agar surface using a sterile bent glass rod or wire loop. pre-treatment procedure: add 1 ml of water sample in 5 test tubes containing 9 ml distilled water and make 10 fold dilution (1:100 – 1:100,000). 0.1 ml from the last tube is inoculated on nutrient agar plate. incubate the plate at 37 °c for 24 hours. all the samples were run using above procedure for pre-treatment. post-treatment procedure: sufficient amount of water sample is poured to biosand filter and collect in a sterile container. add 1 ml of filtered water sample in 5 test tubes containing 9 ml distilled water and make 10 fold dilution (1:100 – 1:100,000). 0.1 ml from the last tube is inoculated on nutrient agar plate. incubate the plate at 37 °c for 24 hours. all the samples were run using above procedure for post-treatment. r e s u l t s the research project was carried out to detect the efficiency of locally designed plastic bottle bio-sand filter to reduce the microbial load and for the removal of impurities present in water sample. results showed that it effectively reduced microbial load up to 98%. 30 samples were tested throughout the project from different areas. from 4 samples microbial load is reduced up to 50% while microbial load is reduced up to 8090% in 19 samples. 1 sample reflected complete removal of to study the efficacy of bio-sand filter for commercial use vol 8 (2), july 2017 issn (print): 2305 – 8722 issn (online): 2521 – 8573 48 r a d s j . b i o l . r e s . a p p l . s c i 48 table i: number of colonies before and after filtration s. no samples no. of colonies before filtration no. of colonies after filtration 1. sabzimandi ++ 2. gulshan ++ + 3 nazimabad # 3 ++ + 4. eissa nagri +++ + 5. lalukhait ++ + 6. north karachi + + 7. buffer zone ++ + 8. north karachi tntc tntc 9. north nazimabad +++ + 10. gulistan-e-johar ++ + 11. dastageer ++ + 12. shadman +++ + 13 nazimabad # 2 tntc tntc 14. f.b area ++ + 15. shahra-e-faisal tntc ++ 16. nazimabad # 4 +++ + 17. nazimabad # 7 ++ + 18. clifton ++ + 19. garden east ++ + 20. karimabad tntc tntc 21. m.a jinnah road ++ + 22. malir ++ + 23. orangi town + + 24. saddar ++ + 25. north nazimabad + + 26. tower + + 27. azizabad tntc + 28. lasbaila ++ + 29. al-noor + + 30. golimar tntc + *tntc: too numerous to count figure 1: graphical representation of number of colonies before and after filtration of the samples to study the efficacy of bio-sand filter for commercial use vol 8 (2), july 2017 issn (print): 2305 – 8722 issn (online): 2521 – 8573 49 r a d s j . b i o l . r e s . a p p l . s c i 49 microorganisms. samples with too heavy growth before filtration showed reduced growth after treatment with filter. results also suggest that biosand filter effectively reduced turbidity from the samples containing turbid water. the randomized trial of the biosand filter suggests that it has the ability of effectively reducing microbial load as well as the turbidity from the water sample. figure 2 figure 3 figure 4 figure (2-4): water samples plated before filtration process. figure 5 figure 6 figure 7 figure (5-7): water samples plated after filtration process. d i s c u s s i o n the results conducted through the study suggest that biosand filter is effective in reducing bacterial load from water. the quality of water improved after filtration. biosand filter reduced microbial load up to 98%. it also reduces the turbidity of the water. different field and laboratory studies were conducted previously by buzunis to study the efficacy of bio-sand filter for commercial use vol 8 (2), july 2017 issn (print): 2305 – 8722 issn (online): 2521 – 8573 50 r a d s j . b i o l . r e s . a p p l . s c i 50 in 1995, palmateer in 1997 baumgartner in 2006, duke and baker in 2005 which indicates the reduction in bacterial load up to 80 to 98%, viral and protozoan reduction up to 99 to 99.9% and turbidity up to 95% (15). all the results indicate that biosand filters are low cost solutions for water treatment as filter improves the quality of water. the water after filtration through biosand filter is safe for drinking and other uses. i have conducted a similar research study in jinnah university for women under the supervision of department of microbiology. the research project was carried out to detect the efficiency of bio-sand filter to reduce the microbial load present in water samples. 30 samples were tested throughout the project from different areas. from 4 samples microbial load is reduced up to 50% while 80-90% from 19 samples. 1 sample reflected complete removal of microorganisms. results also suggest that biosand filter effectively reduced turbidity from the samples containing turbid water. the overall results suggest that biosand filter is effective in reducing the number of microorganisms, suspended particles and other impurities. this makes the water after filtration safe to use for drinking, cooking, sanitation and other household purposes. r e f e r e n c e s 1. who/unicef joint water supply, sanitation monitoring programme. water for life: making it happen. world health organization; 2005 jun 30. 2. mahlangu to, mpenyana-monyatsi l, momba mn, mamba bb. a simplified cost-effective biosand filter (bsfz) for removal of chemical contaminants from water. j chem eng mater sci., 2011 nov 30;2(10):156-67. 3. unicef and world health organization. drinking water equity, safety and sustainability. 2011. available: http://www.wssinfo.org/fileadmin/user_upload/resourc es. 4. world health organization. meeting the mdg drinking water and sanitation target: the urban and rural challenge of the decade, 2006. available from: www.who.int/water_sanitation_health/monitoring/jmp2 006/en/www.who.int/water_sanitation_health/monitori ng/jmp2006/en/ 5. bhalme sp and nagarnaik pb. analysis of drinking water of different places-a review. int j eng res appl 2012;2:3155-58. 6. kikkawa i. modification of a biosand filter in the northern region of ghana (doctoral dissertation, massachusetts institute of technology), 2008. 7. cawst. 2010a biosand filter manual: design, construction, installation, operation and maintenance. center for affordable water and sanitation technology. university of calgary, ca. available from: http://www.sswm.info/sites/default/files/reference_atta chments/cawst%202009%20biosand%20filter%20 manual.pdf 8. kubare m, haarhoff j. rational design of domestic biosand filters. j water supply res t., 2010 feb 1;59(1):1-5. 9. manz dh, concrete biosand water filter constructions manual book 7. installation and commissioning, 2008. website available: http://manzwaterinfo.ca/cmanz.html. 10. bipin d (environment and public health organization, enpho), spuhler d (second international gmbh). biosand filter. http://www.sswm.info/content/biosandfilter 11. zaman s, yeasmin s, inatsu y, ananchaipattana c, bari ml. low-cost sustainable technologies for the production of clean drinking water—a review. j environ prot, 2014;5(01):42. 12. unger, m. and m. r. collins. assessing escherichia coli removals in the schmutzdecke of slow-rate biofilters. journal awwa, 2008;100(12): 60-73. 13. cawst. 2010b summary of field and laboratory testing for the biosand filter. center for affordable water and sanitation technology. calgary, ca. assessable http://www.cawst.org/en/resources/biosand-filter. 14. sisson a, wampler pj, rediske r, mcnair jn, and frobish dj. long-term field performance of biosand filters in the artibonite valley, haiti. annis water resource institute, grand valley state university, muskegon, michigan; department of geology and department of statistics, grand valley state university, allendale, michigan, 2013;88(5): 862–867. 15. cawst (editor) (2009): biosand filter manual, design, construction, installation, operation and maintenance. alberta: center for affordable water and sanitation technology (cawst). available from: https://www.cawst.org/assets/file/biosand_filter_%2 0manual_version_10_no_appendices_sep09.pdf [accessed: 07.10.2015]. alteration in hepatic enzyme activity of tilapia mossambica vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 30 r a d s j . b i o l . r e s . a p p l . s c i . 30 op en ac ces s f u l l l e n g t h a r t i c l e alteration in hepatic enzyme activity of tilapia mossambica upon exposure to fluoride farha aziz*, afshan zeeshan wasti and farah jabeen department of biochemistry, jinnah university for women, karachi, pakistan a b s t r a c t fluoride, as the super reactive element fluorine, is found naturally throughout earth's crust. it has been identified as a strong, persistent powerful cumulative toxic agent, commonly distributed in the rivers, lakes, seas of earth. it is highly mobile and biologically active element in aquatic systems. fish are considered as the excellent and valuable bioindicator of ecosystem pollution. the present study was designed to estimate acute fluoride toxicity on enzyme activity of liver of freshwater fish tilapia mossambica. the major enzymes of carbohydrate protein metabolic pathways are alkaline phosphatase (alp), alanine transaminase (alt), aspartate transaminase (ast) in association with carbohydrate, protein, lipid in the liver of tilapia mossambica, from kalri lake (keenjhar lake), sindh, pakistan at low amount of fluoride (sub-lethal) was estimated by using uvvisible spectrophotometer. results showed alp, ast and alt enzymes present in the liver tissue were significantly changed (p < 0.001). finally, it is concluded that fluoride produces the adverse poisonous effect on liver functioning which may be associated with altered or elevated enzyme activity of protein-carbohydrate metabolism. keywords: fluoride, carbohydrate-protein metabolic pathways, alkaline phosphatase (alp), alanine transaminase (alt), aspartate transaminase (ast), tilapia mossambica, liver, keenjhar lake. *address of correspondence: farahtariq2@hotmail.com, farah786star@yahoo.com article info. received: april 18, 2018 accepted: april 23, 2018 cite this article: aziz f, wasti az, jabeen f. alteration in hepatic enzyme activity of tilapia mossambica upon exposure to fluoride. rads j. biol. res. appl. sci. 2018; 9(1): 30-34. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n the liver is a chief central metabolic organ, essential for life in vertebrates and invertebrates. the liver plays a main role in the metabolism of carbohydrate, protein, amino acid, and lipid. it acts as a detoxifying organ which may accumulate bio-transform and excretes a variety of toxicants or their by-products1,2. toxicants can produce disorders in the physiology of animal in association with altered enzyme activity. several studies reported the changes in enzyme activity in the freshwater fish when exposed to various pollutants3-6. fluoride is highly mobile and biologically active element in aquatic systems. fish are considered as an excellent and valuable bioindicator of ecosystem pollution. a number of cellular and biological processes including enzymatic reactions, cessation of protein secretion and synthesis, production of reactive oxygen species (ros), and variation of gene expression can be affected by fluoride7,8. fluoride as an anabolic agent or toxicant promotes cell proliferation and acts as an enzyme inhibitor for lipases, phosphatases and esterases, atp production cycle and cellular respiration at the sublethal concentrations. fluoride showed a significant alteration in enzymes activities of alkaline phosphatase (alp), alanine transaminase (alt), aspartate transaminase (ast) in fish gills at the sublethal concentration of fluoride9,10. o r i g i n a l a r t i c l e alteration in hepatic enzyme activity of tilapia mossambica vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 31 r a d s j . b i o l . r e s . a p p l . s c i . 31 m a t e r i a l a n d m e t h o d s normal alive tilapia mossambica (weight 90.2g and length 8.7cm) were collected from the keenjhar lake, with the help of local fisherman. fish were divided into two groups under normal condition. group i was considered as control (non-treated) while group ii marked as experimental treated with 1.5 gm naf / 70 l of water. each group contains twelve fishes and acclimated in a glass aquarium, filled with tap water. aquarium was aerated by using air pumps and filters. the commercial pellet as the fish food was used for both groups (i and ii) per a day. aquarium water was changed after 2 or 3 days. chemical study of water was performed according to approved procedures of apha11. study of biochemical constituents and enzymatic assays fishes of both groups (control and treated) were dissected and livers of all fishes were removed and cleaned with water. a glass homogenizer was used to homogenized tissues in cold solution of saline (0.89% nacl) and centrifuged in a refrigerated laboratory centrifuge. a clear supernatant was used for alkaline phosphatase (alp) estimation by p-nitro phenol method however aspartate transaminase (ast), alanine transaminase (alt) were measured by randox kit methods. estimation of glucose was done by gop-pap method and protein by lowry et al. method12,13. estimation of total lipid by sulphophospho-vanilline (spv) method14. data were represented as mean ± s.e.m. a paired student t-test at 95% confidence interval of the difference was applied to find out the level of significance. p value < 0.05 was considered significant. r e s u l t s a n d d i s c u s s i o n the effect of fluoride on important and essential enzymes of carbohydrate protein metabolism were measured in the liver of tilapia mossambica, at 1st, 2nd, 3rd and 4th week upon exposure to fluoride. aminotransferases are important group of enzymes participating in the shifting of the amino group from alpha-amino acids to the alpha-keto acid without the liberation of ammonia. the alpha-keto acid enters into the citric acid cycle for the release of energy. in this process alpha-keto acids changed into amino acids might be used in protein synthesis and take part in carbohydrate-protein metabolism regulation 10, 15, 16. alkaline phosphatase (alp) removing phosphate groups from nucleotides, proteins, and alkaloids, a process called dephosphorylation. the results showed significant increase in the enzymatic action of alkaline phosphatase (alp), alanine transaminase (alt), aspartate aminotransferase (ast) (table 1, fig. 1) in treated liver under fluoride when compare to control fish (p < 0.001) due to utilization of glucose and amino acid in carbohydrate-protein metabolism (table 2)10,17-21. high levels of ast, alt, alp upon long-term exposure (4th week) to fluoride might be due to altered liver function or liver dysfunction under stress22, 23. table 1: enzymatic activity of ast, alt, alp in the liver of tilapia mossambica at 1st, 2nd, 3rd and 4th week upon exposure to fluoride (1.5g/70l). biochemical parameter (u/l) control fish treated fish 1st week treated fish 2nd week treated fish 3rd week treated fish 4th week ast 633.65±0.077 823.54±0.009 876.42±0.013 856.14±0.008 734.12±0.051 alt 755.6±0.068 890.4±0.073 974.6±0.005 1005.6±0.009 798.34±0.034 alp 308.4±0.090 677.13±0.007 790.85±0.007 859.26±0.007 467.47±0.074 values expressed as mean ± s.e.m; *: significant at p< 0.05; **: highly significant at p<0.01; ***; very highly significant (p<0.001) compared with control alteration in hepatic enzyme activity of tilapia mossambica vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 32 r a d s j . b i o l . r e s . a p p l . s c i . 32 fig. 1: fluoride effect (1.5g/70l) on the activity of asp, alt, alp enzymes in the liver of tilapia mossambica at 1st, 2nd, 3rd and 4th week. table 2: biochemical constituents protein, lipid, cholesterol, glucose in the liver of tilapia massombica at 1st, 2nd, 3rd and 4th week upon exposure to fluoride (1.5g/70l). biochemical parameter (mg/g) control fish treated fish 1st week treated fish 2nd week treated fish 3rd week treated fish 4th week protein 6.745±0.089 4.553±0.098 3.497±0.068 3.300±0.067 2.756±0.023 lipid 86.063±0.018 72.55±0.008 48.4±0.105 52.637±0.056 49.327±0.016 cholesterol 82.805±0.078 308.612±0.066 177.6±0.077 131.625±0.055 101±0.078 glucose 63.418±0.084 37.418±0.037 32.495±0.050 12.947±0.008 10.650±0.097 values expressed as mean ± s.e.m; **: highly significant at p<0.01; ***; very highly significant (p<0.001) compared with control fig. 2: fluoride effect (1.5g/70l) on protein, lipid, cholesterol glucose in the liver of tilapia mossambica at 1st, 2nd, 3rd and 4th week. alteration in hepatic enzyme activity of tilapia mossambica vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 33 r a d s j . b i o l . r e s . a p p l . s c i . 33 quantitative measurements of biochemical constituents in the liver of freshwater fish tilapia massombica at 1st, 2nd, 3rd and 4th week are presented in table 2 and fig. 2. results showed decreased in protein-carbohydrate and lipid contents due to changes in carbohydrate–protein metabolism (p < 0.001). this condition might be due to the decrease oxidation of glucose while decreased in protein might be associated with energy production. the whole process is needed to overwhelm the tension for the endurance of fish24. decreased lipid content in fish liver exposed to fluoride may be due to the decrease in enzyme acyl co-a synthetase activity, play an essential role in fatty acid synthesis. an increase in the cholesterol level in the liver due to fluoride dose, measurements of these important clinical biochemical parameters serve as excellent biomarkers of the aquatic environmental condition. c o n c l u s i o n the liver is a fundamental organ of various metabolism and different process in fishes. this study has revealed that the liver of tilapia mossambica upon exposure to fluoride may result in the alteration in the enzyme activities might be due to a defensive mechanism to overcome the lethal stress caused by fluoride in freshwater fish tilapia mossambica. the present study demonstrates the disorder of biochemical processes occurring in the fish body caused by the fluoride, the mobile persistent toxic agent producing the adverse effect on fish health, growth and number in the freshwater reservoir. r e f e r e n c e s 1. hinton de, segner h, braunbeck t. toxic responses of the liver. in: schlenk d, bensen wh (eds) toxicity in marine and freshwater teleosts. taylor & francis, london 2001; 1: 224-8. 2. pedlar rm, klaverkamp jf. the accumulation and distribution of dietary arsenic in lake whitefish (coregonus clupeaformis). aquat toxicol. 2002; 57(3):153-6. 3. begum g, vijayaraghavan s. carbohydrate metabolism in hepatic tissue of freshwater catfish clarias batrachus l during dimethoate exposure. food chem toxicol. 1995; 33(5):423-6. 4. begum g, vijayaraghavan s. in vivo toxicity of dimethoate on protein and transaminases in the liver tissue of fresh water fish clarias batrachus (linn). bullet environ contamin toxicol. 1995; 54(3):370-5. 5. begum g, vijayaraghavan s. alterations in protein metabolism of muscle tissue in the fish clarias batrachus (linn) by commercial grade dimethoate. bulletin of environmental contamination and toxicology. 1996 aug 1;57(2):223-8. 6. humtsoe n, dawoodi r, kulkarni b, chavan b. effect of arsenic on the enzymes of the rohu carp, labeorohita (hamilton, 1822). raffles bull zool. 2007; 14: 17-19. 7. kaur r, saxena a, batra m. a review study on fluoride toxicity in water and fishes: current status, toxicology and remedial measures. ijeab. 2017; 2(1), 456-6. 8. barbier o, arreola-mendoza l, del razo lm. molecular mechanisms of fluoride toxicity. chem-biol interac. 2010; 188(2):319-33. 9. aziz f., study of physiological and biochemical parameters of farm raised fish with toxicants [ph.d. dissertation], jinnah university for women, pakistan (2012) 10. aziz f, akhtar y, bilal b, parveen n. effect of fluoride exposure on key enzymes activity of protein carbohydrate metabolism in gills of fresh water fish tilapia mossambica, keenjhar lake, thatta, sindh, pakistan. ijeab. 2013; 2(8), 24-7. 11. apha. standard methods for the examination of water and wastewater, 20th ed.washington, dc, american public health association. 1998 12. trinder p. determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. annals of clinical biochemistry. 1969; 6(1):24-7. 13. lowry oh, rosenberg nj, farr al randall rj. protein measurement with the folin phenol reagent. j biol chem. 1951; 193(1): 265-75. 14. frings cs, dunn rt. a colorimetric method for determination of total serum lipids based on the sulfophosphovanillin reaction. am j clin pathol 1970; 53(1), 89-91. 15. knox we. the regulation of some enzymes of nitrogen metabolism. an introduction to enzyme physiology. adyan en regnl. 1965; 3, 247-313. 16. martin dw, mayes pa. harper's review of biochemistry, 18th ed, lange medical publications, california. 1981. 17. miszta h, dabrowski z, szygula z, spodaryk k. the effect of intrarenal nickel subsulfide injections upon the activity of selected erythrocyte and bone marrow enzymes in rats. basic clin pharmacol toxicol.1986; 59(5), 425-9. alteration in hepatic enzyme activity of tilapia mossambica vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 34 r a d s j . b i o l . r e s . a p p l . s c i . 34 18. singh m. biochemical and cytochemical alterations in liver and kidney following experimental fluorosis. fluoride. 1984; 17: 81-3. 19. chenoy nj, narayana mv, sequerra e, joshp sm, bard jm, purohit rm, et al. studies on effects of fluoride in 36 villages and mehsana district, north gujarat. fluoride, 1992, 25, 101-10. 20. chitra t, reddy mm, rao jvr. levels of muscle and liver tissue enzymes in channa punctatus bloch exposed to naf. fluoride. 1983; 16, 48-51. 21. reddy sln, venugopal nbrk. reddy an, rao jvr. fluoride induced changes in carbohydrate metabolism in the tissues of fresh water carb barytelphusa guerini. ecotoxicol environ saf. 1989; 18(1): 59-66. 22. devi j, shankar r. effect of fluoride on aspertate and alanine amino transferase activities in the fresh water fish, clarias batrachus (linn.). bullet pure & appl sciences-zoology 2006. 23. gabriel lp, william rh. toxicology of the liver / editors, gabriel l. plaa, william r. hewitt. new york: raven press, c1982. 24. ganeshwade rm, rokade pb, sonwane sr. impact of dimethoate on protein content in the freshwater fish puntius ticto (ham). the bioscan. 2012; 7(1), 153-5. modification of process parameters for enhanced lipase induction from bacillus sr1 vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 14 r a d s j . b i o l . r e s . a p p l . s c i . 14 op en ac ces s f u l l l e n g t h a r t i c l e modification of process parameters for enhanced lipase induction from bacillus sr1 sana ahmad1, *, aliya riaz1, hina abbasi2, reeja eijaz1, muhammad noman syed3 1department of biochemistry, jinnah university for women, karachi, pakistan 2department of biomedical engineering, sir syed university of engineering and technology, karachi, pakistan 3department of biochemistry, university of karachi, karachi, pakistan a b s t r a c t the enzymes catalyze the cleavage of triacylglycerols into fatty acids and glycerols are referred to as lipases (ec 3.1.1.3). lipases are widely distributed in flora and fauna. microbial lipases are of great importance than lipases from plants and animals due to their catalytic activity, ease of production and optimization. lipases have tremendous industrial applications such as in the processing of fats and oils, detergents and degreasing formulations, food processing, the synthesis of fine chemicals, paper manufacture, and production of cosmetics, and pharmaceuticals. therefore, a potential lipase producing bacterial strain was isolated and identified as gram +ve bacillus sr1. among different oils tested, olive oil was found to be the favorable substrate for lipase induction. additionally, lipase induction was observed highest in 24 hours of fermentation at 37⁰c and ph 7.5. keywords isolation, lipase, bacillus, fermentation, triacylglycerol. *address of correspondence sanaahmad_185@yahoo.com article info. received: april 27, 2018 accepted: february 28, 2019 cite this article: ahmad s, riaz a, abbasi h, eijaz r, syed mn. modification of process parameters for enhanced lipase induction from bacillus sr1. rads j. biol. res. appl. sci. 2019; 10(1): 14-17. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n triacylglycerol and fatty acids require lipases (ec 3.1.1.3) for their degradation. lipases have been defined as carboxylesterases that catalyze the hydrolysis, esterification and transesterification of acylglycerides. lipases are serine hydrolases and hence not required addition of cofactors1. the active site consists of three catalytic residues: a nucleophilic residue (serine, cysteine, or aspartate), a catalytic acid residue (aspartate or glutamate), and a histidine residue2. lipases are stereoselective as well as regioselective biocatalysts3. consequently, lipase reactions show more selectivity under mild conditions and become the factor for increasing demands of lipases. lipases are produced by animals, plants and microorganisms, the majority of lipases used for biotechnological purposes have been isolated from bacteria and fungi4. microbial lipases are more stable and their recovery is comparatively easy4. among microbial lipases, bacterial lipases are of great interests due to easy and inexpensive production5. fermentation is the classical bioprocess used for lipase production by bacteria. bacterial lipases can be produced by submerged fermentation (smf) in addition to solid state fermentation (ssf)6. fermentation is used to produce enzymes on industrial scale7. economic fermentation techniques, low energy consumption and greater productivity are the reasons for preference of microbial lipases in industrial sector. lipases have several industrial applications7,8. bacterial lipases can be used in many processes such as in the processing of fats and oils, detergents and degreasing formulations, food processing, the synthesis of fine chemicals, paper manufacture, and production of cosmetics, and pharmaceuticals8,9. besides this well established role; still the demand for novel lipase with specific properties such as specificity, stability, ph, and temperature is increasing. this has drawn the greater interest in isolation of new and unique lipase producing micro-organisms from diverse habitat and modulation of the process for maximum lipase o r i g i n a l a r t i c l e modification of process parameters for enhanced lipase induction from bacillus sr1 vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 15 r a d s j . b i o l . r e s . a p p l . s c i . 15 yield10-12. the present study was therefore aimed to isolate novel lipase producing bacterium, its cultivation using submerged fermentation and optimization of fermentation parameters for maximum lipase yield. m a t e r i a l s a n d m e t h o d s collection of sample for present research, greasy scrapping of kitchen was selected as it is greasy and has a long term oil exposure. greasy scrapping was collected from a depth of 5-10cm by means of a sterilized spatula and stored in a sterilized vial. sample was transferred to the laboratory soon after collection and processed immediately13. isolation of lipolytic bacteria collected sample was enriched in nutrient broth supplemented with 1% olive oil. for this purpose 1g of sample was suspended in 50 ml of enrichment medium and then agitated at 120 rpm; 37⁰c for 30 minutes on a rotary shaker. the sample was then incubated at 37⁰c for 48 hours. consequently, serial dilutions (10-1-10-4) of the sample were prepared using sterilized 0.9% nacl. 200μl of each dilution were inoculated on nutrient agar plates by spread plate method and incubated at 37⁰c for up to 72 hours. isolated microbial colonies were picked and examined for lipolytic activity and further subjected to strain identification14. screening of bacterial isolates for lipolytic activity and identification screening media used for lipolytic activity consisted of nutrient agar with olive oil (1%) and tween 80 (0.1%). isolated colonies showed growth on screening medium were selected on the basis of hydrolytic zones were further purified and identified through gram staining15. storage and maintenance of culture the isolated strain was stored at 4⁰c and subcultured after 15 days. the culture was revived on weekly bases in enrichment medium. lipase production extracellular lipase from the isolated strain was harvested through submerged fermentation. the process was carried out in 100ml erlenmeyer flask. a 10% v/v seed culture was inoculated in medium and incubated at 37⁰c for 48 hours. consequently, the fermented media was centrifuged at 0⁰c and 10,000xg to pallet cells and the cell free extract was served as source of crude lipase. lipase assay lipase activity was monitored spectrophotometrically by using para-nitrophenyl palmitate (pnpp) as substrate16 with slight modifications. the reaction was initiated with 1ml of substrate (40μm) and 0.1 ml of crude enzyme. after 30 minutes incubation naoh (5%) was added to cease the reaction and absorbance was monitored at 410nm for release of para-nitrophenol. one unit of lipase activity was defined as μmoles of para-nitrophenol released in one minute per assay conditions. optimization of fermentation parameters lipase production was optimized with variation in one factor at the constant level of other variables. the parameters tested were time course, ph, temperature and different oil substrates. effect of time on lipase production in order to observe time period for maximum lipase production, isolated strain was allowed to ferment for different time periods (18, 24, 48 and 72 hours) effect of ph on lipase induction different ph values ranging from 6-8.5 were tested to select the one with higher lipase yield. effect of temperature on lipase production temperature effect on lipase production was detected by varying temperature from 30 to 60⁰c. evaluation of different substrates additionally, lipase production was also examined with different oil substrates such as castor oil, mustard oil, canola oil, palm oil, almond oil, olive oil. the selected oil substrate with maximum lipase induction was further tested in presence of tween 80 in order to examine the further enhancement in lipase production. influence of substrate concentrations on lipase induction the selected substrate was further tested with different concentrations (1-5%) to optimize the suitable concentration for enhanced lipase yield. r e s u l t s a n d d i s c u s s i o n the colony represent greater zone of hydrolysis was selected for this study. the isolated strain was observed as gram positive, rod shaped and spore former bacterium and identified as bacillus sp. sr1 (figure 1a and b). the production process of lipase from bacillus sp. modification of process parameters for enhanced lipase induction from bacillus sr1 vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 16 r a d s j . b i o l . r e s . a p p l . s c i . 16 figure 1. isolation and identification of bacillus sr1. a: growth of isolated strain on tributyrin agar. b: gram staining of isolated strain bacillus sr1 showing gram positive rods. sr1 was further modified with respect to time, ph, temperature and different substrates. time course study of lipase production revealed that bacillus sp. sr1 produce maximum lipase (32.6 u/ml) in 24 hours of fermentation. figure 2 describes that after 24 hours of fermentation lipase activity got decreased gradually up to 72 hours of fermentation. bacillus sp. figure 2. effect of fermentation time on lipase synthesis. results are expressed as mean+sd (n=3). sr1 produce highest lipase yield in its exponential growth phase. according to current study lipase activity decreased subsequently 24 hours which may be due to concomitant production of various proteases after log phase. similar results were obtained in bacteria isolated from palm oil contaminated waste17. the production of proteases after 20 hours of fermentation and release of ammonia from deamination of amino acid caused alkalinisation of the media. together these two factors are responsible for decreased lipase activity after 24 hours of fermentation and above ph 7.518,19. influence of various ph values on hyper production of lipase was presented in figure 3. it was observed that greater lipase yield was achieved at ph 7.5. this is in accordance with the reported literature16,17 and reflected the neutral nature of bacillus sp. sr1 lipase. figure 3. effect of ph on lipase synthesis. results are expressed as mean+sd (n=3). reported literature showed that usually bacteria preferred ph 7.0 for lipase production20. temperature has a profound effect on a protein and bacterial growth. in present study it was observed that the lipase yield was highest (40.987 u/ml) at 37⁰c and gradually decreased after it (figure 4) which indicative of enzyme unstability at higher temperature ranges17,19. figure 4. effect of temperature on lipase synthesis. results are expressed as mean+sd (n=3). figure 5. lipase synthesis with different substrates. results are expressed as mean+sd (n=3). modification of process parameters for enhanced lipase induction from bacillus sr1 vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 17 r a d s j . b i o l . r e s . a p p l . s c i . 17 bacillus lipase is an inducible enzyme and its induction is directly affected by oil substrates21. therefore, different oil substrates were tested to assess their effect on lipase induction. it was observed that all the tested oils were induced lipase synthesis by bacillus sp. sr1 varyingly (figure 5). however, lipase synthesis reaches to its peak i.e. 45.23 u/ml when olive oil was used as substrate. utilization of olive oil and simultaneous production of lipase indicates the stimulation of lipase operon by olive oil and also the preference of bacillus sp. sr1 for lipase production. however, palm oil, almond oil and castor oil were also found to stimulate lipase production by bacillus sp. sr-1. lipase induction was also affected by concentration of substrate and for this purpose; different concentrations of olive oil (0.5-5%) were also investigated. lipase yield was found to be greater i.e. 57.97 u/ml at 1% olive oil concentration and further increase in concentration showed significant decrease in lipase synthesis by bacillus sp. sr1 which may be due to increase in viscosity of the medium which in turn is a cause of low aeration20. c o n c l u s i o n a potential lipase producing bacterial strain was isolated and identified as bacillus sp. sr1. it produces a neutral enzyme with 57.97 u/ml of lipolytic activity in presence of 1% olive oil. on the basis of results obtained the isolated enzyme seems a good addition in industrially important bacterial lipases. future research will focus on the further characterization of lipase according to industrial needs. r e f e r e n c e s 1. sunna a, hunter l, hutton ca, bergquist pl. biochemical characterization of a recombinant thermoalkalophilic lipase and assessment of its substrate enantioselectivity. enzyme microb. technol. 2002;31(4):472-6. 2. wang cs, hartsuck ja. bile salt-activated lipase. a multiple function lipolytic enzyme. bba-lipid lipid met1993;1166(1):1-9. 3. hasan f, shah aa, hameed a. industrial applications of microbial lipases. enzyme microb. technol. 2006;39(2):235-51. 4. jaeger ke, eggert t. lipases for biotechnology. curropin biotechnol. 2002;13(4):390-7. 5. treichel h, de oliveira d, mazutti ma, di luccio m, oliveira jv. a review on microbial lipases production. food bioproc tech. 2010;3(2):182-96. 6. el-mansi em, bryce cf, allman ar, demain al. fermentation microbiology and biotechnology. crc press; 2011. 7. kumar a, dhar k, kanwar ss, arora pk. lipase catalysis in organic solvents: advantages and applications. biolproced online. 2016;18(1):2. 8. sharma s, kanwar ss. organic solvent tolerant lipases and applications. sci. world j. 2014;2014. 9. choudhury p, bhunia b. industrial application of lipase: a review. biopharm journal. 2017;1(2):41-7. 10. bhattacharya c, pandey b, sarkar ak. study of lipase producing bacterial strains from oil contaminated soil. j. basic appl. res. 2016;2(4):512-5. 11. mazhar h, abbas n, hussain z, sohail a, ali ss. extracellular lipase production from bacillus subtilis using agro-industrial waste and fruit peels. punjab univ. j. zool. 2016;31(2):261-7. 12. alhamdani ma, alkabbi hjj. isolation and identification of lipase producing bacteria from oil-contaminant soil. j biolagrichealthc. 2016; 6 (20): 2016. 13. sagar k, bashir y, phukan mm, konwar bk. isolation of lipolytic bacteria from waste contaminated soil: a study with regard to process optimization for lipase. international journal of scientific &technology research. 2013;2(10):214-8. 14. claus d. a standardized gram staining procedure. world j. microbiol. biotechnol. 1992;8(4):451-2. 15. habibollahi h, salehzadeh a. isolation, optimization, and molecular characterization of a lipase producing bacterium from oil contaminated soils. pollution. 2018;4(1):119-28. 16. pencreac'h g, baratti jc. hydrolysis of p-nitrophenyl palmitate in n-heptane by the pseudomonas cepacia lipase: a simple test for the determination of lipase activity in organic media. enzym microb technol. 1996;18(6):417-22. 17. hasan na, nawahwi mz, yahya n, othman na. identification and optimization of lipase producing bacteria from palm oil contaminated waste. rev scifondam appl. 2018;10(2s):300-10. 18. alkan h, baysal z, uyar f, dogru m. production of lipase by a newly isolated bacillus coagulans under solid-state fermentation using melon wastes. appl biochembiotechnol. 2007;136(2):183-92. 19. gombert ak, pinto al, castilho lr, freire dm. lipase production by penicilliumrestrictum in solidstate fermentation using babassu oil cake as substrate. process biochem. 1999;35(1-2):85-90. 20. iqbal sa, rehman a. characterization of lipase from bacillus subtilisi-4 and its potential use in oil contaminated wastewater. braz arch biol technol. 2015; 58(5):789-97. 21. amin m, bhatti hn, zuber m, bhatti ia, asgher m. potential use of agricultural wastes for the production of lipase by aspergillus melleus under solid state fermentation. j anim plant sci. 2014. 24:1430-7. nutritional and antioxidant potential of common vegetables in pakistan vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 36 r a d s j . b i o l . r e s . a p p l . s c i . 36 op en ac ces s f u l l l e n g t h a r t i c l e nutritional and antioxidant potential of common vegetables in pakistan sumra naseer, shabbir hussain*, zark zahid department of chemistry, lahore garrison university, dha phase vi, lahore, pakistan a b s t r a c t vegetables contain many important antioxidants and nutrients. daucus carota l (carrot) is rich in hydrophilic and lipophilic antioxidants and is a good source of carbohydrates, vitamins, and minerals such as ca, mg, p, and fe. it shows anti-carcinogenic, antioxidant, and immune boosting properties. it also provides protection against muscular degradation and development of senile cataract and reduces the risk of many diseases in humans. the nutritional value of cucurbita pepo linn (pumpkin) is attributed to the high level of carotenoids, carbohydrates, vitamins, other minerals. pumpkin due to the presence of vitamin b complex, ca and p, provides protection against many acute and chronic diseases. pumpkin peel and pulp are the good source of antioxidants, polyphenols, carotenoids, dietary fiber and minerals such as p and fe. pumpkin extract shows antioxidant, antimicrobial and other activities. spinacia oleracea (spinach) is a good source of essential nutrients such as carotenoids, vitamins, and minerals (p, k, mg). its leaves contain carotenoids, polyphenols, flavonoids, and ascorbic acid. fresh spinach extract shows antioxidant and antimicrobial activity. solanum tuberosum (potato) is rich in starch, alkaloids, flavonoids, dietary fiber, and some other minerals (k, mg, fe). potato helps to overcome chronic and acute diseases and has an impact on blood pressure and gut health. keywords carrot, pumpkin, spinach, potato, nutrients, antioxidant, vitamins. *address of correspondence shabchem786@gmail.com article info. received: june 9, 2018 accepted: november 29, 2018 cite this article: naseer s, hussain s, zahid z. nutritional and antioxidant potential of common vegetables in pakistan. rads j. biol. res. appl. sci. 2019; 10(1): 36-40. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n vegetables and fruits are not only the source of nutrition for human beings but also very important for maintaining health. due to the presence of essential minerals and vitamins, the vegetables play a vital role in a diet of mankind1. they are an important source of vitamins, minerals, and fibers for human lives. however, vegetables are different from fruits due to their antioxidant activities as well as their fiber and vitamin contents. vegetables are bioavailable because of the presence of antioxidants. different vegetables show different antioxidant activities and provide protection against oxidative stress1. vegetables and fruits contain a high concentration of antioxidants that help to treat many diseases such as aging, brain dysfunction, inflammation, heart disease, arteriosclerosis, arthritis, and cancer etc. the substance which has the capacity to prevent oxidative damage to fats, proteins and nucleic acid with the help of reactive oxygen species are known as antioxidants. these reactive oxygen species primarily are reactive free radicals including alkoxyl, peroxyl, hydroxyl, superoxide, and non-radicals, for example, hypochlorous, hydrogen peroxide2. vegetables contain no of antioxidants such as vitamins a, c, and e as well as carotene all of which play a vital role in good health. they also play a significant role in different mechanisms including co-factors for certain enzymes, involvement in oxidation and reduction reaction3. in fruits and vegetables, three major groups are present which are ascorbic acid, phenolic compounds, and carotenoids. beta carotenoids are responsible for the antioxidant defensive system. ascorbic acid and phenolic r e v i e w a r t i c l e nutritional and antioxidant potential of common vegetables in pakistan vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 37 r a d s j . b i o l . r e s . a p p l . s c i . 37 compounds are also called hydrophilic antioxidants and carotenoids are called lipophilic antioxidants4. in the present review, a focus has been made on the nutritional and antioxidant value of four common vegetables i.e., daucus carota (carrot), cucurbita pepo linn (pumpkin), spinacia oleracea (spinach) and solanum tuberosum (potato) (figure 1). figure 1. a: daucus carota (carrot); b: cucurbita pepo linn (pumpkin); c: spinacia oleracea (spinach); d: solanum tuberosum (potato). daucus carota l the most important crop of apiaceae family is daucus carota commonly called carrot. carrot is a root vegetable and has the worldwide distribution. carrot shows different colors such as white, red, orange, yellow, purple and dark purple; yellow and purple colors carrot are the first cultivation of this family. carrot is one of the most important vegetables all over the world and its phytochemicals and polynutrients are beneficial for mankind5. carrots are rich in lipophilic and hydrophilic antioxidants and a good source of carbohydrates, vitamins, and minerals such as mg, ca, p and fe6. it is also rich in carotenoids especially beta carotenoids. due to the presence of vitamins and antioxidants carrot shows beneficial health effects on mankind as it possesses immune boosting, antioxidant and anti-carcinogenic properties. the chemical structure of carrot contains a polyene chain having 11 conjugated double bonds and a β-ring at each end of the chain7. carrots on the basis of their nutrition are ranked on 38 number1. nitrogen balance in our body and the quantity of urine can also be controlled by carrot8. sometimes eyes photoreceptors start to deteriorate which is an eye vision problem due to the deficiency of vitamin a. beta carotenoid (the major source of vitamin a) in carrots helps to protect the vision and also provides protection against muscular degradation and the development of senile cataract, the leading cause of blindness in aged people. carrots one of the richest sources of provitamin a and its high intake is associated with a significant decrease in postmenopausal breast cancer due to the presence of carotenoids. carrot plays a vital role in human health because of enrichment of carotenoids and phenolic compounds which also reduce the risk of many diseases5. cucurbita pepo linn pumpkin is the common name of cucurbita pepo linn which belongs to the cucurbitaceae family. pumpkin is also called elegede in southwest and nigeria regions. the word pumpkin is derived from a greek word pepon which means a large melon. in the plant kingdom, this family is one of the largest families which consist of a large number of edible plant species9. in pumpkin family melon, cucumber, gourds and summer winter squash also included10. pumpkin is used as food due to its high nutritional value as it contains a high level of carotenoids. pumpkins have many varieties which are grown in brazil11. pumpkins are a good source of carotenoids, carbohydrates, vitamins, and other minerals12; they provide protection against many acute and chronic diseases due to the presence of b complex, calcium, and phosphorous13. after the nutritional analysis, it has been noted that pumpkin wastes (both peel and pulp) are the richest source of antioxidants, nutrients, polyphenol compounds, carotenoids dietary fiber, and minerals such as p and fe. the pulp of pumpkin is comprised of the high level of beta carotenoids as well as many other vitamins. pumpkin extract shows antioxidant activity, antimicrobial activity, as well as various other activities. this extract consists of active compounds having the ability to scavenge different free radicals12. spinacia oleracea spinacia oleracea (spinach) belongs to the family amaranthaceae which has about 102 genera and 1400 species worldwide. it also belongs to the leafy green family. the word spinach has been derived from the persian word ispanai which means the “green hand”. in latin, it is called spanachia and in english, it is called as nutritional and antioxidant potential of common vegetables in pakistan vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 38 r a d s j . b i o l . r e s . a p p l . s c i . 38 spinach. the range of color of spinach is from light green to dark green. spinach can be classified into two categories: spinach with crinkle leaf and the spinach with smooth leaf. although there are a lot of varieties, however, all of these have many characters which are similar to each other14. spinach is a leafy green vegetable and is a rich source of vitamins and antioxidants. it is not only a common raw material in the food processing industry but also finds importance as a dietary vegetable. spinach is a good source of essential nutrients for example carotenoids, vitamins (vitamin a, c) and minerals (p, k, mg). it also shows antioxidant and various activities but it does not exhibit hypolipidemic activity15. the leaf of this vegetable contains carotenoids, polyphenol, flavonoids, and ascorbic acid. fresh spinach extract shows antioxidant and antimicrobial activities16. solanum tuberosum the word potato is derived from spanish word patatas and in english, it is called potato which grows down the earth about 6 cm. it is the third most important crop in the united states. the production of potato crop is more than 300 million metric tons17. according to their shapes and color, it is categorized into many varieties which include round whites, yellow flesh, long whites, round red etc18. there are 5000 varieties of potatoes present in all over the world. the major variety of potato is solanum tuberosum which is a starchy vegetable and consist of alkaloids and flavonoids in a large amount. potatoes are polynutrients and contain carbohydrates, dietary fiber, and some other minerals (k, mg, and fe etc). it is used for allergic reactions as well as it plays a vital role in the production of the antioxidant defense system due to the presence of essential nutrients (vitamins, polyphenol, beta carotene, and minerals). polynutrients in potatoes help in chronic and acute diseases18. one of the most important vitamins c (ascorbic acid) in potatoes tuber has a significant role in human nutrition. the highest content of ascorbic acid (about 20.75 mg/kg) is present in marble potato (a variety of potato)19. potato nutrients also show a favorable impact on satiety, blood pressure, gut health and other acute and chronic diseases in children teen adults18. comparative studies studies investigate that vegetables, such as pumpkin, spinach, shallots, potato, carrots, and cabbage, have high antioxidant activities. beside antioxidant nutrients such as ascorbic acid, tocopherols, and carotenoids, these vegetables are also a good source of polyphenol components20. among these vegetables, carrots show highest antioxidant activity followed by pumpkin, spinach, and potatoes. carrot peel and juice are rich in pro-healthy antioxidants both of lipophilic (carotenoids) and hydrophilic (phenolic compounds) characters21. pumpkin seeds have a high content of vitamin e (tocopherol)22 and the pulp was found to be a rich source of β-carotene (142.38 mg/100 gm). the peel and pulp samples present almost similar values of polyphenols and antioxidants23. spinach is rich in antioxidants such as beta-carotene and lutein. these have antioxidant and anticancer properties24. spinach is a rich source of polyphenols. it contains high levels of flavonols unique to spinach. they are mainly quercetagetin derivatives. other compounds in spinach were scarcely studied, although some like pcoumaric acid contribute significantly to its antioxidant activity25. potato is not only a source of antioxidants source such as ascorbic acid, tropocoal, phenolic acid and flavonoids in human diet but it consists of high amount of polyphenolic compounds. potatoes are good sources of phenolic compounds, with total phenolic content higher than other widespread fruits and vegetables like carrots, pumpkin, or tomatoes because of their high consumption rates26. the aqueous and methanolic extracts of spinach (spinacea oleracia), pumpkin (cucurbita pepo), carrots and potatoes were evaluated for antimicrobial activity against bacterial strains27. potatoes exhibits well-known antimicrobial activity against different microbes. the methanolic and aqueous extracts of carrot (leaves, stems, and roots) on four microorganism species showed that the root, leaf, and stem were active against b. cereus, s. aureus, and e. coli; it shows that carrot shows maximum antimicrobial activity28. leaves of spinach was extracted with methanol extract. the extract of spinach (20mg/ml) showed maximum activity against the escherichia coli (32mm), staphylococcus aureus (29mm) zone of inhibition respectively whereas the minimum activity is reported against the bacillus subtilis nutritional and antioxidant potential of common vegetables in pakistan vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 39 r a d s j . b i o l . r e s . a p p l . s c i . 39 (12mm) and penicillium chrysogenum (15mm). antimicrobial activity of pumpkin: the fruity body of cucurbita pepo (pumpkin) was extracted with methanol extract. methanolic extract of pumpkin shows the sufficient zone of inhibition against the microorganisms as 23 mm in escherichia coli, 22 mm in staphylococcus aureus29. c o n c l u s i o n s vegetables contain many important antioxidants and nutrients. these antioxidants provide protection against diseases and reduce the rate of risk in mankind. the use of these vegetables helps to maintain the health of human being. daucus carota l is a good source of antioxidants, carbohydrates, vitamins, and minerals such as ca, mg, p, and fe. it shows anti-carcinogenic, immune boosting properties and protection against muscular degradation. the cucurbita pepo linn (pumpkin) contains carotenoids, carbohydrates, vitamins, antioxidants, polyphenols, carotenoids, dietary fiber and minerals such as p and fe. spinacia oleracea (spinach) is a good source of carotenoids, vitamins, and minerals (p, k, mg). its leaves contain carotenoids, polyphenols, flavonoids, and ascorbic acid. solanum tuberosum (potato) is rich in starch, alkaloids, flavonoids, dietary fiber, and some other minerals (k, mg, fe). potato helps to overcome chronic and acute diseases and has an impact on blood pressure and gut health. r e f e r e n c e s 1. owolade so, akinrinola ao, popoola fo, aderibigbe or, ademoyegun ot, olabode i. study on physicochemical properties, antioxidant activity and shelf stability of carrot (daucus carota) and pineapple (ananas comosus) juice blend. int food res j. 2017;24(2):534–40. 2. rice-evans c. flavonoids in health and disease. boca raton: crc press; 2003. 3. shetty a, magadum s, managanvi k. vegetables as sources of antioxidants. j food nutr disord. 2013;2(1):1–5. 4. arshiya s. the antioxidant effect of certain fruits: a review. 2013;5(12):265–8. 5. da jc, dias s. nutritional and health benefits of carrots and their seed extracts. food nutr sci. 2014;5(5):2147–56. 6. sharma kd, karki s, thakur ns, attri s. chemical composition, functional properties and processing of carrot-a review. j. food sci tech mys. 2012;49:22– 32. 7. augspole i, rakcejeva t, kruma z, dimins f. shredded carrots quality providing by treatment with hydrogen peroxide. 9th baltic conference on food science and technology. food for consumer wellbeing. foodbalt 2014, jelgava, latvia. 2014;2014:150–4. 8. noella j, umuhoza k, sylvestre h, philippe s. nutritional quality of carrot (daucus carota l.) as influenced by farmyard manure. wjas. 2014;2(5):102–7. 9. oloyede f. growth, yield and antioxidant profile of pumpkin (cucúrbita pepo l.) leafy vegetable as affected by npk compound fertilizer. j soil sci plant nutr. 2012;12(3):379–88. 10. bratsch a. specialty crop profile: horseradish. virginia coop ext. 2009;10. 11. nawirska-olszanska a, kita a, biesiada a, sokolletowska a, kucharska az. characteristics of antioxidant activity and composition of pumpkin seed oils in 12 cultivars. food chemistry. 2013;39:155–61. 12. mala s. nutritional composition and antioxidant activity of pumpkin wastes. int j pharm chem biol sci. 2016;6(3):336–44. 13. priori d, valduga e, castelo j, villela b, mistura cc, vizzotto m, et al. characterization of bioactive compounds, antioxidant activity and minerals in landraces of pumpkin (cucurbita moschata) cultivated in southern brazil. food sci tecnhology. 2016;37(1):1–8. 14. kiple kf, ornelas kc. cambridge world history of food. united kingdom: cambridge, 2002; 1857: 288. 15. chu y, sun j, wu x, liu rh. antioxidant and antiproliferative activities of common vegetables. j. agric. food chem. 2002;50(23):6910-6916. 16. ligor m, trziszka t, buszewski b. study of antioxidant activity of biologically active compounds isolated from green vegetables by coupled analytical techniques. food anal. method. 2013;6:630–6. 17. camire me, kubow s, donnelly dj. critical reviews in food science and nutrition potatoes and human health potatoes and human health. crit rev food sci nutr. 2009;49:823–40. 18. zaheer k, akhtar mh. potato production, usage, and nutrition—a review. crit rev food sci nutr. 2016; 56(5):711-21. nutritional and antioxidant potential of common vegetables in pakistan vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 40 r a d s j . b i o l . r e s . a p p l . s c i . 40 19. love sl, salaiz t, mosley ar, shafii b, price wj, thornton re. ascorbic acid concentration and stability in north american potato germplasm. acta hortic. 2003;619:87-93. 20. ismail a, marjan zm, foong cw. total antioxidant activity and phenolic content in selected vegetables. food chem. 2004;87(4):581-6. 21. hager tj, howard lr. processing effects on carrot phytonutrients. in hort science. 2006;41(1):74-9. 22. nor n, carr a, hardacre a, brennan c. the development of expanded snack product made from pumpkin flour-corn grits: effect of extrusion conditions and formulations on physical characteristics and microstructure. foods. 2013;2(2):160-9. 23. kwonyi, apostolidis e, kim yc, shetty k. health benefits of traditional corn, beans, and pumpkin: in vitro studies for hyperglycemia and hypertension management. j. med. food. 2007;10(2). 24. hatamjafari f, tazarv vm. study of antioxidant activity of spinacia oleracea l. ojc. 2013;29(2):4515. 25. bajcan d, tomas j, uhlirova g, árvay j, trebichalsky p, stanovic r, simansky v. antioxidant potential of spinach, peas, and sweetcorn in relation to freezing period. czech j.food sci. 2013;31:613-8. 26. akyol h, riciputi y, capanoglu e, caboni mf, verardo v. phenolic compounds in the potato and its byproducts: an overview. int. j. mol. sci. 2016;17(6):835. 27. routray r, kar m, sahu rk. evaluation of an antioxidant potential in selected leafy vegetables of odisha, india. int j pharm pharm sci. 2013;1(4):21-7. 28. dib ma, bendahou m, bendiabdellah a, djabou n, allali h, tabti b et al. partial chemical composition and antimicrobial activity of daucus crinitus desf. extracts. grasasy aceites. 2010;61(3). 29. routray r, kar m, sahu rk. evaluation of an antioxidant potential in selected leafy vegetables of odisha, india. int. j. pharm. pharm. sci. 2013;5(1):232-35. screening assay of antimicrobial compounds and enzymatic activity vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 24 r a d s j . b i o l . r e s . a p p l . s c i . 24 op en ac ces s f u l l l e n g t h a r t i c l e a simplistic screening assay of antimicrobial compounds and enzymatic activity from local soil microbes kashmala zainab* and hira batool department of microbiology, jinnah university for women, karachi, pakistan a b s t r a c t antibiotics production is the most emerging field worldwide with a constant need for the new ones to fight the microbial resistance. in this context, the research was pursued to isolate, characterize and screen for promising antibioticproducing microbes from local soil. the soil bacterial isolates (s1, s2, s3,s4, and s5) and fungal isolates (f1, f2, f4, f6, and f7) were selected and screened for antimicrobial activity against the test bacteria by agar well diffusion and disc diffusion methods. assays for extracellular enzymes including protease, lipase, lecithinase, cellulase, and amylase following the substrate hydrolysis were performed on different agars such as casein agar, tween 80 agar, egg yolk agar, carboxymethylcellulose agar, and starch agar respectively. the isolated microorganisms which produced antimicrobial compounds were identified as bacillus, actinomycetes, streptomycetes, h. werneckii, a. niger, a. flavus, a. fumigatus and p. notatum on the basis of their cultural and microscopic characteristics and their optimum growth. the antimicrobial activity was determined by varying ph and nacl concentrations. the research work revealed that among all isolates actinomycetes (21%), p. notatum (29%) and h. werneckii (21%) showed maximum bioactivities against the test organisms and all isolates exhibited at least four of the tested enzymes. keywords: antibiotics, soil microorganisms, enzymatic assay, optimization. *address of correspondence: kashm955@gmail.com hb_heer@hotmail.com article info. received: march 25, 2018 accepted: june 30, 2018 cite this article: zainab k, batool h. a simplistic screening assay of antimicrobial compounds and enzymatic activity from local soil microbes. rads j. biol. res. appl. sci. 2018; 9(1): 24-29. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n the term 'antibiotic' means ‘against life'. an antibiotic was initially characterized as a substance, produced by microorganism 1, 2, which at low concentrations can inhibit the growth and development of other microorganisms1,3. soil is a primary source of diverse type microorganisms. most of the novel antibiotics have been detected by screening of "wild isolates" from the soil. first best-known and most broadly exploited antibiotic was penicillin2,3. after the discovery of penicillin, different antibiotics were procurer. in 1939, work begins on the isolation of potential anti-microbial products from the soil microorganisms such as streptomycin. other antibiotics that have been discovered since including bacitracin, polymyxin, chloramphenicol, and tetracycline. scientists have discovered several mechanisms of action of antibiotics. these antibiotics target cell wall, proteins, and nucleic acid synthesis1,5. soil microorganism synthesizes antibiotics and show excellent enzymatic activity by consuming nutrients degraded by various commercially important enzymes. enzymes are biocatalysts produced by living cells for specific biochemical reactions and metabolic processes of the cell. enzymes are present in o r i g i n a l a r t i c l e screening assay of antimicrobial compounds and enzymatic activity vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 25 r a d s j . b i o l . r e s . a p p l . s c i . 25 each living cell, including all microorganisms4. single strain of microorganisms may produce a galaxy of enzymes, hydrolyzing, oxidizing or reducing, and are metabolic in nature. consequently, it is logical to choose strains for the industrial enzymes which are produced insignificant amount. commercial enzymes are produced by molds, bacteria, and yeast etc3. ever since the possibilities of industrial uses of microbial enzymes have increased significantly in 21st century increasing as such enzymes have great potential for many industries to meet the demand of humans1. among hydrolytic enzymes, proteases play a pivotal role with respect to their applications in both physiological and commercial fields. proteolytic enzymes catalyze the cleavage of peptide bonds in proteins, and microorganisms produce a large array of intracellular and/or extracellular proteases. casein agar is a medium utilized for the recognition of hydrolytic microorganisms. proteins are comprised of different chains of amino acids held together by peptide bonds, and hydrolytic enzymes hydrolyze these peptide bonds10. starch particles are hydrolyzed by amylases to yield assorted products, like dextrin and dynamic polymers composed of the units of glucose10. alpha-amylases are the starch-converting enzymes which have the great importance in industries. for amylases, starch agar is widely used that manifest the capacity of a microorganism to produce certain hydrolytic exo-proteins, including alpha-amylase and oligo-1, 6-glucosidase8. carboxymethylcellulose (cmc) including (cellobiohydrolase and beta-glucosidases, which are broadly known as cellulases, hydrolyze the glycosidic bonds of cellulose molecules. cmc screening by microorganisms was performed on different agar plates having selective substrates like cmc. 4 in these cases the cellulolytic actions were checked by staining or having zones like precipitation of substrate observed in cmc plates. whereas clear zones of restraint encompassing the wellspring of the enzymes. an assortment of colors has been utilized for the differential staining, the most well-known stain being the congo red8,10. bacterial lecithinases are of extraordinary intrigue on account of the conceivable part of these proteins in pathogenicity. probably, the most critical contaminants associated with nourishment poisonous quality are lecithins. the bacterial compound is a zinc protein. egg yolk agar (eya) is a differential medium. the incorporation of lecithin in the egg yolk brings about a misty precipitation around the colonies10. lipases catalyze the hydrolysis of long-chain triglycerides. tweens, for example, tween 80 (unsaturated fat esters of polyoxyethylene sorbitan) sought after have been the most extensively sought after substrates for the area of lipolytic microbes in a chromogenic culture media and as fluoro-genic substrates. the methodology relies upon the precipitation, (as the calcium salt), of the unsaturated fat released out of hydrolysis of tween. concerning the character of tweens as lipolytic substrates, there are a few reports of tweens for measuring the lipase trial of esterase, and at times for a ''tweenase'' or ''tweenhydrolyzing'' activity. enzymes applications in pharmaceutical industry are as broad and fast developing8. the biosynthesis of anti-infection agents like other microbial metabolites is controlled by various factors like growth conditions, carbon, nitrogen, mineral salt levels and physical parameters like temperature, ph, and agitation during production9. moreover, the evolution of drug-resistant microorganisms warrants for an enhanced search for new secondary drugs with the new structure6. in this manner, new antibiotic-producing microorganisms and new resources must be tapped the screening program. in this context, local soil samples were collected and analyzed7. m a t e r i a l s a n d m e t h o d s sample collection: a total of 10 soil samples (20 grams of each) collected from different locations in karachi. each sample was scooped from a larger volume and was put in a separate plastic bag under aseptic conditions. the plastic bags containing soil samples were marked and stored at 4°c for further work1. test microorganisms included: escherichia coli (gram-negative), proteus vulgaris (gramnegative), pseudomonas aeruginosa (gram-negative), staphylococcus aureus (gram-positive), candida albicans (yeast). all cultures were preceded from dr. essa laboratory and diagnostic centre1. screening assay of antimicrobial compounds and enzymatic activity vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 26 r a d s j . b i o l . r e s . a p p l . s c i . 26 isolation of soil microorganisms: one gram of soil sample was diluted into 15 ml falcon tube to which 9 ml of distilled water for serial dilution. tenfold serial dilution was carried out, 0.1 ml of 10-3 and 10-4 were poured in respective plates of potato dextrose agar, nutrient agar, czapek dox agar. the plates were incubated at 37°c, 25°c for 24 hours and 72 hours respectively1,2. screening for antibiotic producing bacteria: soil microorganisms which were grown on potato dextrose agar and nutrient agar were overlaid by soft agar (0.75%) seeded with test strains. incubated the plates at 37°c for 24 hours. the zones of inhibition (mm) were observed after 24 and 48 hours. the strains which inhibited the test strains by forming clear zones were isolated, purified and maintained in nutrient agar slants at 40°c for further use. 2 based on the zones of inhibition in preliminary screening of isolates having potential antimicrobial activity were selected for further work. solvent extraction was done by centrifugation and bioactivity of the extracts was assessed following agar well diffusion method. the lawns of each test organisms were prepared on mha plates. the wells of (6mm) were made by using sterile borer on mha. a volume of 100μl of the supernatant of the culture as added into wells and left for 30 minutes until it was diffused. the plates were incubated for respective time (for bacteria 24 hours at 37°c and 7 days for fungi at 28°c). zones of inhibition were recorded3,4. enzymatic assay of isolated stains: for fast track assay for monitoring for production of extracellular enzymes by microbes, distinctive substrates were added into agar medium. presence of extracellular enzymes namely protease, lipase, lecithinase, cellulose, and amylase following the substrate hydrolysis was monitored on different agars such as casein agar, tween 80 agar, egg yolk agar, carboxymethylcellulose agar, and starch agar respectively. for this a sterile wire loop was used to pick colonies from a pure culture, streaked on selected agar plate by dividing it into four quadrants followed by incubation (37°c for 48°c hours)1,10. optimization of growth and antibiotic production: this was done in broth isolates by varying their physical and chemical properties such as ph, nacl (percentile) etc. then the growth and antibacterial activity of the isolates were observed. for this 25ml of nutrient broth and sda broth were prepared in different tubes with the ph values changed in the tubes7,8. the ph varies from 6.0-9.0 and the nacl concentrations of 0.5%, 1.0%, 3.0% and 5.0% were used. then added culture in equal amount in all tubes and incubated for 48hrs at 37°c. after incubation, the growth (o.d) was measured by a spectrophotometer. then centrifuged the broth and examined the bioactivity of supernatant by agar well diffusion method6-8. statistical analysis: all the data obtained from secondary screening were analyzed by one way anova. the level of significance was determined using spss version 15 and the results having a p-value <0.05 were defined to be significant. r e s u l t s bioactive compounds such as antibiotics are widely used. basically, this study is focused on the isolation of antibiotic-producing microorganisms from soil. initial isolation of soil microorganisms was done by spared plate method on na, sda, pda, cza, tsa. screening of antibiotic-producing bacteria was done by agar overlay method. soil microorganism that suppresses the growth of test bacteria by (as the zone of inhibitions) were selected for further confirmation of antimicrobial activity measured by agar well diffusion method (as shown in fig. 1 interestingly). enzyme activity by bacterial assay was performed on different agars (as shown in table 1) all the isolated strains did hydrolyze four of the given substrates. fig. 1: bar diagram of agar well diffusion method, in which different colors represent isolated strains activity against test microorganisms e. coli, p. aeroginosa, s. aureus, p. vulagris, c. albicans (on the basis of their zones of inhibition in (mm). among all isolates s5 (21%), f7(29%) and f1(21%) showed maximum zones of inhibition against the test organisms. screening assay of antimicrobial compounds and enzymatic activity vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 27 r a d s j . b i o l . r e s . a p p l . s c i . 27 table 1: enzymatic activities of the isolated strains on different agars substrates. isolates cellulase activity cmc agar amylase activity starch agar lipase activity tween 80 protease activity casein agar lecithinase activity egg yolk agar s1 +++ +++ +++ +++ +++ s2 +++ +++ +++ +++ + s3 +++ +++ + +++ + s4 + ++ +++ ++ s5 +++ +++ + +++ f1 +++ +++ +++ ++ f2 ++ ++ + +++ f4 ++ +++ +++ + f6 ++ ++ +++ +++ + f7 +++ +++ +++ +++ ++ -: no growth, +: growth, ++: growth and zone, +++ growth and zone very good. optimization for antibiotic production was done and the growth of the isolates was studied by a spectrophotometer while antibacterial activity was monitored by using supernatant in agar well diffusion. it was found that optimal ph for the antimicrobial activity of the isolated strains ranged from ph 6 to ph 7 (as shown in figs. 2 and 3). the antibiotic-producing microbes actinomycetes, streptomyces, bacillus, hortea werneckii, aspergillus flavus, aspergillus fumigatis, penicillium notatum, aspergilus niger were identified on the basis of their growth characteristics (as shown in figs. 4 and 5). fig. 2: effect of ph on antibiotic-producing microbes. fig. 3: effect of nacl conc. on antibiotic-producing microbes. screening assay of antimicrobial compounds and enzymatic activity vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 28 r a d s j . b i o l . r e s . a p p l . s c i . 28 table 2: culture characteristics of the isolated antibiotic-producing soil microbes. isolates growth characteristics microscopic characteristics microbial strains identification s1 white creamy, large opaque, raised and margined colonies gram-positive rods in chains bacillus s2 off-white, large opaque, raised, irregular colonies gram-positive rods in short chains bacillus s3 thin, transparent colonies with red soluble pigment gram-positive filamentous rods streptomyces s4 white thin, transparent colonies gram-positive branches, spider-like streptomyces s5 white, powdery pinpoint colonies gram-positive, diphtheroid or filamentous rods actinomycetes f1 shiny black, slimed or mucoid colonies round yeast. aerial mycelia. septate hyphae and hyphae conidia hortea werneckii f4 greenish-yellow color, overall velvety to woolly texture. vesicles are spherical, septate hyphae, long conidiophores, and biseriate structure. aspergillus flavus f6 blue-green color, white edged. powdery texture. hyphae septate, hyaline. phialides brush like conidia unicellular, ovoid in chains. penicillium notatum f6 blue-green color, white border powdery texture subclacate vesicle, hyphae septate smooth walled aspergillus fumigatus f7 black color with velvety or cottony texture. terviticillate, conidia vary in shape i.e. ovoid to fusiform. aspergillus niger d i s c u s s i o n evolution emergence and widespread dissemination of multi-resistant pathogens genuinely warrant novel and sustainable methodologies and approaches for the development of new antibiotics with a wide range of activities, against infectious agents1,3,7. the study illustrates that the antimicrobial substances give promising results against pathogens. it is essential to search for antibiotics and metabolite producing microbes from different ecological niches such as soil1,2. in the present research work, different microorganisms were isolated from soil. a sample collected from various locations of karachi. primary screening (using agar overlay method methods) that 10 isolates showed manifested antimicrobial activity against test isolates that have potent antimicrobial activity were selected then the antimicrobial activity of the extracts was examined following agar well diffusion methods1,9. extracellular enzymes produced by microorganisms play a vital role in the cycling of biological compounds10. functional enzymes potentially can be determined by estimating of enzymatic activities by using target substrates. assays of extracellular enzymes protease, lipase, lecithinase, cellulase, and amylase following the substrate hydrolysis were performed on different agars. all bacterial and fungal strains hydrolyze the provided substrates as given in table 11,10. fig. 4: macroscopic and microscopic characteristics of isolated antibiotic-producing microbial isolates. fig. 5: enzymatic activity of the isolated strains on different substrate less agar medium. screening assay of antimicrobial compounds and enzymatic activity vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 29 r a d s j . b i o l . r e s . a p p l . s c i . 29 all the bacterial and fungal isolates exhibited at least four of the tested enzymes1,9. the optimum nacl concentrations for the antibacterial activity for all the isolated strains range from (1% to 3%) as shown in fig. 3 & discussed in table 26. revealed that among all isolates actinomycetes (21%), p. notatum (29%) and hortea werneckii (21%) showed appreciable bioactivity against test organisms and all the isolates produces at least three of the tested enzymes such as amylases, lipases, and proteinases. so, the isolated strains carry commercial value and their bioactivity potential as important for industrial point of view1,2,10. c o n c l u s i o n s the potential of local soil microbial isolates to produce antimicrobial substances hydrolyze the enzymes that can utilize varied substrates has been demonstrated. these isolates were daily identified and characterized. further studies will be used by analysis of protein electrophoresis and ms/ms mass-spectrometry that may help to identify and characterize the enzyme protein1,10. r e f e r e n c e s 1. lihan s, lin cs, ahmad i, sinang fm, hua nk, sallehin aa. antimicrobial producing microbes isolated from soil samples collected from nanga merit forest in sarawak, malaysian borneo. euro j experi biol. 2014;4(1):494501. 2. sudha sk, hemalatha r. isolation, and screening of antibiotic-producing actinomycetes from the garden soil of sathyabama university, chennai. asian j pharma clini res. 2015;8:110-4. 3. amin a, khan ma, ehsanullah m, haroon u, azam sm, hameed a. production of peptide antibiotics by bacillus sp: gu 057 indigenously isolated from saline soil. brazi j microbi. 2012 dec;43(4):1340-6. 4. gebreyohannes g, moges f, sahile s, raja n. isolation and characterization of potential antibiotic-producing actinomycetes from water and sediments of lake tana, ethiopia. asian p j tropi biomedi. 2013 jun 1;3(6):42635. 5. manivasagan p, venkatesan j, sivakumar k, kim sk. pharmaceutically active secondary metabolites of marine actinobacteria. microbiolo res. 2014; 169(4):262-78. 6. riya banerjee, l. m). optimization of different parameters for antimicrobial compound production by soil microorganisms. frontiers in biomedical sciences, 2016,1, 1-5. 7. merina paul das*, m. b.optimization of culture conditions for production of antibacterial metabolite by marine bacteria. interna j pharmaceu sci rev res ( 0976 – 044x), 1-5. 8. jackson cr, tyler hl, millar jj. determination of microbial extracellular enzyme activity in waters, soils, and sediments using high throughput microplate assays. j visuali experi: jove. 2013(80). 9. mashoria a, lovewanshi hs, rajawat bs. isolation of antimicrobial producing bacteria from soil samples collected from bhopal region of madhya pradesh, india. int. j curr microbiol app sci 2014;3(12):563-9. 10. underkofler la, barton rr, rennert ss. production of microbial enzymes and their applications. app microbi. 1958;6(3):212. expression of growth form factors during morphogenesis in candida albicans amna shafiq1,aziz fatima1, qudsia hussain1, shazia tabassum hakim1, sayyada ghufrana nadeem1* 1department of microbiology, jinnah university for women, karachi -74600, pakistan abstract the transition of candida albicans from unicellular yeast form to filamentous form i.e, pseudohyphae and hyphae is referred to as morphogenesis. c.albicans has the ability to respond to environmental conditions and accordingly changing its cell morphology. three main morphological forms of c.albicans are unicellular yeast, pseudohyphae and hyphae. environmental factors are important in selectively favouring yeast or hyphal form, most important being the growth medium, incubation temperature and external ph value. cell morphology in c.albicans is associated with changes in the expression of specific factors that are expressed exclusively in particular growth form. these growth form factors are an important target in the investigation of morphogenesis in c. albicans and in the discovery of antifungal drugs that targets the specific growth form of c. albicans. keywords: candida albicans, environmental factors, growth specific form, morphogenesis, introduction morphogenesis is an essential trait in the pathogenic fungus c. albicans and it is clearly required for virulence (lo et al., 1997; braun et al., 2000; braun et al., 2001, murad et al., 2001; saville et al., 2003). morphogenesis in c. albicans is defined as transition from unicellular yeast form to filamentous form (pseudohyphae or hyphae) (khan et al., 2010). it can grow in a variety of morphological forms such as yeast, pseudohyphae and hyphae (sudbery et al., 2004; merson-davies et al., 1989). c. albicans also can form chlamydospores that is to say; thick walled spherical cells or asexual spores which develop over pseudohyphal support cells and appear under unfavourable environmental conditions (sudbery et al., 2004). in pseudohyphae daughter bud elongates and, after septum formation, the daughter cell remains attached to the mother cell. the elongation of buds in pseudohyphae can be so extreme that these filaments can superficially resemble hyphae. because of this, it is often useful to be able to refer to pseudohyphae and hyphae collectively and we will use the term ‘filamentous’ for this purpose. (sudbery et al., 2004). however, hyphae are narrower than pseudohyphal cells ("2 mm) and have parallel walls with no obvious constriction at the site of septation (sudbery et al., 2004). germ tube are the initial projections observed when c. albicans switches from yeast form to hyphal growth (yang, 2003). growth is polarized in c. albicans hypha, with continuous apical growth throughout the cell cycle and parallel cell walls at the septal junctions. in contrast, the growth of pseudohypha and blastophores is only limited to the apical tip during the initial part of the cell cycle (court et al., 2007). pseudohypha can also be distinguished from true hypha on the basis of their morphological index which quantifies the dimensions of cell compartments. alternatively they can be distinguished on the basis of the positions of their septal junctions. these lie at the bud neck for pseudohypha, and within the germ tube for emerging hypha (sudbery et al., 2004). hyphae, pseudohyphae and yeast differ from each other in the rate and order of cell cycle events (berman, 2006). *corresponding author: huma_45@hotmail.com 75 vol 4 (2), july 2013; 75-79 morphogenesis is interrelated to the pathogenesis of c. albicans. adherence has been shown to play critical role in the pathogenesis of infections. germ tubes, that are short hyphal elements, are important in the adherence of organism to the host epithelium. (kimura et al., 1978, lee et al., 1938, sobel el al., 1984). hypha of c. albicans are also important for tissue destruction and host invasion (berman and sudbery, 2002). the yeast form of c. albicans also have virulence attributes and is thought to promote dissemination within the blood stream and establishment of infection at distant sites (braun et al., 2000; gow, 2002; sudbery et al., 2004; sundstrom, 2006). several researchers have investigated that hyphal form is more virulent than yeast form. the principal determinant in the development of disease is the ability of c. albicans to switch between yeast and hyphal forms rather than the individual morphologies (saville et al., 2003). morphological changes between the yeast and the various filamentous forms occur in response to alterations in the growth conditions. parameters that promote hyphal development in vitro include a growth temperature above 35 °c, a ph greater than 6 . 5 , n i t r o g e n a n d / o r c a r b o n s t a r v a t i o n , nonfermentable carbon sources, low oxygen concentrations, and a wide range of chemicals including n-acetylglucosamine, proline (and other amino acids) and alcohols. serum is one of the most potent inducer of hyphal development (brown, 2002). the effect of serum is complex but it is proposed to act, in part, by conferring amino acid starvation. two known inducers of hyphal formation, nacetylglucosamine (glcnac) and proline, may contribute to the serum effect since they are generated by degradation of serum (glyco-) proteins. it is well established that a ph around neutrality favours hyphal development of c. albicans in vitro, while a low ph (ph <6.5) blocks hyphal formation and stimulates growth of the yeast form. growth of cells in the yeast form is promoted by an inoculums above 106 cells/ml, a growth temperature below 35°c, a ph of less than 6.5, glucose and ammonium salts (odds, 1988). compared to liquid medium, induction on solid media appears to represent a weaker hyphainducing condition, because minor defects in filamentation show a defective phenotype on solid but not in liquid media. so the mycelial tendency is stronger in liquid than on solid media (mcclary, 1952) and stationary phase cells are most responsive to hyphal and pseudohyphal induction signals (berman, 2006). it is generally assumed that changes in cell morphology in c. albicans are associated with changes in the expression of specific factors that are expressed exclusively in particular growth form. a total of 5 true hyphae specific genes have been identified. all appear to encode structural proteins, as opposed to regulatory proteins, and four of the five appear to encode cell surface proteins. (brown, 2002). ece1 (extent of cell elongation 1) was the first hyphae specific gene to be identified. ece1 is probably an intracellular protein; having a predicted molecular size of 28,886 da. this protein consists of eight degenerate repeats, 34 amino acids in length. ecei was highly expressed when hyphae were formed, regardless of the induction signal, and ecei expression occurred soon after the stimulus to form hyphae was given. (birse et al., 1993) in previous work (marot-leblond et al., 1995), a c. albicans germtube-specific antigen 3d9 antigen was recovered by the use of a mab. the relative molecular mass ranged from 120 to 220 kda. later (beucher et al., 2009) it was concluded that antigen 3d9 is als3 with same molecular mass. als3 is a hypha specific protein (coleman et al., 2009) member of one of the major families of c. albicans adhesins als (agglutinin-like sequence) that encode cell-surface glycoproteins.(hoyer et al., 2008). als3 have molecular size of 119,927 da, it consists of 10 degenerate repeats and 36 amino acids in length (hoyer et al., 1998). als3 has been shown to be required for mature-biofilm formation, binding 76 vol 4 (2), july 2013; 75-79 extra cellular matrix, adhesion to host cells, and internalization of c. albicans by endothelial cells (hoyer et al., 2008; sheppard et al., 2004; zhao et al., 2006). als3 is activated under serum, nacetylglucosamine and proline-inducing conditions (hoyer et al., 1998). als8 has been mentioned several times in the literature as an als3-like gene (leng et al., 2001; murad et al., 2001) als8 gene was also shown to encode a hypha-specific cell surface agglutinin. more detailed analysis confirmed that als8 is expressed under serum, n-acetylglucosamine and ph-inducing conditions and hence that als8 is a hypha-specific factor (leng & brown, unpublished data). als8 have a molecular mass of 111,945 da uniprotkb (http://www.uniprot.org/uniparc/upi000006a36a). hwp1 (hyphal wall protein 1) is expressed during hyphal development (kim et al., 2007; staab et al., 1996). the gene was tentatively called ece2 (sharkey et al.,1999). it functions as hypha-specific cell surface adhesion (sharkey et al., 1999; staab et al., 1999) molecular mass of hwp1 is 65,342 da. peptide mass obtained from candida db (http://genolist.pasteur.fr/candidadb/). hwp1 (hyphal wall protein) encodes an outer surface mannoprotein on the hyphal wall; the amino terminal sequences of this adhesin are recognized as mammalian transglutaminase substrate (tgase) and form covalent binding with hbec (chaffin et al.,1998; staab et al.,1999) its mrna being expressed during ph and cell culture mediumi n d u c i n g c o n d i t i o n s ( s t a a b s e t a l . , 1 9 9 6 ) hyr1 (hyphally regulated gene) is putative hyphaspecific cell surface glycoprotein. the hyr1 sequence revealed a 2,810-bp open reading frame capable of encoding a 937-amino-acid protein with a predicted molecular mass of 94,000 da. hyr1 is induced specifically during hyphal development during,serum,ph, or n-acetylglucosamine-induction (bailey et al.,1996) als3, als8 and hyr1 are all members of multigene families, and a second ece locus appears to exist in c. albicans (cgd). these are genes that are expressed specifically during hyphal development (swoboda et al., 1994). conclusion several experimental approaches have been applied to the investigation of morphogenesis in c. albicans. the key 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cellular dimorphism. mol. microbiol., 13:663–672. yang yl. 2003. virulence factors of candida species. j. microbiol. immunol. infect., 36:223-228 zhao x, daniels kj, oh sh, green cb, yeater km, soll dr and hoyer ll. 2006. candida albicans als3p is required for wild-type biofilm formation on silicone elastomer surfaces. microbiology, 152:2287–2299. “rads” publishes articles within the whole field of physical science. generally these articles will be in or related to need of the country. manuscripts or data will be considered that have not been previously published or submitted elsewhere for publication. however, re-analysis of previously published data can be accepted. 1. all articles submitted for publication will be reviewed by referees. submit three copies of the article / manuscripts along with a floppy d i s k e t t e ( i b m c o m p u t e r ) c o n t a i n i n g t h e manuscript in duplicate. while submitting diskette it should only the latest version of the manuscript. name the 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volume,number and finally first and the last page number such as carling de & brown mf. 1980. relative effect of vesicular arbuscular mycorrhizal fungi on growth and yields of soybeans. soil sci. soc. amer. j., 44: 528-532. 5. the manuscript should be accompanied by a covering letter signed by corresponding authors. instruction to authors frequency & antibiotic resistance of e. coli in neonatal sepsis vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 113 op e n ac c e s s f u l l l e n g t h a r t i c l e frequency and antibiotic resistance profile of escherichia coli in neonatal sepsis javaid akhter hashmi1, aaliya javaid2, agha syed ali haider naqvi3,*, muhammad umer javaid hashmi4 1department of community medicine, shahida islam medical and dental college, lodhran, pakistan. 2department of gynaecology, syed hospital, bahawalpur, pakistan. 3federal medical and dental college, islamabad, pakistan. 4hbs medical and dental college, islamabad, pakistan. a b s t r a c t background: in developing countries, the most common cause of neonatal mortality is neonatal sepsis. currently, the most significant and common hospital acquired pathogen is escherichia coli, associated with many problems such as septicemia, pneumonia and meningitis in the neonates. due to emerging antibiotic resistance in microorganisms, the current antibiotics which are extensively used are insufficient to resolve the issues. objectives: the aim of the study was to find out the frequency and antibiogram assay of escherichia coli in neonatal sepsis. methodology: the research study was cross sectional study directed by the department of neonatology, bahawal victoria hospital, bahawalpur. the study duration for the current study was from september 2019 to january 2021. blood samples were collected from all the suspected neonates for isolation and identification of e.coli, and standard microbiological protocols were used for the identification. further a modified kirby bauer method of disk diffusion was used for antibiotic sensitivity testing. results: during the defined study duration, total 150 blood cultures were found positive for neonatal sepsis. prevalence of e.coli in these 150 positive blood cultures was 70% (n = 105). the most effective antibiotics observed in our study were gatifloxacin, imipenam and amikacin, while the least effective antibiotics were vancomycin, ampicillin, gentamycin and linezolid. gatifloxacin shows 100% sensitivity against all isolates of e.coli, while all e.coli isolates show 100% resistance to vancomycin. conclusion: our study concluded that e.coli is a major cause for neonatal sepsis in neonates admitted at bahawal victoria hospital, bahawalpur. the resistance pattern was alarmingly increased as observed in the currently available antibiotics. therefore, surveillance of this emerging resistance is needed in these antibiotics. furthermore, in order to limit the resistant strains of the pathogens, there is a need of effective infections control program. keywords antibiotics, escherichia coli, frequency, neonatal sepsis, susceptibility pattern, sepsis. *address of correspondence aghasyedalihaidernaqvi@gmail.com article info. received: february 5, 2021 accepted: november 22, 2021 cite this article hashmi ja, javaid a, naqvi asah, hashmi muj. frequency and antibiotic resistance profile of escherichia coli in neonatal sepsis. 2021; 12(2):113-119. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n neonatal sepsis denotes to an infection of the bloodstream in newborn infants of around <28 days old. it is a disseminated disease with positive blood culture, and considered more common in underdeveloped countries than developed countries1. annually, about 30% to 50% of the death of neonates occur due to neonatal sepsis, hence making it renowned as the most common cause of death in neonates2. neonatal sepsis has been classified into eos (early onset sepsis) and los (late onset sepsis). eos takes place within first 7 days of life while los take place o r i g i n a l a r t i c l e frequency & antibiotic resistance of e. coli in neonatal sepsis vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 114 after first 7 days of life3. the associated risk factors for eos include labor and delivery, while the risk factors for los include care providers or hospital staff. even with the advancement in the health care system, the significant cause of neonatal mortality and morbidity remains neonatal sepsis, instigated by gram-negative bacilli4. recently in nepal, sepsis caused by gram-negative bacteria have been identified5. out of total sepsis cases in neonates, about 18% to 78% of neonatal sepsis has been reported to be caused by gram-negative bacteria6, 7. the most common bacteria in under-developed countries, for eos are escherichia coli, klebsiella species and staphylococcus aureus, while s. aureus, streptococcus pneumoniae, and streptococcus pyogenes are common bacteria that cause los8. however, the bacteria causing sepsis in neonates in underdeveloped countries are different as compared to developed countries. in welldeveloped states, the leading bacteria that cause eos are group b streptococcus and e. coli, while coagulasenegative staphylococci trailed by group b streptococcus and s. aureus are responsible to cause los. additionally, in developing countries, the bacteria that cause eos and los sepsis are alike, particularly in the same hospital setup9, with reported bacteria including salmonella spp, klebsiella spp, e. coli, pseudomonas aeruginosa, coagulase negative staphylococci, s. aureus, s. pyogenes and s. pneumonia10, 11. the reported incidence of neonatal sepsis are 1-8 cases/thousand live births. among these, meningitis have been stated to occur in 1/6 neonatal sepsis patients12. increased morbidity, mortality, and prolong hospital stay have been observed in neonates with neonatal sepsis than neonates having no sepsis13. due to infections, approximately 1.6 million neonates die every year in developing countries14. in pakistan, approximately 7% of the global neonatal deaths occur15, out of which 33% deaths occur due to sepsis infection16. currently, amongst these, the most significant and common hospital acquired pathogen is escherichia coli, associated with many problems such as septicemia, pneumonia, and meningitis in the neonates. the life of the neonate with neonatal sepsis can be saved by early diagnosis, early treatment with antibiotics and proper supportive care. the bacteria responsible for neonatal sepsis have developed antimicrobial resistance and therefore cannot be easily treated with the commonly used antibiotics12. hence, for the selection of appropriate antibiotics, it is essential to know the causative agent for neonatal sepsis which can be determined by the antimicrobial susceptibility testing. additionally, geographical variations have also been reported depending upon the bacteria causing sepsis and the antibiotics used for their treatment at that specified geographical location. hence, continuous surveillance is needed to observe the variation in the epidemiology of microorganisms, sensitivity of antibiotics, and antibiotics used to determine the emerging resistance13. since, limited data is available about the neonatal sepsis, causative organisms for neonatal sepsis, and their antimicrobial profile. therefore, our study was directed to describe the frequency and antibiotic susceptibility pattern of e. coli for better management of neonatal sepsis. m a t e r i a l s a n d m e t h o d s this research study was cross sectional study, directed by the department of neonatology, bahawal victoria hospital, bahawalpur. the duration of this study was from september 2019 to january 2021. the study was approved by the hospital committee for research and ethics for neonatal sepsis sample collection. a consent form was also signed from the guardians of all included neonates. only the cases having positive blood culture were included in our study while negative blood culture cases and premature neonates were excluded from our study. blood samples (5ml) were taken from all suspected neonates and sent to the diagnostic laboratory of the hospital for further investigation. the reports of blood culture were divided into positive and negative reports and only positive reports of blood culture were included in this study. standard blood culture bottles were used for inoculation in the same laboratory and all the samples were incubated for 5 days. e.coli was isolated from all positive blood culture samples by standard microbiological procedures. for identification of the bacteria, gram staining and biochemical tests were done. after confirmation of e. coli, all isolates were processed for antibiogram assay. modified kirby bauer method of disk diffusion was used for antibiogram assay. mueller-hinton agar was used for disc diffusion method and different antibiotic discs were used in antimicrobial sensitivity testing that includes ciprofloxacin (5µg), frequency & antibiotic resistance of e. coli in neonatal sepsis vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 115 amikacin (30µg), ampicillin (10µg), netilmycin (30µg), cefotaxime (30µg), ceftazidime (30µg), imipenem (10µg), piperacillin (100µg), gatifloxacin (5µg), gentamicin (10µg), tobramycin (10µg), linezolid (30µg) and vancomycin (30µg). the inhibitory zones were measured according to guidelines of clinical and laboratory standards institute (clsi)17. data was entered and analyzed by using spss version 23. mean and standard deviation were documented for continuous variables while for categorical data, frequency, and percentages were reported. all the data were presented in figures and tables. r e s u l t s during this one-year study, 150 sepsis positive blood cultures were found. in 60% (n=90) cases late onset sepsis (los) was observed, while in 40% (n=60) early onset sepsis (eos) was observed in our study (table 1). out of 150 positive blood cultures, 53.33% (n=80) were baby boys, while 46.66% (n=70) were baby girls (fig. 1). the prevalence of e.coli in these 150 positive blood cultures was 70% (n=105) (fig. 2). on the basis of antibiotic susceptibility testing, ampicillin & cefotaxime, currently used as empirical therapy at bahawal victoria hospital, bahawalpur were observed resistant in 80% and 65.71% respectively, to most of the e.coli isolates in our study. the most effective antibiotics observed in our study were gatifloxacin, imipenem, and amikacin and these were effective against e.coli as 100%, 80% and 80%, respectively. while, the antibiotics to which e.coli shows more resistance were vancomycin, gentamycin and linezolid and resistance observed in these antibiotics was 100%, 80% and 80%, respectively. in other antibiotics like piperacillin, ciprofloxacin, netilmycin, and tobramycin the resistance observed was 40%, 60%, 65.71% and 31.42%, respectively (table 2). table 1. distribution of patients on the basis of types of sepsis. type of sepsis number of patients (n) percentage (%) early onset sepsis 60 40% late onset sepsis 90 60% figure 1. gender-wise distribution of neonates. frequency & antibiotic resistance of e. coli in neonatal sepsis vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 116 table 2. antibiotic susceptibility pattern of e.coli in neonatal sepsis. s. no. antibiotics used concentrations antibiotics susceptibility e.coli isolates 1 ampicillin 10µg s 21 (20) r 84 (80) 2 cefotaxime 30µg s 36 (34.28) r 69 (65.71) 3 ceftazidime 30µg s 42 (40) r 63 (60) 4 piperacillin 100µg s 63 (60) r 42 (40) 5 ciprofloxacin 5µg s 42 (40) r 63 (60) 6 gatifloxacin 5µg s 105 (100) r 00 (00) 7 netilmycin 30µg s 36 (34.28) r 69 (65.71) 8 tobramycin 10µg s 72 (68.57) r 33 (31.42) 9 imipenam 10µg s 94 (89.52) r 11 (10.47) 10 linezolid 30µg s 21(20) r 84 (80) 11 vancomycin 30µg s 00(00) r 105 (100) 12 gentamicin 10µg s 21 (20) r 84 (80) 13 amikacin 30µg s 94 (89.52) r 11 (10.47) figure 2. frequency of isolated e.coli in neonatal sepsis. frequency & antibiotic resistance of e. coli in neonatal sepsis vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 117 d i s c u s s i o n in spite of significant progress in hygienic conditions, new antimicrobial agent introduction and new advanced techniques for early diagnosis and treatment of neonatal sepsis is still considered as a major cause of neonatal mortality and morbidity13. sepsis of neonate has high prevalence around the globe and is a leading health issue in neonates. according to previous studies, neonatal sepsis incidence were 1-10/thousand normal live births, while the incidence of neonatal sepsis in premature neonates is 1/250 premature live births3. the best technique for sepsis diagnosis is blood culture9. during one year study, total 150 positive sepsis samples from blood culture were found. in 60% (n = 90) cases, late onset sepsis (los) was observed, while in 40% (n = 60) early onset sepsis (eos) was observed in our study. these results are in contrast with the studies reported in other underdeveloped countries like iran, where the los:eos is 22.5%:77.5%, and bangladesh with the ratio as 29.3%:70%18, 19. our findings are in accordance with the previous reports from saudi arabia, where the ratio of eos:los was 39%:61%, pakistan 42%:58% and in libya 31%:69% was observed20-22. moreover, the prevalence of e.coli in these 150 positive blood cultures was 70% (n = 105). this finding is consistent with the early reports from underdeveloped countries where they reported that the major sepsis causing bacteria is gram-negative rods. our results are in accordance with the earlier research which reported 77.1% prevalence of e.coli 23. similarly, in our study, out of 150 positive blood cultures, 53.33% (n = 80) were baby boys, while 46.66% (n = 70) were baby girls. this finding is consistent with the earlier study done in dow university, karachi where 1:0.9 male to female ratio was reported24. antibiotic susceptibility testing revealed that ampicillin and cefotaxime, which are presently utilized as empirical treatment at bahawal victoria hospital in bahawalpur, were resistant to 80% and 65.71% of the e.coli isolates in our study, respectively. gatifloxacin, imipenem, and amikacin were the most effective antibiotics in our study, with 100%, 80%, and 80% efficacy against e.coli, respectively. vancomycin, gentamycin, and linezolid were the antibiotics to which e.coli showed the most resistance, with resistance rates of 100%, 80%, and 80%, respectively. resistance to other antibiotics such as piperacillin, ciprofloxacin, netilmycin, and tobramycin was found to be 40%, 60%, 65.71 %, and 31.42 %, respectively. earlier study which was done by jhoshi et al., in neonates reported high resistance in gram negative bacteria to the majority of penicillin’s and cephalosporin’s, which might be due to beta-lactamase production25. in this study, all the e.coli isolates show sensitivity to imipenem. this high sensitivity of e. coli to imipenem might be due to lack of selective pressure to rare prescription of this antibiotic. these findings are in accordance with the study done by marzban et al. that high sensitivity of all gram-negative bacteria to imipenem (95%), with resistance to imipenem shown by only one e.coli strain26. a previous study done by anwer et al. reported the effectiveness of aminoglycosides against gram negative bacteria and they suggested the reservation of aminoglycosides only for severe infections27. c o n c l u s i o n our study concluded that e. coli is a major causative agent for neonatal sepsis in neonates admitted at bahawal victoria hospital, bahawalpur. the resistance pattern was alarmingly increased which was observed in the currently available antibiotics. therefore, surveillance of this emerging resistance is needed in currently used antibiotics. furthermore, in order to limit the resistant strains of the pathogens, there is a need of effective infections control program. our study proposed amikacin in the empirical antibiotic therapy, to decrease the morbidity & mortality from neonatal sepsis. to control this antimicrobial resistance, there should be judicial use of antibiotics with perfect dosage and duration. e t h i c a l a p p r o v a l the study was approved by the hospital committee of bahawal victoria hospital, bahawalpur, for research and ethics of neonatal sepsis sample collection. a consent form was also signed from the guardians of all included neonates. c o n f l i c t s o f i n t e r e s t none. frequency & antibiotic resistance of e. coli in neonatal sepsis vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 118 f u n d i n g s o u r c e none. a c k n o w l e d g m e n t s none. l i s t o f a b b r e v i a t i o n s clsi eos clinical and laboratory standards institute early onset sepsis los late onset sepsis r e f e r e n c e s 1. aletayeb smh, khosravi ad, dehdashtian m, kompani f, aramesh mr. identification of bacterial agents and antimicrobial susceptibility of neonatal sepsis: a 54month study in a tertiary hospital. afr j microbiol res. 2011; 5(5):528-31. 2. bang at, reddy hm, deshmukh md, baitule sb, bang ra. neonatal and infant mortality in the ten years (1993 to 2003) of the gadchiroli field trial: effect of home-based neonatal care. j perinatol. 2005; 25(1):92-107. 3. ansari s, nepal hp, gautam r, shrestha s, neopane p, chapagain ml. neonatal septicemia in nepal: early-onset versus late-onset. int j pediatr. 2015; 1-8. 4. khanal b, shariff m, deb m. neonatal septicaemia: a hospital based study in eastern nepal. j nepal med ass. 2004; 43(155)8-17. 5. yadav ns, sharma s, chaudhary dk, panthi p, pokhrel p, shrestha a, et al. bacteriological profile of neonatal sepsis and antibiotic susceptibility pattern of isolates admitted at kanti children’s hospital, kathmandu, nepal. bmc res notes. 2018; 11(1):1-6. 6. macharashvili n, kourbatova e, butsashvili m, tsertsvadze t, mcnutt l-a, leonard mk. etiology of neonatal blood stream infections in tbilisi, republic of georgia. int j infect dis. 2009; 13(4):499-505. 7. zaidi ak, thaver d, ali sa, khan ta. pathogens associated with sepsis in newborns and young infants in developing countries. ped inf dise j. 2009; 28(1): 10-18. 8. sundaram v, kumar p, dutta s, mukhopadhyay k, ray p, gautam v, et al. blood culture confirmed bacterial sepsis in neonates in a north indian tertiary care center: changes over the last decade. jpn j infect dis. 2009; 62(1):46-50. 9. karthikeyan g, premkumar k. neonatal sepsis: staphylococcus aureus as the predominant pathogen. ind j ped. 2001; 68(8):715-7. 10. hyde tb, hilger tm, reingold a, farley mm, o’brien kl, schuchat a. trends in incidence and antimicrobial resistance of early-onset sepsis: population-based surveillance in san francisco and atlanta. ped. 2002; 110(4):690-5. 11. isaacs d. a ten year, multicentre study of coagulase negative staphylococcal infections in australasian neonatal units. archives of disease in childhood-fetal and neonatal edition. 2003; 88(2):89-93. 12. hussain m, aurakzai aa, irshad m, ullah i. neonatal sepsis. prof med j. 2018; 25(11):1683-8. 13. aggarwal r, sarkar n, deorari ak, paul vk. sepsis in the newborn. ind j ped. 2001; 68(12):1143-7. 14. black re, morris ss, bryce j. where and why are 10 million children dying every year? lancet. 2003; 361(9376):2226-34. 15. jehan i, harris h, salat s, zeb a, mobeen n, pasha o, et al. neonatal mortality, risk factors and causes: a prospective population-based cohort study in urban pakistan. bull world health organ. 2009; 87:130-8. 16. jalil f, lindblad b, hanson l, khan s, yaqoob m, karlberg j. early child health in lahore, pakistan: ix. perinatal events. acta paed. 1993; 82:95-107. 17. jorgensen jh, hindler jf, reller lb, weinstein mp. new consensus guidelines from the clinical and laboratory standards institute for antimicrobial susceptibility testing of infrequently isolated or fastidious bacteria. clin infect dis. 2007; 44(2):280-6. 18. movahedian a, moniri r, mosayebi z. bacterial culture of neonatal sepsis. iran j public health. 2006; 35(4):84-9. 19. rasul ch, hassan ma, habibullah m. neonatal sepsis & use of antibiotic in a tertiary care hospital. pak j med sci. 2007;23(1):78-85. 20. dawodu a, al umran k, twum-danso k. a case control study of neonatal sepsis: experience from saudi arabia. j trop pediatr. 1997; 43(2):84-8. 21. aftab r, iqbal i. bacteriological agents of neonatal sepsis in nicu at nishtar hospital multan. jcpsp. 2006; 16(3):216-9. frequency & antibiotic resistance of e. coli in neonatal sepsis vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 119 22. misallati a, el bargathy s, shembesh n. blood-cultureproven neonatal septicaemia: a review of 36 cases. emhj. 2000; 6(2-3):483-6. 23. aurangzeb b, hameed a. neonatal sepsis in hospitalborn babies: bacterial isolates and antibiotic susceptibility patterns. jcpsp. 2003; 13:629-32. 24. rao mh, khan s, waseem t, naeem s, sabir s. sepsis in infants: analysis of bacterial pathogens and their antibiotic susceptibility, a study at government tertiary care hospital, karachi. jduhs. 2013; 7(1):35-40. 25. joshi s, ghole v, niphadkar k. neonatal gramnegative bacteremia. ind j ped. 2000; 67(1):27-32. 26. asghart m, samaee h, hossein pm, amir v. neonatal late-onset sepsis in a nicu: analysis of causative organisms and antimicrobial susceptibility ali asghar children hospital from (2004/5-2007/5), tehran, iran. res j biol sci. 2010; 5(5):376-9. 27. anwer sk, mustafa s, pariyani s, ashraf s, taufiq k. neonatal sepsis: an etiological study. jpma. 2000; 50(3):91-3. age, gender, and seasonal effects on thyroid profile vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 17 op e n ac c e s s f u l l l e n g t h a r t i c l e age, gender, and seasonal effects on thyroid profile in adults with normal thyroid functions sajjad ahmad1,*, haji muhammad rashid2, hiza hassan3, muhammad mujahid4, maryam gul5 1department of pathology, quaid-e-azam medical college, bahawalpur, pakistan. 2department of chemical pathology, university of health sciences, lahore, pakistan. 3department of medical lab technology, university of haripur, pakistan. 4pathology department, bsl-3 lab, dhq hospital sargodha, pakistan. 5microbiology department, kohat university of sciences and technology, kohat, pakistan. a b s t r a c t background: thyroid hormone variations have been observed according to age and sex. seasonal effects also induce variations in thyroid function even in people with normal thyroid levels. the majority of clinical laboratories follow a single reference range without the consideration of age, gender, and especially seasonal variations in thyroid function tests. objectives: to evaluate age, gender, and seasonal variations in thyroid function tests in adults with normal thyroid functions. methodology: this cross sectional study was carried out at pathology department, quaid-e-azam medical college, bahawalpur. one year data from jan 2018 to jan 2019 of thyroid function tests (tsh, t3 and t4) and demographic details were obtained for all those patients whose thyroid stimulating hormone (tsh) was in normal reference range (0.5-4.5mu/l). we found the data of 418 subjects from 16 to 75 years of age, among them 196 were females and 222 were males. results: serum tsh levels were higher in females (2.11mu/l ±1.54) than serum tsh levels of males (1.59mu/l ±1.2) with a p-value of 0.0002. ft4 levels (normal range: 0.89-1.76ng/dl) were significantly lower (p-value 0.0018) in females (1.33ng/dl ±0.50) as compared to males (1.48ng/dl ±0.48). in <20 years of the age group of both genders, serum tsh was at lowest levels, while highest in 20-40 years of the age group of males and >60 years of females. the mean tsh of all subjects was high (2.98mu/l) in winters and it was low (2.4mu/l) in autumn. ft4 was at the lowest level (1.16ng/dl) in winters and it was highest (1.46ng/dl) in summers. ft3 levels in the winter season were higher than in other seasons. conclusion: age, gender, and season affects the thyroid hormones levels, and these factors should be considered while interpreting the lab results of thyroid function tests. keywords age, ft3, ft4, gender, seasons, tsh. *address of correspondence sajjad.ahmadbwp@gmail.com article info. received: march 29, 2021 accepted: april 20, 2021 cite this article ahmad s, rashid hm, hassan h, mujahid m, gul m. age, gender, and seasonal effects on thyroid profile in adults with normal thyroid functions. rads j biol res appl sci. 2021; 12(1):17-23. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n the most important gland in the endocrine system is the thyroid gland. it is situated below the larynx, on both anterior sides of the trachea1. the thyroid gland secretes two major hormones; triiodothyronine (t3) and thyroxin (t4). the concentration of t3 is 7% and t4 is 93% of total thyroid hormone secretion. both thyroid hormones circulate in the blood in a free and bound form. about 99% of thyroid hormones are bound forms, and the major carrier o r i g n a l a r t i c l e age, gender, and seasonal effects on thyroid profile vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 18 of bound forms of t3 and t4 is thyroid binding globulin, whereas a small amount is carried out by transthyretin and albumin2. free form is the active form of both hormones and free t3 (ft3) is more active than free t4 (ft4)3. the ft4 is involved in the control mechanism of thyroid secretion and both ft3 and ft4 are indicators of thyroid health of an individual4. these hormones are involved in growth and metabolism of the body, therefore, in the absence of thyroid hormones, the metabolism is decreased and bmr fall to 50% below the range of normal5. thyroid hormones are very important in brain development6. iodine and a protein known as thyroglobulin are required for the synthesis of t3 and t4 from the thyroid gland. different factors like age, sex, body weight, nutritional, climate and health and disease condition effect the production of t3 and t47. the thyroid stimulating hormone (tsh) which is also called thyrotropin is released from the anterior pituitary. tsh controls the production of t3 and t4 from the thyroid gland through a negative feedback mechanism7. tsh itself is regulated by thyroid releasing hormone (trh) from the hypothalamus, by positive feedback mechanism8. tsh measurement can accurately assess the functions of the thyroid gland and it is used as a screening test for thyroid disorders9. in patients with normal pituitary function, an accurate inverse association between free ft4 and the logarithm of tsh can be calculated across the range of primary thyroid disorders. according to the logarithmic response in the concentration of tsh to variations in ft4 levels, tsh measurement gives a more accurate estimation of thyroid gland health than thyroid hormones measurement10. reference range for tsh is 0.5-4.5mu/l11. the application of this reference normal range for thyroid function of all normal populations has been much discussed over the last 20 years12. the normal reference range for ft4 is 0.891.76ng/dl and ft3 is 2.3-4.2ng/dl11. age influences t3, t4 and tsh concentrations. it has been observed that the tendency to develop auto-antibodies against the thyroid gland and its components are gradually increased with age, making people more prone to thyroid disorders. higher incidences of thyroid disorders are observed in people exceeding 40yrs of age13. the variations in thyroid hormone levels have also been observed in different genders14. asian women are involved in many activities and they have more domestic responsibilities, therefore malnutrition among poor and illiterate women and in their children are more prevalent. they are at high risk to develop goiter, anaemia and other disorders. the decrease in the concentration of thyroid hormones with age has been observed in both sexes, but the deprivation of thyroid hormone is more in women than men15, 16. the seasonal effect on t3, t4 and tsh has also been observed. t3 and t4 levels in autumn and winter were higher than in spring and summer17. in common clinical lab practices here in pakistan, almost a single reference value of thyroid profile is applied for both genders, without the consideration of age and seasonal variations. this study was carried out to analyze age, gender and season related variations in thyroid profile in adults with normal thyroid functions, to provide sound data to a clinician to consider such factors when interpreting the thyroid profile results. m a t e r i a l a n d m e t h o d s required data for this cross sectional study was obtained from jan 2018 to jan 2019 from pathology department, quaid-e-azam medical college, bahawalpur. during one year, a total of 1034 patients attended the pathology lab for the thyroid profile (tsh, ft3 and ft4) test. in this study, only data of those patients were collected whose tsh was within the normal reference range (0.5-4.5mu/l)11. we found the data of 418 subjects from 16 to 75 years of age, included 196 females and 222 males. inclusion criteria data of all patients with tsh in the normal reference range (0.5-4.5mu/l) from jan 2018 to jan 2019 were included in this study. exclusion criteria the patients with hyper or hypothyroidism, those who were on medication for hyper or hypothyroidism, and those less than 16 years were excluded from the study. measurements acess2, beckman coulter, a sophisticated fully automated immunoassay analyzer was used for the measurement of thyroid function tests. test results of thyroid profile (tsh, ft3 and ft4) of these subjects with age, sex and season were statistically analyzed to explore the age, gender, and seasonal variations. four seasons were considered in one year cycle, winter from dec to feb, spring from march to age, gender, and seasonal effects on thyroid profile vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 19 may, summer from june to aug, and the autumn period from sep to nov. therefore, to present the seasonal variations in thyroid profile, the obtained data were distributed into four seasons according to the date of test reports. r e s u l t s in our study, a total of 418 subjects was selected. the frequency distribution according to age and gender is given in table 1, and gender wise comparison of the thyroid is given in table 2. two sample t-test was used to compare tsh, ft3 and ft4 between male and female groups. tsh and ft4 levels were significantly different in both groups with a p-value <0.05. ft3 levels between both groups were not significantly different with a p-value >0.05. age wise and seasonal variations were presented in charts. variations in thyroid profile in males in different age groups were presented in fig. 1. tsh levels were comparatively low in <20 years and high in >60 years of age. ft3 levels were high in 20 to 60 years of age, while ft4 levels were at a peak in <20 years of age group and were low in >60 years of age group. variations in thyroid profile in a female with age groups were presented in fig. 2. table 1. frequency distribution according to age and gender. gender <20 year 20-40 41-60 >60 total males 38 48 65 71 222 females 29 44 55 68 196 table 2. gender wise compression of thyroid profile. gender tsh (mu/l) ft3 (ng/dl) ft4 (ng/dl) male (222) 1.59 ± 1.2 2.4 ± 1.04 1.48 ± 0.48 female (196) 2.11 ± 1.54 2.48 ± 0.8 1.33 ± 0.50 pvalue 0.0002 0.3754 0.0018 figure 1. variations in thyroid profile of males in different age groups. age, gender, and seasonal effects on thyroid profile vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 20 figure 2. variations in thyroid profile of females in different age groups. in females, tsh levels were low in <20 years and high in >60 years age groups, while high levels were observed in 20 to 60 years of age. the highest ft3 levels were seen in the>60 years of age group while there was little difference in other age groups. ft4 mean levels in the <20 year age group were significantly high. we observed a negative relationship between tsh and ft4 in all groups of both genders. seasonal variations in all participants were presented in fig. 3. subjects were divided into four groups according to the date of test result reports. tsh mean value was high in winters and low in the summer season. ft3 levels were also high in winters and comparatively low in autumn. ft4 levels in all four seasons were slightly different with considerably low levels in winters. figure 3. seasonal variations in thyroid profile. age, gender, and seasonal effects on thyroid profile vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 21 d i s c u s s i o n iodine deficiency disorders (idds) are commonly prevalent in developed and less-developed countries18. pakistan has overcome this problem to some extent but still, there are some areas, where the idds are present with high frequency. we need some serious steps to improve idd control program19. iodine deficiency is the main cause of hypothyroidism in both genders as well as in children and elderly people20. thyroid functions are decreased with the increase of age17. the present study was conducted to explore the effect of age, gender, and season on thyroid profile. gender wise comparison of thyroid profile (table 2) suggests that tsh levels were significantly higher in females than the males, and ft4 levels were higher in males as compared to females. ft3 in both genders was not statistically different with a p-value of 0.375. our study presented the picture of slightly decreased thyroid functions in females, similar findings about the variations in thyroid hormone in two sexes were in accordance with previous work by razzak et al21 and chaurasia et al. 201122. there is already evidence for the decrease of thyroid functions during menstrual abnormalities. elevated tsh level in one of eight females during child bearing age, high prevalence of hypothyroidism that is 4.8% in females, and higher tendency towards primary hypothyroidism of females with the increase of age support our findings23-25. some other studies also are in favor of our high ft4 results in males, according to the findings of these studies, male sex hormones increases the level of thyroxin binding globulin (tbg), which leads to increase in level of t417, 22. age-related variations were given in fig. 1 for the male group and fig. 2 for females. in <20 years age group of both genders, tsh level was comparatively low, whereas ft4 was higher than all other age groups of both genders except >60 year age group of males, where tsh was low. a study carried out in india during 2011 also reflects similar findings of>60 years male group22. in females of >60 years of age group, the tsh level was significantly high with low ft4, which indicates a decrease in the thyroid functions of females after menopause and with an increase of age26 as well as a high prevalence of subclinical and overt hypothyroidism in females of advance age27. seasonal variations in the concentration of tsh, t3 and t4 is given in fig. 3, which presents a higher level of tsh in winters than in summers, while t3 is nearly similar in all seasons but slightly higher in winters and slightly lower in autumn. t4 is similar in all seasons but slightly lower in winters and slightly high in summers. summer and winter results are comparable with the findings of chaurasia et al. 201122 and are in accordance with a study done by khan et al. 200128. the season in which thyroid tests were performed was independently related to the transition from euthyroid status to subclinical hypothyroid status. seasonal variations in tsh concentration should be taken into account before deciding on treatment for subclinical hypothyroidism, especially in areas with a wide range of annual temperatures29. the climatic components contributed to the slight variance in hormone levels during the different seasons and the effect was mainly on the peripheral conversion of ft4 to ft3, rather than on the pituitary-thyroid axis, leading to slightly more ft3 high in winters30. seasonal changes in tsh occurred independently of the changes in peripheral thyroid hormone, gender, age, and environmental temperatures. the underlying physiological mechanism remain uncertain and specific studies are necessary to clarify its impacting role in humans31. serum-free t3 was significantly correlated with the previous month's average outside temperature. serum tsh did not show any correlation with the average temperature of the month or with free t3. a low level of free t3 serum in winters suggests that the elimination of thyroid hormones is accelerated by the cold, as described in polar t3 syndrome. elevations in serum tsh are not taken into account by changes in circulating thyroid hormones, suggesting that other influences, such as photoperiod may mediate this fluctuation32. c o n c l u s i o n from our study, it is concluded that gender, age, and seasons have a significant effect on thyroid profile, and females have high tsh levels as a whole, especially >60 years of age, while tsh is high in both genders during winters. these factors should be observed while interpreting the lab results of the thyroid profile. c o n f l i c t s o f i n t e r e s t none. age, gender, and seasonal effects on thyroid profile vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 22 f u n d i n g s o u r c e none. a c k n o w l e d g m e n t s we are thankful to professor dr. asma shaukat, head of pathology department, quaid-e-azam medical college, bahawalpur, for technical support. l i s t o f a b b r e v i a t i o n s ft3 free t3 ft4 free t4 t3 triiodothyronine t4 thyroxin tsh thyroid stimulating hormone trh thyroid releasing hormone r e f e r e n c e s 1. franjic s. in shortly about thyroid gland. clin surg. 2021; 4(9):1-5. 2. silva jf, ocarino nm, serakides r. thyroid hormones and female reproduction. biol reprod. 2018; 99(5):907-21. 3. wejaphikul k, groeneweg s, hilhorst-hofstee y, chatterjee vk, peeters rp, meima me, et al. insight into molecular determinants of t3 vs t4 recognition from mutations in thyroid hormone receptor alpha and beta. j clin endocrinol metab. 2019; 104(8):3491-500. 4. roef gl, rietzschel er, van daele cm, taes ye, de buyzere ml, gillebert tc, et al. triiodothyronine and free thyroxine levels are differentially associated with metabolic profile and adiposity-related cardiovascular risk markers in euthyroid middle-aged subjects. j thyroid res. 2014; 24(2):223-31. 5. ettleson md, bianco ac. individualized therapy for hypothyroidism: is t4 enough for everyone?. j clin endocrinol metab. 2020; 105(9): 3090-104. 6. ahmed rg. non-genomic actions of thyroid hormones during development. app clin pharmacol toxicol: 2018;10-18. 7. soundarrajan m, kopp pa. thyroid hormone biosynthesis and physiology. thyroid dis reprod. 2019; 1-17. 8. nillni ea. regulation of the hypothalamic thyrotropin releasing hormone (trh) neuron by neuronal and peripheral inputs. front neuroendocrinol. 2010; 31(2):134-56. 9. soldin op, chung sh, colie c. the use of tsh in determining thyroid disease: how does it impact the practice of medicine in pregnancy? j thyroid res. 2013; 22-31. 10. stockigt j. assessment of thyroid function: towards an integrated laboratory-clinical approach. clin biochem rev. 2003; 24(4):109-16. 11. lewandowski k, editor reference ranges for tsh and thyroid hormones. j thyroid res. 2015; 8(1):1-3. 12. sheehan mt. biochemical testing of the thyroid: tsh is the best and, oftentimes, only test needed–a review for primary care. clin med res. 2016; 14(2):83-92. 13. franco j-s, amaya-amaya j, anaya j-m. thyroid disease and autoimmune diseases. autoimmunity: from bench to bedside: el rosario university press; 2013; 18-27. 14. lamichhane tr, pant sp, lamichhane b, gautam c, paudel s, yadav bk, et al. age-and gender-specific changes in thyroid size and thyroid function test values of euthyroid subjects. jbsm. 2018; 6(11):59-73. 15. rakov h, engels k, hönes gs, brix k, köhrle j, moeller lc, et al. sex-specific phenotypes of hyperthyroidism and hypothyroidism in aged mice. biol sex differ. 2017; 8(1):1-10. 16. fox el, davis c, downs sm, schultink w, fanzo j. who is the woman in women's nutrition? a narrative review of evidence and actions to support women's nutrition throughout life. cdn. 2019; 3(1):76-84. 17. dabla pk, sharma s, sinha n. effect of age, gender and season on thyroid hormones status in children of east delhi-a hospital based study. front endocrinol. clin chem lab med. 2018; 54-63. 18. lazarus j. iodine status in europe in 2014. eur thyroid j. 2014; 3(1):3-6. 19. khattak rm, khattak mnk, ittermann t, völzke h. factors affecting sustainable iodine deficiency elimination in pakistan: a global perspective. int j epidemiol. 2017; 27(6):249-57. 20. chung hr. iodine and thyroid function. ann pediatr endocrinol metab. 2014; 19(1):8-12. 21. abdel r. effect of age and sex on thyroid function tests. establishment of norms for the egyptian population. develop radioimmunoassay related pro. 1992; 353-8. age, gender, and seasonal effects on thyroid profile vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 23 22. chaurasia p, modi b, mangukiya s, jadav p, shah r. variation in thyroid hormones level among people of different age, gender and seasons, piparia, gujarat. j int med res. 2011; 1(2):57-9. 23. velayutham k, selvan ssa, unnikrishnan a. prevalence of thyroid dysfunction among young females in a south indian population. indian j endocrinol metab. 2015; 19(6):781-84 24. li x, meng z, tan j, liu m, jia q, zhang g, et al. gender impact on the correlation between thyroid function and serum lipids in patients with differentiated thyroid cancer. exp ther med. 2016; 12(5):2873-80. 25. meng z, liu m, zhang q, liu l, song k, tan j, et al. gender and age impacts on the association between thyroid function and metabolic syndrome in chinese. med. 2015; (94):50-8. 26. suzuki s, nishio s-i, takeda t, komatsu m. genderspecific regulation of response to thyroid hormone in aging. j thyroid res. 2012; 5(1):1-8. 27. al eidan e, ur rahman s, al qahtani s, al farhan ai, abdulmajeed i. prevalence of subclinical hypothyroidism in adults visiting primary health-care setting in riyadh. j community hosp intern med perspect. 2018; 8(1):11-5. 28. khan a, akhter s, siddiqui mm, khan mma, nawab g. effect of age, sex and seasons on the concentration of thyroid and thyroid stimulating hormones. res j med sci. 2001; 1(4):224-37. 29. kim th, kim kw, ahn hy, choi hs, won h, choi y, et al. effect of seasonal changes on the transition between subclinical hypothyroid and euthyroid status. j clin endocrinol metab. 2013; 98(8):3420-29. 30. mahwi to, abdulateef ds. relation of different components of climate with human pituitary-thyroid axis and ft3/ft4 ratio: a study on euthyroid and sch subjects in two different seasons. int j endocrinol. 2019; 12-21. 31. santi d, spaggiari g, brigante g, setti m, tagliavini s, trenti t, et al. semi-annual seasonal pattern of serum thyrotropin in adults. sci rep. 2019; 9(1):1-7. 32. leppäluoto j, sikkilä k, hassi j. seasonal variation of serum tsh and thyroid hormones in males living in subarctic environmental conditions. int j circumpolar health. 1998; 57:383-5. storage conditions for soymilk vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 120 op e n ac c e s s f u l l l e n g t h a r t i c l e effect of different storage conditions on the sensory attributes of soymilk muhammad dawood1, shahzor gul khaskheli1, asif irshad1,*, amir ali2, pawan kumar1, muhammad ishaq1, abdul raheem1 1institute of food sciences and technology, sindh agriculture university, tandojam, hyderabad, pakistan. 2institute of horticultural sciences, university of agriculture, faisalabad, pakistan. a b s t r a c t background: soymilk is a plant-based protein-rich beverage that is produced from soybeans. it is a steady emulsion of water, oil, and protein. along with similar vegetable-based milk, soymilk can be utilized as an alternative for dairy milk by individuals who are lactose intolerant or vegan. it can also be a decent swap for cow's milk where cow milk is not accessible in adequate amounts. objectives: to evaluate the sensory characteristics of soymilk under different storage conditions i.e., refrigerator, chiller, and room temperature. methodology: examined soybean seeds were rinsed and soaked in the water multiple times the volume of the beans, for around 10hrs. the soaked beans were parboiled (partial cooking) in boiling water for a few moments with steady fomentation. the boiled beans were cooled, de-husked, washed, and after that, the seeds were homogenized with clean water into a stable paste. after that obtained paste was drawn out, (sieved) by utilizing a perfect muslin cloth to isolate the filtrate from the paste. then the obtained milk was boiled to eliminate the beany flavor and was packed in sterilized glass bottles, stored, and coded as t1 (refrigerator temperature, 7°c ±1°c), t2 (chiller temperature, 4°c ±1°c), and t3 (room temperature, 25°c ±2°c). results: the results of the study showed that the maximum score of color, flavor, taste, aroma, and overall acceptability of soymilk recorded as 8.06, 8.22, 8.45, 8.54, and 8.57, respectively on chiller temperature (4°c ±1°c). while the lowest score of color, flavor, taste, aroma, and overall acceptability of soymilk was recorded 7.36, 7.55, 7.78, 7.65, and 7.65, respectively at room temperature (25°c ±2°c). conclusion: it was observed from the present study that in between the stored samples, sensory attributes such as color, flavor, taste, aroma, and overall acceptability perceived better scoring for t2, t1, and t3, respectively. also, during storage, a little decrease in sensory attributes was observed among all treatments mostly from treatments stored at room temperature. keywords soybean, soymilk, sensory attributes, storage conditions, temperature, aroma. *address of correspondence asifirshadbaloch@gmail.com article info. received: march 21, 2021 accepted: october 23, 2021 cite this article dawood m, khaskheli sg, irshad a, ali a, kumar p, ishaq m, raheem a. effect of different storage conditions on the sensory attributes of soymilk. 2021; 12(2):120-127. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n from the last few decades, increased consumption of plant-based protein as the substitution from animal sources has been seen from all over the world. soybean (glycine max) which contains a high protein content (up to 40%) is an excellent plant protein source. from this, several soybean-based items have been acquainted with the business sectors of western nations, out of which soy drink is considered as an appropriate substitute of milk, with high significance for the individuals who are milk protein hypersensitive, lactose intolerant and vegan persons1. o r i g i n a l a r t i c l e storage conditions for soymilk vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 121 soybeans are known to have beneficial effects on human wellbeing which contain a substantial quantity of dietary fiber (both soluble and insoluble fiber) and polyunsaturated fatty acids (pufa) and have a small amount of cholesterol, saturated fatty acid, and sodium2,3. soybeans contain various nutrients, proteins, carbohydrates, lipids, vitamins, and minerals. among foods grown from the ground, soybeans abundantly comprise bioactive constituents of phenolic nature and non-steroidal with chemical structure like 17β-estradiol4. soymilk is obtained from the extracted water of soaked soybeans from the time it is being prepared by crushing water-soaked soybeans. it is known to have a mixture of balanced nutrients as cow milk, but it is deficient in lactose, gluten, and cholesterol, and rich in great phytochemical compounds related to human wellbeing. soymilk is known to emphatically influence bone mineral density (bmd) and bone strength by upgrading intestinal retention of calcium2. in comparison to cow milk, soymilk contains a larger number of proteins, carbohydrates, polyunsaturated fatty acids, and fibers. it is additionally a decent source of potassium, iron, and manganese. furthermore, it has slighter fat, calories, and sugar as compared to cow milk5. it is considered lactose-free milk so, it can be alternative milk for those people who are associated with lactose intolerance disease6. the eating habits of individuals in many developing countries involve starch, cereals, and some legumes. unfortunately, protein derived from animal sources especially milk, that is utilized to supplement the starchbased diets are costly and far off for poor families7. the shortage of milk supply in non-industrial nations may be prompted by the improvement of elective milk from vegetable sources8, and soymilk is one such vegetable extracted milk that can be available to people at a low cost to fulfill their nutrients requirements especially protein. in such situations, soymilk can be the ideal product to meet the nutrition requirements of people. plain soymilk is packed in 200/500ml polythene-made bags, glass containers, and tetra packs. it has the ease of use of a half year when stuffed in tetra packs or likely for a few weeks under refrigerated conditions. it should be put away and dispersed at ordinary temperatures9. the shelf life of soymilk is of great consideration to make the product acceptable and safe for human consumption. shelf life is influenced by mean factors, such as heating and preparation method, its packaging, and how the milk alternatives are being stored to lengthen the storability factor and to reach the final consumers with utmost quality10. soybean is one of the main and important legumes concerning the world's total grain production and is most frequently consumed due to its high protein content and moderately low cost. soymilk in rural households has stayed a problem if not consumed soon after its production and lose its appealing quality. hence, there is a need to efficiently evaluate the impact of storage techniques on the sensory quality of soymilk. although several studies have been reported on soy however very little information is available on the changes in sensory properties during the storage of soymilk. considering the above, the present investigation is proposed to study the sensory evaluation at different storage techniques. m a t e r i a l s a n d m e t h o d s procurement of the sample the soybean seeds were bought from the neighborhood market of hyderabad and an experiment was conducted in the research laboratories of the institute of food sciences and technology, sindh agriculture university, tandojam. later the soybeans were soaked in water to prepare soymilk. preparation of soymilk soymilk was extracted from soybean according to the method described by shurtleff and aoyagi11 as described in fig. 1. first, soybeans were cleaned and arranged (to eliminate broken, harmed, and stained seeds), furthermore, winnowed soybean seeds were rinsed and soaked in the water around multiple times the volume of the beans for around 10-12hrs. the soaked beans were parboiled in water for a few moments with steady fomentation. after that, the boiled beans were cooled and de-husked, altogether washed, and after that, the seeds were homogenized with clean water into a stable paste. after that, the obtained paste was drawn out (sieved) by utilizing a perfect muslin cloth to isolate the filtrate (milk) from the paste. then the obtained milk (soymilk) was boiled to eliminate the beany flavor which is normal for soymilk. after that, the milk was packed in sterilized glass bottles, storage conditions for soymilk vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 122 stored, and coded as t1 (refrigerator temperature, 7°c ±1°c), t2 (chiller temperature, 4°c ±1°c), and t3 (room temperature, 25°c ±2°c). sensory evaluation the sensory evaluation of soymilk was performed by the panel of twenty judges including professors and senior students of the institute of food sciences and technology, sindh agriculture university, tandojam to measure the degree of preference among the different treatments for various sensory attributes i.e. color, flavor, taste, aroma, and overall acceptability. this was conducted by using a nine-point hedonic scale as described by iwe12 representing as, (9=like extremely, 8=like very much, 7=like moderately, 6=like slightly, 5=neither like nor dislike, 4=dislike slightly, 3=dislike moderately, 2=dislike very much, 1=dislike extremely). all three treatments (t1 to t3) were presented to the panel of judges to assess the sensorial attributes. statistical analysis the recovered data from the present findings were tabulated and analyzed according to the statistical procedure, and significant differences of the mean were further computed using the least significant difference (lsd) at 0.05% level of probability. the final data was analyzed using statistix 8.1 computer software13. figure 1. flow diagram for processing of soybeans to soymilk. storage conditions for soymilk vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 123 r e s u l t s a n d d i s c u s s i o n the results showed that the impact of different temperatures on the score of sensory attributes was statistically significant (p < 0.05) affected by distinct temperatures, storage periods, and interactions of different temperatures. color the color of any food is vital because it gives customers an early introduction and thusly, it can impact acceptance and rejection of the product. generally, the color of fresh soymilk is slightly yellow or creamy14. the color of soymilk stored under different storage conditions varied significantly among different treatments and during storage (fig. 2). the maximum score of color was recorded in t2 (8.06) followed by t1 (7.81), while the lowest score of color was noticed in t3 (6.36). however, during the storage period, a decrease in the color of all treatments was observed and the maximum score of color 7.98 was recorded on 0 day, followed by 20th day 7.32, 10th day 7.26, and 30th day 6.01, respectively. present results correlate with the findings of al-nabulsi15 who reported that the values of color enzymatic and non-enzymatic reaction in the soybeans are accelerated when soybeans are stored under high temperature, thus brown pigment are formed which are carried over into the soymilk. flavor soymilk contains a beany flavor and researchers are trying to eliminate the beany flavor in soymilk utilizing different flavors and enzymes16. the results for the score of the flavor of soymilk stored under different storage conditions are given in fig. 3. the maximum score for flavor was recorded in t2 (8.22) followed by t1 (8.21) while the lowest score for flavor was observed in t3 (7.55). however, during the storage period, the flavor of soymilk changed slightly among all the treatments, and the highest score for flavor 7.78 was observed on the 20th day followed by 30th day 7.75, 10th day 7.33, and 0 days 7.15, respectively. similar findings were also recorded by ugochi17 who recorded results in the range of 7.4 to 6.30 and observed a decrease in flavor at a higher temperature that may be due to the maillard reaction and enzymatic activities. taste taste consists of those properties of a product that is judged visually or by touch. the results for the score of the taste of soymilk stored under different storage conditions varied significantly among different treatments and during storage (fig. 4). the maximum score of taste was recorded in t2 (8.45) followed by t1 (8.05), while the lowest score of taste was recorded in t3 (6.55). however, during the storage period, the taste of soymilk varied among all treatments, and maximum results of 7.73 were observed on the 30th day followed by 20th day 7.66, 0 days 7.60, and 10th day 7.20, respectively. the present findings are in accord with the observed values of vanga18 who also observed relevant results in their study. a similar study of lawrence19 observed meaty/breathy flavor and better taste in shelf-stable soymilk. this could be because of higher heat treatment got by shelf-stable soymilks contrasted with refrigerated soymilks. aroma fresh soymilk is known to have a mild smell, so if a difference in smell is noticed, then milk is probably off. significantly different scores for the aroma of soymilk stored under different storage conditions were observed and are given in (fig. 5). the highest score of aroma was recorded in t2 (8.54), followed by t1 (8.25), while the lowest score of aroma was recorded in t3 (6.65). whereas during the storage period changes occurred among all treatments and the maximum score of aroma 8.80 was recorded on 0 days followed by 10th day 8.33, 30th day 7.86, and 20th day 7.53, respectively. the data observed from the present study correlates with the observations of odu20 who also observed changes in aroma with the storage period and obtained better results for refrigerated soymilk as compared to the soymilk stored at room temperature. overall acceptability acceptability is a subjective measure dependent on hedonics or pleasures, which thusly is impacted by the sensory properties of the food. the significantly different scores of overall acceptability of soymilk stored under different storage conditions are given in fig. 6. the maximum score of overall acceptability was observed in t2 (8.52), followed by t1 (8.30), while the lowest score of overall acceptability was recorded in t3 (6.65). in the case of the storage duration, the highest score of overall storage conditions for soymilk vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 124 acceptability 8.80 was observed on 0 days followed by 10th day 8.26, 20th day 8.06, and 30th day 7.46, respectively. these obtained results are in close agreement with the findings of niyibituronsa21 who also examined similar results of decreasing trends (8.00 to 6.20) in overall acceptability in soymilk. figure 2. average score of color of soya milk as affected by various storage temperature. treatments storage t x s se± 0.0705 0.819 0.1415 lsd 0.05 0.1467 0.1696 0.2935 t1 = refrigeration (7 ±1°c) t2 = chiller (4 ±1°c) t3 = room temperature (20 ±2°c) figure 3. average score of flavor of soya milk as affected by various storage temperature. temperature days t x d se± 0.2137 0.2756 0.4767 lsd 0.05 0.4272 0.5512 0.9539 t1 = refrigeration (7 ±1°c) t2 = chiller (4 ±1°c) t3 = room temperature (20 ±2°c) storage conditions for soymilk vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 125 figure 4. average score of taste of soya milk as affected by various storage temperature. temperature days t x d se± 0.2049 0.2645 0.4582 lsd 0.05 0.4104 0.5299 0.9178 t1 = refrigeration (7 ±1°c) t2 = chiller (4 ±1°c) t3 = room temperature (20 ±2°c) figure 5. average score of aroma of soya milk as affected by various storage temperature. temperature days t x d se± 0.2240 0.2888 0.4996 lsd 0.05 0.4477 0.5776 0.9998 t1 = refrigeration (7 ±1°c) t2 = chiller (4 ±1°c) t3 = room temperature (20 ±2°c) storage conditions for soymilk vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 126 figure 6. average score of overall acceptability of soya milk as affected by various storage temperature. temperature days t x d se± 0.1848 0.2385 0.4129 lsd 0.05 0.3698 0.4776 0.8268 t1 = refrigeration (7 ±1°c) t2 = chiller (4 ±1°c) t3 = room temperature (20 ±2°c) c o n c l u s i o n the increasing demand for proteins in the developing countries opened new ways to develop an elective source of protein particularly from plant origin which would be cheaper than animal protein, and still provide protein content close to the animal protein. production of soymilk from soybean is being practiced for the last few decades as it contains a high protein content and has close characters to animal milk. it can be concluded from the present study that in between the stored samples, sensory attributes such as color, flavor, taste, aroma, and overall acceptability perceived better scoring for t2, t1, and t3, respectively. also, during storage, a little decrease in sensory attributes was observed among all treatments mostly from treatments stored at room temperature. based on conclusions, it is suggested that the different flavoring agents and fruits can be used in soy products to finish their beany smell and taste. different methods for the preparation of soymilk can also be tried for the better quality of soymilk. c o n f l i c t s o f i n t e r e s t none. f u n d i n g s o u r c e none. a c k n o w l e d g m e n t s the authors are grateful to the authorities of institute of food sciences and technology, sindh agriculture university, tandojam for providing facilities to conduct this study. l i s t o f a b b r e v i a t i o n s bmd bone mineral density lsd least significant difference pufa polyunsaturated fatty acids r e f e r e n c e s 1. kaczmarska kt, chandra-hioe mv, frank d, arcot j. aroma characteristics of lupin and soybean after storage conditions for soymilk vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 127 germination and effect of fermentation on lupin aroma. lwt-food sci technol. 2018; 87:225-33. 2. jeewanthi rk, lee nk, lee ka, yoon yc, paik hd. comparative analysis of improved soy-mozzarella cheeses made of ultrafiltrated and partly skimmed soy blends with other mozzarella types. j food sci technol. 2015; 52(8):5172-9. 3. hu c, wong wt, wu r, lai wf. biochemistry and use of soybean isoflavones in functional food development. crit rev food sci nutri. 2020; 60(12):2098-112. 4 . ahn h, park yk. soy isoflavone supplementation improves longitudinal bone growth and bone quality in growing female rats. nut. 2017; 37:68-73. 5. usda. food composition database. 2017. 6. rinaldoni an, campderros me, padilla ap. physicochemical and sensory properties of yogurt from ultrafiltreted soymilk concentrate added with inulin. lwt-food sci technol. 2012; 45(2):142-7. 7. kolapo al, oladimeji gr. production and quality evaluation of soy-corn milk. j appl bio sci. 2008; 1(2):40-5. 8. onweluzo jc, nwakalor c. development and evaluation of vegetable milk from treculia africana (decne) seeds. pak j nutri. 2009; 8(3):233-8. 9. gandhi ap. development of haccp procedure for the production of full-fat soy flour. inter food res j. 2008; 15(2):141-54. 10. caluza g. shelf life and acceptability of different fruity flavored soy milk under two types of storage method. int j manag. 2019; 02(1):107-13. 11. shurtleff w, aoyagi a. history of soymilk and other non-dairy milks: including infant formulas, calf milk replacers, soy creamers, soy shakes, soy smoothies, almond milk, coconut milk, peanut milk, rice milk, sesame milk, etc. soy info center. 2013; 1226-9. 12. iwe mo. the science and technology of soybean. rejoint comm ser ltd. 2003; 145-9. 13. statistix. statistix version 8, user’s manual. analytical software, tallahassee, fl, usa. 2006. 14. khodke su, shinde ks, yenge gb. a study on the storage of sterilized soya milk. int j farm sci. 2015; 4(4):166-79. 15. al-nabulsi a, shaker r, osaili t, al-taani m, olaimat a, awaisheh s, holley r. sensory evaluation of flavored soy milk-based yogurt: a comparison between jordanian and malaysian consumers. j food sci eng. 2014; 4(1):27-32. 16. laswai hs, thonya n, yesaya d, silayo vck, kulwa k, mpagalile jj, et al. use of locally available flavouring materials in suppressing the beany taste in soymilk. african j food agr nut develop. 2009; 9(7):1548-60. 17. ugochi nf, chukwuma um, nwanneoma oj, ndako kj, nwabugo ma. nutrient and sensory quality of soymilk produced from different improved varieties of soybean. pak j nutri. 2015; 14(12):898-906. 18. vanga sk, wang j, raghavan v. effect of ultrasound and microwave processing on the structure, in-vitro digestibility and trypsin inhibitor activity of soymilk proteins. lwt-food sci technol. 2020; 131:10970815. 19. lawrence se, lopetcharat k, drake ma. preference mapping of soymilk with different us consumers. j food sci. 2016; 81(2):463-76. 20. odu nn, egbo nn, okonko io. assessment of the effect of different preservatives on the shelf-life of soymilk stored at different temperatures. res. 2012; 4(6):62-9. 21. niyibituronsa m, onyango an, gaidashova s, imathiu s, ming z, ruinan y, peiwu l. evaluation of five essential oils by gas chromatography-mass spectrometry and their effect on fungal growth inhibition and sensory acceptability of soymilk. j food res. 2020; 9(2):36-47. identification and antibiotic susceptibility testing of esbl producing klebsiella strains vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 8 r a d s j . b i o l . r e s . a p p l . s c i . 8 op e n ac c e s s f u l l l e n g t h a r t i c l e identification and antibiotic susceptibility testing of esbl producing klebsiella strains by phenotypic methods taqdees malik*, asma naim* and asma saeed department of microbiology, university of karachi, karachi, pakistan a b s t r a c t multi drug resistance has now become a worldwide therapeutic challenge due to the widespread use of broad spectrum antibiotics. klebsiella species have significant importance in clinical field as they cause various infections in human and are considered as potential pathogens that express antibiotic resistance through their strong enzymatic activity. extended spectrum beta lactamases (esbls) are plasmid mediated enzymes produced mostly because of mutation and few other factors. these enzymes confer resistance against various β-lactam drugs including cephalosporins and monobactams. among the genus klebsiella, esbls are highly prevalent in k. pneumoniae followed by k. oxytoca. this study was conducted in pakistan to assess the distribution of esbl producers among klebsiella spp., an important member of the family enterobacteriaceae. from january 2010 to january 2012, a total of 236 gram-negative isolates were collected from different renowned microbiological laboratories. out of the 236 gram-negative isolates, 125 were found as klebsiella spp. by using standard microbiological techniques. antimicrobial susceptibility profiling of these strains was performed by using kirby bauer disk diffusion method. phenotypic detection of the production of extended spectrum beta lactamase enzyme was performed using double disc synergy method and combination disc method. it has been identified that klebsiella strains are highly resistant against amoxicillin, tetracycline, nalidixic acid, cephradine, gentamicin, co-amoxyclav with the percentage of 100%, 86%, 86%, 82%, 82% and 80% respectively. the most effective antibiotics for klebsiella spp. were found to be amikacin, meropenem and piperacillin-tazobactam, with highest sensitivities of 96%, 94% and 91%. phenotypic detection of extended spectrum beta lactamase production by double disc synergy test was able to identify 28% esbl producers among klebsiella isolates whereas 64% were detected by combination disc test. keywords: esbl, klebsiella, ddst, cdt. *address of correspondence: anaeem@uok.edu.pk taqdees_13@hotmail.com article info. received: june 21, 2018 accepted: july 06, 2018 cite this article: malik t, naim a, saeed a. identification and antibiotic susceptibility testing of esbl producing klebsiella strains by phenotypic methods. rads j. biol. res. appl. sci. 2018; 9(1): 8-13. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n klebsiella is a well-known genus of the family enterobacteriaceae associated with nosocomial and community-acquired pneumonia with high morbidity and mortality rates, if not treated timely and properly. klebsiella as an opportunistic pathogen is mainly involved in nosocomial infections affecting immunocompromised individuals more frequently1. klebsiella pneumoniae followed by klebsiella oxytoca are the most important species of the genus klebsiella, found to be associated o r i g i n a l a r t i c l e identification and antibiotic susceptibility testing of esbl producing klebsiella strains vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 9 r a d s j . b i o l . r e s . a p p l . s c i . 9 with human clinical specimens, accounting for 75% to 86% and 13% to 25% isolates respectively2. the continuous spread of drug resistance among pathogenic microbes has become a therapeutic challenge all over the world. the indiscriminate and widespread use of extended spectrum antibiotics has played a significant role for the spread of multiple drug resistance among pathogenic strains3. klebsiella isolates are gaining importance due to the emergence of their multidrug resistant strains, specifically extended-spectrum beta lactamase(esbl)-producing strains, capable of hydrolyzing beta lactam drugs1. the first strain capable of producing esbl was detected in germany in 1983 and later several other outbreaks were reported in usa and other regions due to these esbl producers4. esbls are plasmid mediated enzymes that are capable of transferring resistance from one strain to another, not only for beta lactam drugs, they may also carry resistance genes of aminoglycosides, fluoroquinolones, tetracyclines, chloramphenicol and sulfamethoxazoletrimethoprim. these esbls can hydrolyze third generation cephalosporins including cefotaxime, ceftriaxone, ceftazidime and monobactams but could be inactivated by serine β-lactamase inhibitors such as clavulanate, sulbactam and tazobactam when used in combination with third generation cephalosporins3. the timely and accurate detection of esbl producers can prevent several outbreaks and endemics. although various molecular techniques are now known for the detection of esbl producers, however, in routine lab practice, they are rarely followed. it is therefore suggested to use two or more phenotypic detection methods for identification of esbl producers in routine clinical settings to avoid misuse of antibiotics3.prevalence, susceptibility profile and phenotypic characteristics of esbl producers may vary from country to country and region to region. therefore, in our study, we performed susceptibility testing and phenotypic detection of esbl producer and esbl non-producers among klebsiella strains to establish effective antibiotic strategy and suggest appropriate empirical therapy in high-risk units. the present study was carried out to determine the antibiotic resistance pattern and phenotypic and detection of resistance enzymes in klebsiella species for implementing the strategies and judicial use of drugs for proper therapy. for executing strict infection control policy and to stop the spread of resistant strains, it is important to detect esbl producing isolates more promptly. m a t e r i a l s a n d m e t h o d s bacterial isolates: from january 2010 to january 2012, a total of 236 gramnegative isolates were collected from renowned microbiological laboratories of karachi, pakistan. all the samples were aseptically streaked on nutrient agar slants (oxoid). identification and characterization of bacterial isolates was performed by using standard microbiological techniques7. identified isolates were stored at -70℃ inglycerol stock8. antibiotic susceptibility testing: antibiotic susceptibility testing was performed by kirbybauer method. antimicrobial susceptibilities of the isolates to 25 different antimicrobial agents (µg) viz. amikacin (30), gentamicin (10), tobramycin (10), amoxicillin (20), amoxicillin/clavulanic acid (20/10), piperacillintazobactam (100/10), ciprofloxacin (5), ofloxacin (5), enoxacin (10), sparfloxacin (5), nalidixic acid (30), cephradine/ velosef (30), cefuroxime (30), cefixime (5), cefotaxime (30), ceftazidime (30), ceftriaxone (30), cefoperazone/sulbactam (75/30), imipenem (10), meropenem (10), doxycycline (30), tetracycline (30), trimethoprim/sulphamethoxazole (1.25/23.75), fosfomycin(200) and nitrofurantoin (300) were determined by using commercially available disks (oxoid) and results were interpretedas per national committeefor clinical laboratory standards (nccls) guidelines9. phenotypic detection of esbl producers: extended spectrum beta lactamase producing strains among klebsiella isolates were detected by the methods of double disc synergy and combination disc. i. double disc synergy test: the test was performed by using disks of cefotaxime (30 ug) (oxoid™) and amoxicillin clavulanic acid (20/10 ug) (oxoid™). disc of cefotaximewas placed at 15mm from amoxicillin clavulanic aciddisc and incubated at 37℃ for 24 hours. synergy between the discs was observed and the increased zone of ctx towards amc was identification and antibiotic susceptibility testing of esbl producing klebsiella strains vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 10 r a d s j . b i o l . r e s . a p p l . s c i . 10 considered as a clear indication for esbl production10. ii. combination disc method: cefotaxime (30ug) (oxoid™) and the combined disc of cefotaxime plus clavulanic acid (30/10 ug) (bioanalyse®) were used in this test following protocol recommended by clsi in 2010. both discs were placed at a distance of 24mm to observe the zones of inhibition. esbl producers were detected by comparing the zone sizes of the discs. esbl production can be interpreted by 5 mm increase in the zone size of combination disc as compared to the cefotaxime disc9-11. r e s u l t s out of 125 identified klebsiella strains, 100 strains were isolated from female patients and 25 strains were from male patients. klebsiella isolates were detected higher in the patients of younger age followed by the older group. less number of isolates were detected in the middle-aged patientsand children. distribution of klebsiella isolates with respect to the age group has been shown in fig. 1. fig. 1: age group analysis showing the number of klebsiella isolates collected from selected age groups. susceptibility testing of strains detected that amikacin, meropenem, imipenem and piperacillin-tazobactam are the most effective antimicrobial agents against klebsiella isolates. it was found that only 8% isolates were resistant to imipenem, 6% showed resistance against meropenem, 3% to amikacin, and 2% to piperacillin-tazobactam. we detected resistance against amoxicillin in 100% strains of klebsiella as the isolates were intrinsically resistant to penicillin. highest resistance was observed against nalidixic acid (86%), doxycycline (86%), tetracycline (86%), gentamicin & cephradine (82%) and amoxicillin/clavulanic acid (co-amoxyclav) (80%). resistance pattern of klebsiella strains against other antimicrobials was detected as 63% against cefuroxime, 42% to cefixime; 36% to ciprofloxacin, 33% to sparfloxacin, 30% to enoxacin, 29% to ofloxacin and cefotaxime whereas 26% resistance was observed against ceftazidime, ceftriaxone and cefoperazone/sulbactam and 19% isolates were resistant to tobramycin. complete resistance profile including number of sensitive, intermediate and resistant strains are mentioned in fig. 2. fig. 2: antibiotic resistance pattern of klebsiella species against multiple drugs. out of 125 identified strains of klebsiella, 35 were found as esbl producers by double disc synergy test whereas 80 strains were detected as esbl producers by combination disc method as mentioned in table 1. detection of esbl producers due to size and variation in zones of inhibitions for both methods has been illustrated in fig. 3. table 1: number and percentage of esbl positive klebsiella isolates using ddst and cdt. test no. of esbl positive isolates(n) percentage (%) double disc synergy test 35 (28) combination disc test 80 (64) total no. of esbl producers by both method 81 (64.8) identification and antibiotic susceptibility testing of esbl producing klebsiella strains vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 11 r a d s j . b i o l . r e s . a p p l . s c i . 11 double disc synergy test (a): double disc synergy test positive klebsiella isolate showing keyhole appearance. combination disc test (b): combination disc test positive klebsiella isolate showing extended zone of inhibition in ctx+ca as compared to ctx alone. fig. 3: comparison of (a) double disc synergy test and (b) combination disc test. d i s c u s s i o n several reports and studies have been conducted on klebsiella isolates, describing their occurrence, susceptibility pattern and esbl production. klebsiella strains have gained immense importance among the scientific community due to their multi drug resistance strains with a specific mechanism of resistance i.e. enzyme production. in our study we identified 125 klebsiella strains with high occurrence among females and less in males. our findings confirmed the studies reported from india, nigeria, iran in which klebsiella spp. were detected higher in females as compared to males due to the compressed size of urethra5,6,12-14. we detected that the prevalence of klebsiella isolates was higher in the younger age group. contradictory researches were reported from india and iran in which higher number of isolates were detected in the middle-aged group and the age group of children below 2 years respectively. dissimilarity in the result can be occurred in different regions, especially it depends upon the consumption rate of antibiotics among different age groups5,14. we determined the resistance profile of klebsiella isolates by using twenty-five antimicrobial agents. we found that all klebsiella isolates were found resistant to amoxicillin. nalidixic acid (na), doxycycline, tetracycline, gentamicin, cephradine, and amoxicillin/clavulanate were less effective antimicrobials against klebsiella isolates. moderate resistance was observed against cephalosporins and quinolones. however, carbapenems, amikacin and piperacillin-tazobactam have shown good inhibitory results against klebsiella strains. all strains of klebsiella were resistant against amoxicillin. few other studies reported 100% resistance against amoxicillin however, comparatively less resistance against coamoxiclav was reported in another research15. moderate resistance to third generation cephalosporin and comparatively less resistance against piperacillin tazobactam and aminoglycosides16-18. good susceptibility results were obtained with aminoglycosides and fluoroquinolones6. in our study, we detected carbapenem as an effective drug against klebsiella spp. however, contrary to our research, resistance against carbapenem drugs was also reported in klebsiella isolates19. although we did not obtain good inhibitory results from ciprofloxacin against klebsiella isolates, however, few studies have reported ciprofloxacin as an effective drug against these strains16,20. a study reported that carbapenem should be considered as a drug of choice against the infections caused by klebsiella isolates21. a similar study conducted in australia, revealed that imipenem has the similar susceptibility pattern against esbl producing isolates and esbl non-producing isolates22. klebsiella strains were found to be sensitive against amikacin and imipenem in the study23. comparable results were reported in another study of pakistan in which imipenem, piperacillintazobactam, ampicillin-sulbactam and amikacin were found as effective drugs24. we detected 35 (28%) and 80 (64%) strains of esbl producers by double disc synergy test and by combination disc method respectively. esbl producers were initially reported from europe in 1983, since then their prevalence is increasing day by day. various reports identification and antibiotic susceptibility testing of esbl producing klebsiella strains vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 12 r a d s j . b i o l . r e s . a p p l . s c i . 12 have revealed its prevalence in different regions of the world. esbl production was detected in 30-60% isolates from brazil in a study conducted on the patients of intensive care units21. in another study, 86% of klebsiella strains were detected as esbl producers by both methods17. combination disc method can be considered to be more reliable as it detects higher number of isolates in our research and this method was also verified by the fact that by adding clavulanic acid to third generation cephalosporin, it will increase its effectiveness. similar phenomenon was stated in another research20. comparatively lesser number (24.5%) of esbl producing strains were detected in pakistan 201324. another study from pakistan reported 22% esbl producing strains among klebsiella isolates in 201625. in 2016, a study reported fewer number of esbl producers by using the method of double disc synergy as compared to the combination disc method26. in a study conducted in nepal, higher number of esbl producing klebsiella isolates (18.4%) were reported by using the method of phenotypic confirmatory disc diffusion test27 whereas in 2017, esbl production was reported in relatively less number of klebsiella pneumoniae (3.8%) and klebsiella oxytoca (0.7%) isolates28. therefore, it would be preferable to conduct more than phenotypic screening technique to detect the correct number of esbl producers. c o n c l u s i o n s this is an alarming situation that antibiotic resistance is increasing among pathogenic strains. therefore, now there is an intense need to restrict the widespread use or misuse of antibiotics. antibiotic susceptibility profiling and detection of esbl producing strains must be included in the routine laboratory testing protocols. in the light of our findings, we could suggest that the use of amoxicillin and co-amoxiclav should be restricted. cephalosporins could only be used in case of esbl non-producers. however, carbapenems, amikacin and piperacillin-tazobactam can be the drugs of choice for the treatment of infections caused by klebsiella species. phenotypic screening of esbl producers can be better detected by the combination disc test or it is preferable to use more than one screening procedures for the detection of esbl production. r e f e r e n c e s 1. podschun r, ullmann u. klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. clin microbiol rev. 1998; 11(4):589-603. 2. pokra m, sharma dk, mehta p, verma hr, pundir s, rana j, et al. its alarming, klebsiella spp. towards multidrug resistance. int j curr microbiol app sci 2016; 5(6): 150-60. 3. vinodhini r, moorthy k, palanivel p, punitha t, saranya s, bhuvaneshwari m, et al. detection and antimicrobial susceptibility pattern of esbl producing gram negative bacteria. asian j pharm clin res. 2014;7(1):243-7. 4. behrooozi a, rahbar m, yousefi j.v. frequency of extended spectrum beta-lactamase (esbls) producing escherichia coli and klebseilla pneumoniae isolated from urine in an iranian 1000-bed tertiary care hospital. afr j microbiol res 2010; 4 (9): 881-8. 5. akram m, shahid m, khan a.u. etiology and antibiotic resistance patterns of community-acquired urinary tract infections in jnmc hospital aligarh, india. ann clin microbiol antimicrob 2007; 23; 6: 4. 6. chikwendu ci, amadi es, obi rk. prevalence and antimicrobial resistance in pseudomonas aeruginosa and klebsiella pneumoniae isolates from non-clinical urine samples. ny sci j. 2010; 3(11): 194-200. 7. krieg nr, holt jc. (eds., 1984). bergey’s manual of systematic bacteriology, 1st ed., vol. 1, williams and wilkins, baltimore. 8. cheesbrough m. 2006. a practical and well-illustrated guide to microbiological, hematological, and blood transfusion techniques. district laboratory practice in tropical countries part 2. 9. national committee for clinical laboratory standards (nccls) guidelines 2010. 10. vaidya vk. horizontal transfer of antimicrobial resistance by extended-spectrum β lactamaseproducing enterobacteriaceae. j lab physicians 2011; 3(1): 37-42. 11. lin c-f, hsu s-k, chen c-h, huang jr-r, lo h-h. genotypic detection and molecular epidemiology of extended-spectrum β-lactamase-producing escherichia coli and klebsiella pneumoniae in a regional hospital in central taiwan. j med microb 2010; 59: 665-71. 12. khosravi ad, hoveizavi h, mehdinejad m. prevalence of klebsiella pneumoniae encoding genes for ctx-m-1, tem-1 and shv-1 extended-spectrum beta lactamases (esbl) enzymes in clinical specimens. jundishapur j microbiol 2013; 6(10): e8256. identification and antibiotic susceptibility testing of esbl producing klebsiella strains vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 13 r a d s j . b i o l . r e s . a p p l . s c i . 13 13. babakhani s, derikvand ss, nazer mr, kazemi mj. comparison frequency and determination antibiotic resistance pattern of klebsiella spp. isolated from nosocomial infection in khorramabad shohadayeashayer hospital. bull env pharmacol life sci 2014; 3(12): 14954. 14. akbar da, ali ra, zahra kj. screening and prevalence of shv/ctx-m/tem β-lactamase resistance genes in klebsiella strains isolated bacteria from urinary tract infection in pre-school-age children in jahrom, iran. iioabj 2016; 7 (10): 82-8. 15. wagenlehner fme, niemetz a, dalhoff a, naber kg. spectrum and antibiotic resistance of uropathogens from hospitalized patients with urinary tract infections: 19942000. int j antimicrob agen 2002; 19: 557-64. 16. randrianirina f, soares j-l, carod j-f, ratsima e, thonnier v, combe p, grosjean p, talarmin a. antimicrobial resistance among uropathogens that cause community-acquired urinary tract infections in antananarivo, madagascar. j antimicrob chemother 2007; 59: 309-12. 17. manchanda v, singh np, goyal r, kumar a, thukral ss. phenotypic characteristics of clinical isolates of klebsiella pneumonia & evaluation of available phenotypic techniques for detection of extended spectrum beta-lactamases. indian j med res 2005;122: 330-7. 18. kashef n, djavid ge, shahbazi s. antimicrobial susceptibility patterns of community-acquired uropathogens in tehran, iran. j infect dev ctries 2010; 4(4):202-6. 19. bratu s, tolaney p, karumudi u, quale j, mooty m, nichani s, et al. carbapenemase-producing klebsiella pneumoniae in brooklyn, ny: molecular epidemiology and in vitro activity of polymyxin b and other agents. j antimicrob chemother 2005; 56: 128-32. 20. colodner r. extended-spectrum beta-lactamases: a challenge for clinical microbiologists and infection control specialists. am j infect control 2005; 33(2):104-7. 21. paterson dl, bonomo ra. extended spectrum βlactamases: a clinical update. clin microbiol rev. 2005; 18(4): 657-86. 22. hawser sp, bouchillon sk, hoban dj, badal re, hsueh p-r, paterson dl. emergence of high levels of extended-spectrum-beta-lactamase-producing gramnegative bacilli in the asia-pacific region: data from the study for monitoring antimicrobial resistance trends (smart) program, 2007. antimicrob agen chemother 2009; p. 3280-4. 23. abreu ag, marques sr, monteiro-neto v, gonçalves ag. extended-spectrum beta-lactamase-producing enterobacteriaceae in community-acquired urinary tract infections in são luís, brazil. braz j microb 2013; 44(2): 469-71. 24. ahmed i, sajed m, sultan a, murtaza i, yousaf s, maqsood b, et al. the erratic antibiotic susceptibility patterns of bacterial pathogens causing urinary tract infections. excli j 2015; 14: 916-25. 25. ain nu, abrar s, nazeer k, riaz s. extended spectrum beta lactamases and metallobeta-lactamases: an update from lahore, pakistan eur j pharm med res 2016; 3(9): 92-7. 26. yala j-f, mabika rm, bisseye c, kenguele hm, kama em, dikoumba ac, et al. phenotypic and genotypic characterization of extended-spectrum-beta-lactamases producing-enterobacteriaceae (esble) in patients attending omar bongo ondimba military hospital at libreville (gabon). j mol microbiol biotechnol. 2016; 4(6): 944-9. 27. chaudhary p, bhandari d, thapa k, thapa p, shrestha d, chaudhary hk, shrestha a, parajuli h, gupta bp. prevalence of extended spectrum beta-lactamase producing klebsiella pneumoniae isolated from urinary tract infected patients. j nepal health res counc. 2016;14(33): 111-5. 28. shakya p, shrestha d, maharjan e, sharma vk, paudyal r. esbl production among e. coli and klebsiella spp. causing urinary tract infection: a hospital based study. open microbiol j. 2017; 11: 2330. . lead-tolerant bacteria can minimize lead toxicity in plants vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 1 r a d s j . b i o l . r e s . a p p l . s c i . 1 op en ac ces s f u l l l e n g t h a r t i c l e lead-tolerant bacteria can minimize lead toxicity in plants tehreem aslam and ambreen ahmed* department of botany, university of the punjab, quaid-e-azam campus, lahore 54590, pakistan a b s t r a c t in today’s world, environment is exposed to lead due to various anthropogenic activities. it adversely affects plants as well as microorganisms by disturbing soil health and fertility. bioremediation is a technique used to sequester heavy metals from the contaminated soil and it can be used to decontaminate the polluted soil. lead-tolerant plant health promoting rhizobacteria (phpr) can be used to enhance the efficacy of lead remediation. lead uptake can be enhanced through bacteria by modifying root structure, secreting metal sequestering molecules in rhizosphere and alleviating lead induced phytotoxicity. for this purpose, lead-resistant auxin-producing bacteria were isolated from the rhizosphere of some plants. these auxin-producing lead-tolerant bacteria were used to treat zea mays both in the presence and absence of lead-stress under laboratory conditions and its impact on plant growth and biochemical parameters of zea mays were analysed. keywords: bioremediation, lead, pgpr, zea mays, pmi. *address of correspondence: ambreenahmed1@hotmail.com article info. received: april 23, 2018 accepted: june 25, 2018 cite this article: aslam t, ahmed a. lead-tolerant bacteria can minimize lead toxicity in plants. rads j. biol. res. appl. sci. 2018; 9(1): 1-7. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n increasing global population has lead to increase in industrialization, technological developments and overuse of natural resources and release of contaminants into the environment which in turn resulted in greater number of highly polluted areas. increasing demands of land for housing, infrastructure and recreation reduced the feeling to remediate polluted areas. heavy metals contamination poses a serious threat to plants and animals throughout the whole world. due to their sustainability, they pose a serious threat to agricultural fields1. excess of heavy metals are absorbed by roots which accumulate in various plant organs and reduce growth by disturbing metabolic processes2. thus, soil re-establishment processes are essential for reinvesting in agricultural production. bioremediation is a promising technology which involves environmental friendly processes for remediation of polluted soil and water on a large scale3,4. the advancement of bioremediation strategies, such as the associations of bacteria with the plants rhizosphere that grow in contaminated soils are important because of the biotechnological capability of microorganisms for metal elimination directly from soil or the expected transport of metals to the plants5. lead is among one of the most abundant and widespread heavy metals in the environment. paints, mining sites, gasoline, explosives, paper and pulp industries and municipal sewage are the major causes of its release in the atmosphere1, 4. plant growth is enhanced by bacteria that grow in the rhizosphere which are termed as pgpr6. several studies have investigated that the potential of plants to separate heavy metals from soil is improved by plant growth o r i g i n a l a r t i c l e lead-tolerant bacteria can minimize lead toxicity in plants vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 2 r a d s j . b i o l . r e s . a p p l . s c i . 2 hormones7, 8. plants follow specific growth patterns which are resolved and managed by phytohormones9. auxins play an important role in the plant growth promoting ability of rhizobacteria6. bacterial auxins act as signaling molecules and strengthen bacteria plant relationship. the most significant attributes of plants inoculated with auxin-producing plant growth enhancing bacteria is the alteration in the root structure and growth10. the utilization of pgpb in phytoremediation is also termed as pgpb enhanced phytoremediation (pep) which is considered as a cheaper eco-friendly technique to increase plant stress tolerance, biomass productivity and bioremediation effectiveness during reclamation of polluted lands. some of the lead-tolerant zea mays varieties have been reported. lead mostly accumulates in the root and shoots of zea mays plant, which are not eaten as food. so, chances of lead consumption and its addition into the food chain have reduced. m a t e r i a l s a n d m e t h o d s bacterial strains and mic six lead-resistant bacterial strains i.e., bacillus anthracis (p1iv), bacillus sp. (t4b), sporosarcina sp. (t8bi), bacillus cereus (s4ii), bacillus cereus (t10ai) and t2aii were utilized for the present study. the bacterial strains were cultured at 37°c using l-agar supplemented with various concentrations of lead overnight to know the minimum inhibitory concentration of lead and growth of strains was recorded. biological assay zea mays certified seeds of variety dk-6142 were taken from the punjab seed corporation, lahore, pakistan. autoclaved petriplates (120mm) were taken. two whatman filter paper no. 1 were adjusted in each plate. plates were labeled with different bacterial strains and lead stress as well. seeds of zea mays were washed with 0.1% hgcl2 for 2-3 minutes followed by several washings with autoclaved distilled water. zea mays seeds were dipped in bacterial inoculum. seeds were then placed on the filter paper aseptically and different concentrations of lead solution were added i.e., 0, 400, 800 and 1200µg/ml. all petriplates were kept in dark. root length, number of primary as well as secondary roots of the seedlings were noted after one week. plant-microbe interaction petriplates of 120mm diameter were subjected to washing and drying before use. two layers of whatman filter paper no. 1 were placed in each plate then plates were autoclaved. the autoclaved plates were oven dried. plates were properly labeled for each strain and control (without inoculation) with different concentrations of working solution of lead nitrate (pbno3) i.e., 0 µg/ml (control treatment), 400, 800, 1200, and 1600µg/ml. 10 ml of sterilized salt solution with various concentrations (0, 400, 800 and 1200µg/ml) were added to each respective treatment. inoculated and treated zea mays (var. dk6142) seeds were placed on the moistened filter paper in the petriplate which were placed for three days in dark condition at 25+ 2 ºc. after three days, five germinated seedlings were sown in the labeled pots each with 140 gm sieved soil and lead stress solutions with 0, 400, 800 and 1200µg/ml of lead nitrate (pbno3) was added to the respective pots. the light period of 10 klux, for 16 hours duration was provided to the plants at 25+ 2 ºc. seedlings were grown for 20-25 days and then the growth of the inoculated and non-inoculated treatments was analysed by observing shoot length, root length, fresh wt. and number of leaves. protein test11 and auxin test12 for the inoculated and noninoculated plants were also performed. the experiment was repeated thrice. r e s u l t s biological assay inoculation with the bacterial strain t2aii caused increment in the root length upto 358.3% in the absence of lead stress. the strain bacillus cereus s4ii (20%) caused maximum increase in the number of primary roots without the addition of lead stress. under the lead stress of 400 and 800µg/ml, maximum enhancement upto 55.5 and 75% respectively, was recorded in the number of primary roots of seedlings inoculated with the bacterial strain bacillus sp. (t4b) over control treatments. in the presence of lead stress of 1200µg/ml, inoculation with the bacterial strain bacillus anthracis (p1iv) showed high reduction (80%) in the number of primary roots as compared to control. the number of secondary roots were enhanced by inoculation of seedlings by the bacterial strains bacillus cereus (s4ii), bacillus cereus (t10ai) and t2aii upto 193, 186 and 176%, respectively in comparison lead-tolerant bacteria can minimize lead toxicity in plants vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 3 r a d s j . b i o l . r e s . a p p l . s c i . 3 to control. under the lead stress of 400, 800 and 1200µg/ml, maximum increment in the number of secondary roots i.e., 335, 352 and 160.5% respectively, was exhibited by inoculation with the bacterial strain sporosarcina sp. (t8bi) when compared with control plants (fig. 1). plant-microbe interaction the germination percentage was not affected by bacterial treatment of seeds both in the presence and absence of lead stress. treatment of seeds with the bacterial strains bacillus cereus (t10ai), bacillus cereus (s4ii) and sporosarcina sp. (t8bi) showed enhancement upto 6.2, 5.7 and 5.3%, respectively, in the shoot length in the absence of lead stress. although, shoot length was enhanced by inoculation of seeds with the bacterial strains under the lead stress (400-1200µg/ml) but treatment of seeds with the bacterial strain bacillus cereus (s4ii) showed maximum increase of 13.5, 28.8 and 19.6%, respectively, in comparison to the respective control. treatment of seeds with the bacterial strains t2aii, bacillus cereus (s4ii) and bacillus sp. (t4b) exhibited increment in the root length of plants i.e., 40.9, 29.2 and 28.4%, respectively, without the addition of lead stress. under the lead stress of 400, 800 and 1200µg/ml, increment of 8.9, 19.7 and 8.5% in root length was recorded by inoculation of seeds with the bacterial strain sporosarcina sp. (t8bi), as compared to non-inoculated lead stressed control plants. inoculation of plants with the strain s4ii showed reduction of 13, 17 and 4.2% in the root length as compared to control treatments (fig. 2). in the absence of lead stress, treatment of seeds with the bacterial strains bacillus cereus (s4ii), bacillus cereus (t10ai) and sporosarcina sp. (t8bi) exhibited increment in the number of leaves when compared with control treatments. number of leaves was not much affected by treatment with the lead stress of 400 and 800µg/ml. lead stress of 800µg/ml showed enhancement in number of leaves by inoculation with all the strains and inoculation with the strains bacillus anthracis (p1iv), bacillus cereus (s4ii) and bacillus sp. (t4b) caused maximum increment upto 41.2% over control (fig. 3). although, lead stress did not decrease the number of leaves significantly but the leaves showed chlorosis and tip burns under lead stress which was more prominent in non-inoculated plants compared to inoculated treatments under lead stress. fresh weight of plants was enhanced by inoculation with bacterial strains both in the presence and absence of lead stress. plants treated with the strain bacillus cereus (s4ii) caused enhancement i.e., 39% in fresh weight without the addition of lead stress as compared to control. under the lead stress of 400, 800 and 1200µg/ml, enhancement of 107.8, 76.7 and 13.5% respectively, was exhibited by treatment with the bacterial strain bacillus cereus (s4ii) as compared to non-inoculated control treatments. protein content of plants was reduced by inoculation of seeds with all the bacterial strains (p1iv, s4ii, t10ai, t8bi, t2aii and t4b). maximum reduction of 70.7% was recorded by inoculation with the bacterial strain t2aii without the addition of lead stress. inoculation of seeds with the bacterial strain t2aii also exhibited reduction (29.5, 62.9 and 10.4%, respectively) in protein content under different lead stresses i.e., 400, 800 and 1200µg/ml in comparison to respective control. but, lead stress of 1200µg/ml showed enhancement in plants inoculated with the strain t10ai which showed highest increment of 209.8% in the protein content when compared with control. auxin content showed highest increment upto 30.8% by inoculation of seeds with the bacterial strain bacillus cereus (t10ai) as compared to control plants. under the lead stress of 400µg/ml, auxin content was reduced in all inoculated plants. treatment of seeds with the bacterial strains bacillus cereus (t10ai) and t2aii showed maximum reduction i.e., 52% in the auxin content over respective control. in the presence of lead stress of 800 and 1200µg/ml, increment of 22 and 16% was exhibited in auxin content with the bacterial inoculation of sporosarcina sp. (t8bi) as compared to respective noninoculated control treatments (fig. 4). lead-tolerant bacteria can minimize lead toxicity in plants vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 4 r a d s j . b i o l . r e s . a p p l . s c i . 4 fig. 1: influence of bacterial treatments in the presence and absence of lead stress [(pbno3 (µg/ml)] on primary and secondary roots number of zea mays in laboratory experiment. [w.i. = without bacterial inoculation; bacterial strains = p1iv, s4ii, t10ai, t8bi, t2aii and t4b]. data shows mean of fifteen replicates. different letters designate significant difference between treatments using duncan’s multiple range test (p = 0.05). fig. 2: influence of bacterial treatments in the presence and absence of lead stress [(pbno3 (µg/ml)] on shoot length (cm) and root length (cm) of zea mays in laboratory conditions. [w.i. = without bacterial inoculation; bacterial isolates = p1iv, s4ii, t10ai, t8bi, t2aii and t4b]. data represent mean of fifteen replicates. different letters designate significant difference between treatments using duncan’s multiple range test (p = 0.05). lead-tolerant bacteria can minimize lead toxicity in plants vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 5 r a d s j . b i o l . r e s . a p p l . s c i . 5 fig. 3: influence of bacterial treatments in the presence and absence of lead stress [(pbno3 (µg/ml)] on fresh weight (g) and leaves number of zea mays in laboratory conditions [w.i. = without bacterial inoculation; bacterial isolates = p1iv, s4ii, t10ai, t8bi, t2aii and t4b]. data represent mean of fifteen replicates. different letters designate significant difference between treatments using duncan’s multiple range test (p = 0.05). fig. 4: influence of bacterial treatments in the presence and absence of lead stress [(pbno3 (µg/ml)] on protein and auxin concentration of zea mays under laboratory conditions. [w.i. = without bacterial inoculation; bacterial isolates = p1iv, s4ii, t10ai, t8bi, t2aii and t4b]. data represent mean of fifteen replicates. different letters designate significant difference between treatments using duncan’s multiple range test (p = 0.05). lead-tolerant bacteria can minimize lead toxicity in plants vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 6 r a d s j . b i o l . r e s . a p p l . s c i . 6 d i s c u s s i o n the current research deals with the study of bacterial treatment of zea mays for reducing hazardous impact of lead on plant growth and development under lead stress. the biological assay was conducted to study the effect of auxin-producing bacteria on the growth of zea mays in the presence and absence of various lead concentrations (0, 400, 800 and 1200µg/ml). root length, number of primary roots and number of secondary roots were enhanced by inoculation of seeds with the bacterial strains bacillus cereus (s4ii) and t2aii in the absence of lead stress while inoculation with the strain sporosarcina sp. (t8bi) showed reduction when compared with control treatment. the selected six auxin producing lead-resistant bacterial strains were used to assess their plant health promoting potential under lead stress for which seeds of zea mays variety dk-6142 were inoculated with the selected auxinproducing lead resistant bacterial strains in the presence of lead stress. inoculation with the bacterial strain bacillus cereus (s4ii) caused prominent increment in growth parameters like shoot length, germination percentage, root length, fresh weight, number of leaves, and auxin content of zea mays in the absence of lead stress in comparison to control. under the lead concentration of 400µg/ml, the germination percentage was decreased in inoculated plants whereas lead stresses of 800 and 1200µg/ml does not affect germination percentage of plants treated with various bacterial strains as compared to respective control treatments. the shoot length of plants inoculated with the bacterial strains bacillus anthracis (p1iv), bacillus cereus (s4ii), t10ai, t8bi and bacillus sp. (t4b) showed enhancements under the lead stresses of 400, 800 and 1200µg/ml as compared to control. similarly, hassan et al.13 have reported increment in the height (shoot length) of wheat by inoculation with pseudomonas moraviensis and bacillus cereus upto 32%. the increment in plant height in the presence of pgpr is attributed to increase iaa synthesis that improve cell elongation and cell division13. under various lead stresses (400-1200µg/ml), treatment with the bacterial strain t8bi caused increment in root length over control treatments while inoculation with the bacterial strain bacillus cereus (s4ii) caused reduction in root length under the different lead stresses i.e., 400, 800 and 1200µg/ml when compared with the respective control treatments. likewise, cathrine and navab3 stated that lead accumulate more rapidly in the roots as compared to shoots. so, the roots of lepidium sativum l. are much more sensitive to phytoremediation of lead than the shoots. number of leaves showed no significant effect by inoculation with bacterial strains under lead stress of 400 and 1200µg/ml but inoculation of seeds with the bacterial strains enhanced the number of leaves under the lead stress of 800µg/ml as compared to control. although lead stress has shown negligible effect on the number of leaves but increasing lead stress caused chlorosis of leaves indicating the toxic effect of lead on photosynthetic pigments. so, leaves were not healthy and chlorosis of the leaves suggest that the overall biology of the plants was adversely affected by increasing lead stress. idicko and verma14 have reported increment in number of leaves of inoculated maize plants as compared to control. the pseudomonas sp. improved the number of leaves up to 8% whereas azotobacter sp. increased the leaves number upto 7.67% as compared to the non-treated plants. under the lead stress of 400 and 800µg/ml, the fresh weight of plants inoculated with all the bacterial strains i.e., p1iv, s4ii, t10ai, t8bi, t2aii and t4b showed enhancements as compared to the non-inoculated lead stressed plants. the lead stress of 1200µg/ml exhibited enhancement in fresh weight by plants inoculated with all the bacterial strains (bacillus anthracis p1iv, bacillus cereus s4ii, t10ai, t2aii and bacillus sp. t4b) except plants inoculated with the strain sporosarcina sp. (t8bi) in comparison to control. similar results were obtained showing increment in fresh weight of groundnut plant inoculated with bacterial strains at various stages of its growth as compared to control15. the protein concentration of zea mays plants treated with all the bacterial strains showed enhancements under the lead stress of 400 and 800µg/ml whereas the lead stress of 1200µg/ml exhibited enhancements in the protein content of all the inoculated plants except plants treated with the bacterial strain t2aii when compared with the respective non-stressed treatments. reduction in protein production is an essential biochemical demonstration of lead toxicity which is showed by others also, such as bano et al.16 have also reported that instead of the reports of stress lead-tolerant bacteria can minimize lead toxicity in plants vol. 9 (1), july 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 7 r a d s j . b i o l . r e s . a p p l . s c i . 7 protein synthesis, the protein content generally decreases due to stress as the rate of photosynthesis declines due to unavailability of raw material for protein production which leads to the reduction or complete cessation of the process. the increase in protein degradation as compared to their production can also be the reason of reduction in protein content16. reduction in auxin content of inoculated plants was observed under the lead stress of 400µg/ml over the control treatment. in the presence of lead stress of 800 and 1200µg/ml, treatment of plants with the bacterial strains, sporosarcina sp. t8bi bacillus anthracis p1iv and bacillus sp. t4b showed enhancements in auxin content when compared with the control. according to zhu et al.17, auxin production showed negative association with zinc concentration under the influence of pgpr isolates. by increasing zinc concentration, the auxin production must be decreased by bacterial isolates due to reduction in root growth of wheat plant under zinc stress17. they also observed that instead of reducing the auxin synthesis under zinc stress, bacterial isolates exhibit metal transport from roots to shoots and retarded the zinc accumulation in the root17 so a similar relationship may exist between some of the bacterial isolates and lead stress which caused reduction in the auxin content. c o n c l u s i o n thus the current study suggests that lead stress adversely affects the photosynthetic pigments in plants leading to chlorosis in the leaves under lead stress which results in poor plant growth. treatment with lead-tolerant bacterial isolates improve plant growth under lead stress and thus can be effectively applied in lead-contaminated areas for plant growth improvement. r e f e r e n c e s 1. singh n, yadav a, varma a. effect of plant growth promoting activity of rhizobacteria on cluster bean (cyamopsis tetragonoloba l.) plant growth and biochemical constituents. int j agri res. 2015; 4(5): 1071-82. 2. ahsan mt, najam-ul-haq m, idrees m, ullah 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sci j. 2014; 31 (10): 1734-43. 14. idicko ah, verma rk. effect of growth promoting microbes on initial growth of maize. ind j trop biodiver. 2014; 22(1): 64-9. 15. mathivanan s, chidambaram ala, sundramoorthy p, baskaran l, kalaikandhan r. effect of combined inoculations of plant growth promoting rhizobacteria (pgpr) on the growth and yield of groundnut (arachis hypogaea l.). int j curr microbiol appl sci. 2014; 3(8): 1010-20. 16. bano q, ilyas n., bano a, zafar n, akram a, hassan f. effect of azospirillum inoculation on maize (zea mays l.) under drought stress. pak j bot. 2013; 45(s1): 13-2. 17. zhu d, ouyang l, xu z, zhang l. rhizobacteria of populus euphratica promoting plant growth against heavy metals. int j phytoremed. 2015; 17(10): 973-80. comparison of fpg, lipid profile and sd-ldl vol.12 (2), december 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 128 op e n ac c e s s f u l l l e n g t h a r t i c l e comparison of fasting plasma glucose, lipid profile and small dense low density lipoprotein in severe persistent asthmatic and non-asthmatic adults haji muhammad rashid1,*, nadia awan1, hiza hassan2, anjum rashid3, rabia arshad3, saffora shoukat3 1department of chemical pathology, university of health sciences, lahore, pakistan. 2department of medical lab technology, university of haripur, kpk, pakistan. 3shikh zaid hospital, lahore, pakistan. a b s t r a c t background: the most effective and commonly used control therapy for asthma is oral or parenteral corticosteroids, which are quite effective. but at the same time, they are considered notorious for their side effects like contributing to increasing rates of related metabolic disorders for eg: obesity and type 2 diabetes. studies of blood glucose and lipid profiles in relation to severe persistent asthma are still a few, and the results are ambiguous. objectives: the aim of current study was to evaluate the changes in fasting plasma glucose (fpg), lipid profile (lp) and small dense lipoprotein cholesterol (sd-ldl-c) in severe persistent asthmatic (spa) patients in comparison with non-asthmatic adults, and their correlations with absolute eosinophil count. methodology: this study was a cross sectional comparative research conducted at medsol clinical lab, blue area, islamabad. in this study, 40 pre-diagnosed spa and 40 non-asthmatic adults were enrolled. blood absolute eosinophil count (aec) was performed on haematology analyser (mindray bc 50), fpg and lipid profile were measured by commercially available kits of spin react on microlab 300, and sd-ldl-c were measured by precipitating lipoproteins using heparin-mncl2 solution and measuring sd-ldl-c from supernatant by spectrophotometric method in spa and non-asthmatic adults. data was analyzed by spss 20.2. results: pair-wise comparison between spa and non-asthmatic group was performed by two sample t-test. in spa group, fpg (95 ± 8mg/dl), triglycerides (162 ± 14mg/dl), ldl (97 ± 10mg/dl) and sd-ldl-c (48 ± 4mg/dl) were significantly higher (p < 0.05) than non-asthmatic adults, while hld in spa (38 ± 4.4mg/dl) was significantly lower than non-asthmatic adults (42 ± 3.9mg/dl). we also observed strong positive association of fpg (0.54), triglycerides (0.38) ldl (0.23) and sd-ldl-c (0.60) with aec of spa group and strong negative correlation for hdl (0.50) and aec in spa. conclusion: dyslipidaemia, hyperglycaemia and elevated levels of sd-ldl-c are associated complication of severe persistent asthma and high levels of sd-ldl-c in severe persistent asthma are a potential risk factor to induce atherosclerosis. keywords asthma, glucose, lipid profile, respiratory syndrome, small dense-ldl, cholestrol. *address of correspondence 4849487@gmail.com article info. received: march 10, 2021 accepted: march 30, 2021 cite this article rashid hm, awan n, hassan h, rashid a, arshad r, shoukat s. comparison of fasting plasma glucose, lipid profile and small dense low density lipoprotein in severe persistent asthmatic and non-asthmatic adults. rads j biol res appl sci. 2021; 12(2):128-134. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. o r i g i n a l a r t i c l e comparison of fpg, lipid profile and sd-ldl vol.12 (2), december 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 129 i n t r o d u c t i o n asthma is an inflammatory respiratory syndrome that affects ~350 million people around the world every year1. asthma is characterized by restricted airflow, airway inflammation, and airway hyper responsiveness, including symptoms like shortness of breath, wheezing, and cough2. on the basis of severity, it is classified in 4 classes (intermittent, mild persistent, moderate persistent and severe persistent) by national asthma education and prevention program (naepp), created on the individual's symptoms and spirometer data. asthma prevalence has been increased by 50% every decade, and almost 100 million new asthma cases will be added to this asthmatic population till 20253. as asthma is a chronic inflammatory condition of airways, it induces the production of pro-inflammatory cytokines and oxidative stress which results in osteoporosis, bruising, metabolic abnormalities and psychiatric disturbances4. moreover, the most effective and commonly used controller therapy for asthma is an inhaled corticosteroid which inhibits the production of pro-inflammatory cytokines and reduces bronchial reactivity and frequency of exacerbations. oral and parenteral corticosteroids are effective but are notorious for their side effects like contributing to increasing rates of related metabolic disorders, such as obesity and type 2 diabetes5. asthma induces a number of metabolic variations, but the variations in blood glucose and lipid profile are with conflicting results in various studies6,7. similarly, the variations in small dense low density lipoprotein (sdldl) which is the subclasses of low density lipoprotein (ldl) is also controversial6, 8. keeping in mind the correlation with various blood profile factors, we designed the current study to compare fasting glucose, lipid profile and sd-ldl levels in pre-diagnosed severe persistent asthmatic (spa) and non-asthmatic adults, and their association with blood eosinophil count in spa patients. m a t e r i a l a n d m e t h o d s it was a cross-sectional comparative study conducted at the medsole clinical lab, blue area, islamabad from aug 2019 to feb 2020. ethical approval for the study was obtained from ethical board of pakistan institute of medical sciences (pims), islamabad. we enrolled 40 prediagnosed severe persistent asthmatic and 40 nonasthmatic adults in this study. fasting venous blood samples were collected in edta and plane tubes from spa patients and non-asthmatic adults, after obtaining their consent. blood absolute eosinophil count (aec) were measured on mindray haematology analyser (bc50), fpg and lipid profile were measured on mindray (240pro) auto chemistry analyser, and sd-ldl-c was were measured by spectrophotometric method9 on micro lab 300. the data was entered and analyzed by using spss 20.0 to calculate frequencies, mean, and standard deviations. the pair-wise comparison of the parameters was carried out by two sample t-test and correlations were calculated by pearson’s correlation coefficient. r e s u l t s in the present study, 40 severe persistent asthmatic (spa) patients and 40 non-asthmatic adults were investigated. out of 40 spa patients, 24 (60%) were females and 16 (40%) were males; and amongst them 16% were active smokers and 9% were ex-smokers (smokers or exsmokers 25%) however, 50% were from low socioeconomic class (table 1). pair-wise comparison was carried out with two sample ttest for all tested parameters between spa patients and non-asthmatic group. aec, fpg, trig, ldl and sd-ldl-c levels were significantly higher in spa than non-asthmatic group. hdl levels were significantly lower in spa patients as compared to non-asthmatic adults and there were no significant difference observed in total cholesterol of both groups (table 2). pearson correlation coefficient was calculated for fpg, trig, total-cholesterol, hdl, ldl and sd ldl-c against absolute eosinophil count in spa group. we observed a good positive association of fbg, trig and sd-ldl-c with absolute eosinophil count and strong negative association of hdl was observed with absolute eosinophil count in spa group shown in (table 3). comparison of fpg, lipid profile and sd-ldl vol.12 (2), december 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 130 table 1. demographic characteristics of severe persistent asthmatic patients and non-asthmatic adults enrolled in the study. variables spa (n = 40) non-asthmatic adults (n = 40) gender males females 16 (40%) 24 (60%) 18 (45%) 22 (55%) age (range) years 46.8 ± 7.2 (35-62) 46.6 ± 5.6 (36-56) smoking status non-smokers smokers or ex-smokers 34 (85%) 6 (15%) 12 (30%) 28 (70%) socioeconomic status rich medium low 6 (15%) 14 (35%) 20 (50%) 8 (20%) 16 (40%) 16 (40%) table 2. compression of absolute eosinophil count, lipid profile and small dense ldl-c between severe persistent asthmatic and non-asthmatic adults. variables spa (n = 40) (mean ± sd) non-asthmatic (n = 40) (mean ± sd) p-value aec (cells/µl) 546 ± 411 125 ± 64 <0.001 fasting blood glucose (mg/dl) 95 ± 8 84 ± 7 <0.001 triglycerides (mg/dl) 162 ± 14 154 ±18 0.029 total cholesterol (mg/dl) 168 ± 13 163 ± 16 0.12 hdl (mg/dl) 38 ± 4.4 42 ± 3.9 <0.001 ldl (mg/dl) 97 ± 10 95 ± 8 <0.001 sd ldl-c (mg/dl) 48 ± 4 44 ± 3.5 <0.001 table 3. correlation fpg, lipid profile and sd-ldl-c with absolute eosinophil count in severe persistent asthmatic patients. variables correlation coefficient (absolute eosinophil count) fbg 0.54 trig 0.38 t-chol 0.10 hdl 0.50 ldl 0.23 sd-ldl-c 0.60 here, the negative correlation of fbg, trig, t-chol, hdl, ldl and sd-ldl-c with aec in spa patients indicates that hdl levels are decreased with the increase in aec, while positive correlation shows that fbg, trig, ldl and sdldl-c levels are increased with the increase of aec. comparison of fpg, lipid profile and sd-ldl vol.12 (2), december 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 131 d i s c u s s i o n dyslipidemia, hyperglycemia and high levels of sd-ldl-c are common risk factor for atherosclerosis. asthma is an inflammatory lung disease and it is associated with metabolic variations. in present study, we tried to elaborate the variations in lipid profile, glucose and sd-ldl-c in severe persistent asthmatic adults. for this, we enrolled physician-diagnosed 40 spa patients aged 35-62 years and 40 non-asthmatic adults aged 36-56 years (table 1). there are gender differences in the prevalence of asthma. compared with adults men, adult women have a higher prevalence of asthma due to hormonal difference in both genders; specially low level of testosteron and high level of estrogen make the women more prone for getting th-2 type asthma10, 11. since, the females having spa were at higher ratio than the males, therefeore, out of all the participants in our stdy, there were 16 (40%) males and 24 (60%) females in spa group. however, the role of obesity, sex hormones and other gender-specific factors is yet unclear and may be responsible for such differences11. in the present study, the mean age of spa patients was 46.8 ± 7.2 and range was (35-62 years). throughout the rest of life after the age of 20, lung function is suppose to decline, and this decline rate is affected by air pollution, cigarette smoke exposure, urbanization and climate change. aging is also associated with the increased chest wall stiffness, decreased lung elastic recoil and respiratory muscle weakness12. here, we found that 50% of spa patients come from low socioeconomic status (ses) and these findings are also consistent with previous studies, which concluded that the decrease of ses is linked with the increase in the incidence of asthma and asthma severity13, 14. smoking can have harmful effects on different clinical aspects of asthma, such as accelerated lung function decline, weakened symptom control and weakened response to treatment15. percentage of smokers with spa was higher (20% current smokers, 24% ex-smokers and 2% electric ciggrate-smokers) than the general population in some of the countries16. but in our study, we observed that 15% spa were current smokers or ex-smokers. the reason behind this controversial findings may be the higher ratio of females patients in our study due to consecutive sampling of asthmatic patients, and frequency of females smokers in pakistan (2.8%)17 is less than the world wide percentage of females smokers i.e. 4.3-23%18. kitchen smoking or passive smoking have also significant role in the development of asthma19, however these factors were not investigated in our study. the role of eosinophils in asthma has been extensively studied. increased numbers of eosinophils exist in the airways of most, but not all, persons who have asthma20. in our study we found significantly higher aec (546 ± 411 cells/µl) in spa than non-asthmatic adults (125 ± 64 cell/µl) p value < 0.001. our results are consistent with the findings of badar et al., 2010, where the absolute count of eosinophils found in spa was 684.00 ± 75.58 cells/µl21. a study conducted in india in 2019 also reported similar findings regarding aec (405 ± 83.16 cells/µl) in severe asthma22. we observed clinically significant higher fasting blood glucose levels in spa groups and similar findings were reported by koskela et al., 2013, in which they proposed that medication used to control the asthma may cause hyperglycaemia23. this hyperglycemic effect have also been reported in different other studies on asthmatic patients4, 24. triglycerides and ldl levels were higher and hdl levels were significantly lower in spa group than non-asthmatic adults, while there was no significant difference in the levels of t-cholesterol of both groups in present study. our findings for high ldl and triglyceride are in line with the findings of ko et al., and peng & huang6, 25. t cholesterol levels have no difference in both groups of our study and it is in accordance with the finding of fang et al., 201624. several studies have also reported high t cholesterol in spa group than normal control while low hdl levels were reported by various studies in asthmatic adults similar to our findings26-28. sd-ldl-c is a sub-class of ldl and it is potential atherogenic cholesterol29. in spa patients, we observed significantly raised levels of sd-ldlc and similar findings were reported by scichilone et al., 2013 periously in his study8. at present, the relationship between dyslipidemia and asthma or other allergic diseases is unclear. there are still some possible mechanisms; one of them may be the inflammatory link between asthma and dyslipidemia30. in comparison of fpg, lipid profile and sd-ldl vol.12 (2), december 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 132 addition, dyslipidemia may also enhance eosinophil inflammation, which is related to other conditions in the pathophysiology of asthma, such as excessive mucus secretion, bronchial hyperresponsiveness, and subepithelial fibrosis31. we observed a strong positive association of ldl and sd ldl-c with aec and strong negative association of hdl with aec. patients with decreased lung function have considerably high absolute eosinophil count32. association of triglycerides and hdl have been studied by barochia et al., 2017, and they concluded a positive correlation between hdl and aec of spa group. eosinophilic inflammation in asthma is associated with variation of triglyceride and hdl in asthmatic adults33. sd-ldl-c association (r2=0.60) with eosinophilic inflammatory marker is reported for the first time in present study and further research work is required to validate this association. c o n c l u s i o n from our study, it is concluded that dyslipidaemia, hyperglycaemia and elevated levels of sd-ldl-c are associated complications of severe persistent asthma, and high levels of sd-ldl-c in severe persistent asthma are a potential risk factor to induce atherosclerosis. c o n f l i c t s o f i n t e r e s t none. f u n d i n g s o u r c e technical support and required instruments were provided by ceo medsol clinical lab, and other expenses were selfarranged by the authors. a c k n o w l e d g m e n t s we are thankful to ceo of medsol clinical lab to provide testing facility and technical support. l i s t o f a b b r e v i a t i o n s aec absolute eosinophil count fbg fasting blood glucose hdl high density lipoprotein ldl low density lipoprotein naepp national asthma education and prevention program sd-ldl-c small dense low density lipoprotein cholesterol ses socioeconomic status spa severe persistent asthma r e f e r e n c e s 1. branco accc, sato mn, alberca rw. the possible dual role of the ace2 receptor in asthma and coronavirus (sars-cov2) infection. front cell infect micro. 2020; 10-8. 2. di genova l, penta l, biscarini a, di cara g, esposito s. children with obesity and asthma: which are the best options for their management? nut. 2018; 10(11):1634-9. 3. rashid hm, khan m, jamal m, awan n, waseem m. association of serum sphingosine-1-phosphate with forced expiratory volume and absolute eosinophil count in asthma patients. rawal med j. 2020; 45(4):771-4. 4. torres rm, souza mds, coelho acc, de mello lm, souza-machado c. association 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into rafts. int immunol. 2003; 15(10):1207-18. 31. pham t-h, damera g, newbold p, ranade k. reductions in eosinophil biomarkers by benralizumab in patients with asthma. resp med. 2016; 111:21-9. 32. hancox rj, pavord id, sears mr. associations between blood eosinophils and decline in lung function comparison of fpg, lipid profile and sd-ldl vol.12 (2), december 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 134 among adults with and without asthma. europ resp j. 2018; 51(4). 33. barochia av, gordon em, kaler m, cuento ra, theard p, figueroa dm, et al. high density lipoproteins and type 2 inflammatory biomarkers are negatively correlated in atopic asthmatics. j lip res. 2017; 58(8):1713-21. comparative efficacy of bio-pesticide and synthetic agrochemicals on control of helicoverpa armigera larvae on chickpea vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 75 r a d s j . b i o l . r e s . a p p l . s c i 75 op e n ac c e ss f u l l l e n g t h a r t i c l e comparative efficacy of bio-pesticide and synthetic agrochemicals on control of helicoverpa armigera larvae on chickpea taimur ahmad1,*, rasool khan1, tariq nawaz khattak2, ayaz khan1 and ihsan ullah2 1institute of chemical sciences, university of peshawar, peshawar, pakistan 2nuclear institute for food and agriculture (nifa), peshawar, pakistan a b s t r a c t the comparative efficacy of neem as bio-pesticides and synthetic agrochemicals against the larvae of helicoverpa armigera was carried out during 2016-2017. the experiment was performed using rcbd and consisted of a total of six treatments including the control with four replicates. the results indicated that the synthetic insecticide emamactin was found to be superior in controlling the larval population of pod borer with a % reduction of 63 and 88 after 1st and 2nd application respectively followed by chlorpyrifos after 2nd application. the neem bio-pesticide showed best results next to emamectin after the first application. the acetamiprid was found to be least effective in controlling the pod borer larvae. keywords bio-pesticide, pod borer, neem oil, chickpea, adjuvant. *address of correspondence taimurjf@yahoo.com article info. received: april 13, 2018 accepted: october 23, 2018 cite this article: ahmad t, khan r, khattak tn, khan a, ullah i. comparative efficacy of biopesticide and synthetic agrochemicals on control of helicoverpa armigera larvae on chickpea. rads j. biol. res. appl. sci. 2018; 9(2): 75-81. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n the chickpea is attacked by helicoverpa armigera which is commonly known as gram pod borer and is the main pest of the chickpea1,2. this pest causes a great loss in chickpea3. in most of the chickpea producing area, the gram pod borer has been reported to have developed resistance to most of the commonly used pesticides4. the helicoverpa armigera is a polyphagous insect and attacks more than 200 species of almost 45 crops throughout the world5. the total annual yield loss of different crops by helicoverpa armigera has been reported to be a worth of 2 billion us dollars6. the initial stage of the life cycle, larval stage control is important to prevent the severe damage and yield loss7,8. various control measures have been adopted to control or minimize the attack of chickpea pod borer9. synthetic pesticides are commonly used to control the pests. but the extensive use of synthetic pesticides has created several problems including mammalian toxicity, destruction of useful insects, resistance development in pests etc.10. plant extracts are suitable and safe alternatives to toxic synthetic pesticides. the neem oil is obtained from azadirachta indica and possesses insecticide activities against a wide range of insect pests11-13. the active ingredient in the neem oil is azadirachtin. the neem oil is target specific and insects cannot develop resistance to it. the neem oil is not soluble in water and should be emulsified first before use. usually, surf or detergents are used to emulsify the neem oil. this surf contains synthetic chemicals which may alter the chemistry of plants. furthermore, the emulsified oil is made only when needed and cannot be stored for a long o r i g i n a l a r t i c l e comparative efficacy of bio-pesticide and synthetic agrochemicals on control of helicoverpa armigera larvae on chickpea vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 76 r a d s j . b i o l . r e s . a p p l . s c i 76 time. we have developed an adjuvant at the nuclear institute for food and agriculture nifa, peshawar. this adjuvant is designated as nifa-1 and acts as emulsifier, spreader, and sticker. we have observed that the neem oil emulsified with nifa-1 adjuvant has shown pesticide activity after two years of storage. the total cost for the production of this adjuvant in rs.200 per liter. m a t e r i a l a n d m e t h o d experimental design the experiment was carried out on chickpea variety nifa2005 during 2016-2017. the field was ploughed twice for seed sowing and the weeds and other grasses were also removed from the soil. the plot was divided into twentyfour 24 subplots. the space between the rows was 38 cm and the distance between the plants was 10 cm. there were a total of six treatments including a control with four replications. seven plants were randomly selected in each subplot for the observation of chickpea pod borer larvae. the design used in this experiment was rcbd. the experimental data were analyzed by statistics 8.1 software. the percent data were subjected to square root transformation for statistical analysis. mean comparisons for treatment parameters were compared using tukey's tests at 5% level of significance. application of insecticides the pesticides were applied as a foliar spray with the help of knap sprayer. there were two sprays for each insecticide. the first application was done after the appearance of pod borer larvae and the second was done after one week of the first application. the treatments are shown in the following table 1. table 1: treatments of insecticides and concentration of dose. treatments pesticides the concentration of dose (%) t1 emamectin benzoate 0.5 t2 acetamiprid 0.5 t3 chlorpyrifos 0.5 t4 bifenthrin+neem oil+n.a 1 t5 neem oil+n.a 3 t6 control --- data collection the data was collected regarding the larvae of the pod borer. the data was taken by visual count method of the chickpea pod borer. the pretreatment record of the pod borer was noted 24 hours before the treatment and the post-treatment data was taken after one, two and three days of the applications. seven plants per subplot were selected randomly for the observation of larvae of pod borer. three distinct stages of the pod borer i.e. neonate, the middle and the fully grown larvae were noted. the percent reduction of larvae was determined by using abbot formula developed in 1925. the formula is written as; % reduction = c-t/c x100. where, c = larvae population in control t = larvae population in treatment the percent pod damage and the percent damage reduction of pod over control are given below14. % pod damage = no. of affected pods x100 total no. of pods % reduction in pod damage over control = pod damage in control – pod damage in treatment x 100 pod damage in control the biomass containing the dry chickpea plant was calculated by weighing the chickpea plant at harvest. r e s u l t s & d i s c u s s i o n s first application of pesticides the data in table 2 shows the pretreatment and posttreatment larval population of the chickpea pod borer for the first application of the pesticides. it can be seen from the pretreatment data that chickpea plants contain a large number of the pod borer larvae of neonate stage which is the first distinct stage of the pod borer. there was almost the same population of the neonates before treatments in all the subplots. the number of neonates in the case of emamectin and acetamiprid was 6.2 and 5.8 respectively. the number of neonates was 7.2 and 7.1 in case of chlorpyrifos and bifenthrin +emulsified neem oil combination respectively while the emulsified neem oil and control has a population of 6.5 and 6.8 respectively. the middle stage larval population was 0.3 for emamectin and 0.2 each for chlorpyrifos and neem oil treatment, comparative efficacy of bio-pesticide and synthetic agrochemicals on control of helicoverpa armigera larvae on chickpea vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 77 r a d s j . b i o l . r e s . a p p l . s c i 77 while this middle instar was absent in case of acetamiprid, bifenthrin +emulsified neem oil combination and control treatments respectively. the fully grown instar of the larvae was absent in all the subplots before the application of the 1st spray. so the pretreatment total larval population of all the three instars of the pod borer larvae in case of emamectin and acetamiprid was 6.5 and 5.8 respectively while chlorpyrifos and bifenthrin +emulsified neem oil combinations have total larval population 7.4 and 7.1 respectively. total larval population in case of emulsified neem oil and control treatments were 6.7 and 6.8 respectively. the mean value of the three instars of the chickpea pod borer larvae before the treatment were 2.1, 1.93, 2.4, 2.3, 2.2 and 2.2 for emamactin, acetamiprid, chlorpyrifos, bifenthrin + emulsified neem oil combination, emulsified neem oil and control treatments respectively. the post-treatment data shows that emamectin, the treated plot has a neonate instar population of 5.2 per seven plants while the middle instar (m) has a population of 0.5 and 0.1 fully grown instar. the acetamiprid treated plot has neonate instar population of 7.6 per seven plants. in this treatment, the number of middle instar population was 0.6, while the fully mature instar (f) population was 0.8. the chlorpyrifos treated plot has a post-treatment neonate population of 5.1 per seven plants. in this treatment, the middle instar (m) has a population of 1.6 and 0.8 population of fully mature (f) instar. the bifenthrin + emulsified neem oil combination treatment has a decline in the neonate instar population with a value of 3.2 per seven plants. the middle instar, (m) and the fully grown instar (f) population were found to be 2.7 and 0.2 respectively in case of bifenthrin +emulsified neem oil combinations treatment which consists of neem oil and nifa adjuvant and bifenthrin as a synergist. the last treatment which consists of purely neem oil and nifa adjuvant as emulsifier has a neonate population of 3.4, while the middle instar larval population was found to be 2.7 and no population of the fully grown instar larva was found. the population of neonates were increased in the case of control and was 9.1 per seven plants. the middle instar of larva was also present in large number as compared to all other treatments and their population was 5.3. similarly, the full-grown larval instar (f) population was recorded to be 1.2. it can be seen from table 2. that total larval population in case of emamactin, acetamiprid, chlorpyrifos, bifenthrin + emulsified neem oil combination, emulsified neem oil and control treatments were 5.8, 9, 6.8, 6.1, 6.1 and 15.6 respectively while the mean values of all the three larval populations were 1.93, 3, 2.2, 2.03, 2.03 and 5.2 respectively. second application of pesticides the second application of the pesticides was performed after one week of the first application. in this case, the pretreatment data of the chickpea pod borer was also noted one day before the application of the spray and the post-treatment data was taken after one, two and three days of the application. the pretreatment data shows that the number of neonate population was considerably low and was 0.9 in case of ememactin, the middle instar (m) has a population of 0.2 per seven plants while the fully grown instar (f) was still not found. it can be seen from table 3 that acetamiprid treatment has a neonate instar population of 0.7 and middle instar pod borer population of 0.6 and a fully grown larval population (f) with a value of 0.2. chlorpyrifos treatment has a neonate population of table 2: pretreatment and post-treatment larval population of pod borer after 1st spray. s. no larval population (pretreatment) larval population (post-treatment) treatments n m f total mean n m f total mean t1 6.2±1.6a 0.3±0.21a 0±0 6.5 2.1 5.2±1.8bc 0.5±0.3c 0.1±0.1b 5.8 1.93 t2 5.8±0.9a 0±0b 0±0 5.8 1.93 7.6±1.2ab 0.6±0.4c 0.8±0.3a 9 3 t3 7.2±1.6a 0.2±0.18ab 0±0 7.4 2.4 5.1±0.98bc 1.6±0.47bc 0.1±0.08b 6.8 2.2 t4 7.1±1.6a 0±0b 0±0 7.1 2.3 3.2±0.8c 2.7±0.6b 0.2±0.2b 6.1 2.03 t5 6.5±0.9a 0.2±0.14ab 0±0 6.7 2.2 3.4±0.6c 2.7±0.4b 0±0b 6.1 2.03 t6 6.8±1.1a 0±0b 0±0 6.8 2.2 9.11±1.7a 5.3±0.9a 1.2±0.3a 15.6 5.2 where, n= neonate, m=middle and f=fully grown larval stages respectively comparative efficacy of bio-pesticide and synthetic agrochemicals on control of helicoverpa armigera larvae on chickpea vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 78 r a d s j . b i o l . r e s . a p p l . s c i 78 1.6, middle (m) instar population of 0.7 and the fully grown larval population of 0.1 respectively. the neonates, middle and the fully grown pod borer larvae population values were 0.6, 1.4 and 0.3 respectively for bifenthrin + emulsified neem oil combination treatment. the neem oil emulsified with nifa adjuvant has a neonate population of 0.8, middle (m) stage larval population of 0.7 and 0.1 population of fully mature instar larva of pod borer. in the case of control, the neonate instar population was 1.4, middle instar population (m) was 1.7 while the fully grown instar has a population of 0.6. the pretreatment total larval populations for emamactin, acetamiprid, chlorpyrifos, bifenthrin + emulsified neem oil combination, emulsified neem oil and control were 1.1, 1.5, 2.8, 2.3, 1.6 and 3.7 respectively and the mean values of this larval instar were 0.36, 0.5, 0.9, 0.76, 0.53 and 1.23 respectively. the post-treatment larval population data was conducted after one, two and three days of the application. it can be seen from the table 3 that emamectin treatment has a low population of neonate instar and has a value of 0.4, and the population of the middle (m) instar was 2.7 per seven plants while the fully grown larvae have a population density of 0.6. so the total larval population of all the instars of the pod borer was 3.7 and their mean was 1.23 after the application of the 2nd spray. the acetamiprid treated plot has a greater population of the neonate larvae than that of emamactin and has a value of 3 and the middle instar population of 4.9 and the fully mature larval population of 1 respectively. it can be seen from table 3 that total post-treatment larval population for all the three instars of the pod borer was 8.9 and the mean was 2.96 for acetamiprid. the chlorpyrifos treated plants have a neonate instar population of 0.2 after the application of the 2nd spray. the middle larval stage population has a value of 3.5 and the fully grown larva was found to be 0.5 per seven plants for the posttreatment of the 2nd application. in this case, the total larval population was 4.2 and the mean population of all the three instars was 1.4. the bifenthrin + emulsified neem oil combination treatment consisting of neem oil, nifa adjuvant and bifenthrin as synergist has total neonate post-treatment population of 2.1, and middle and fully grown instar population of 4.2 and 1 respectively. the total larval count for this treatment was 7.3 and the mean value of all the instars was 2.4. the neem biopesticide which was emulsified neem oil treatment has the neonate population of 2.3, middle and fully grown instars population of 3.2 and 0.4 respectively. the total and mean larval population values for this treatments were 5.9 and 1.96 respectively. the largest population of neonates was 11.5 in case of control group while the middle (m) and full grown instars have the population of 15.5 and 5.1 respectively. the total larval population for emamactin, acetamiprid, chlorpyrifos, bifenthrin + emulsified neem oil combination, emulsified neem oil and control were3.7, 8.9, 4.2, 7.3, 5.9 and 32.1 respectively and their mean was 1.23, 2.96, 1.4, 2.4, 1.96 and 10.7 respectively. the results obtained after the application of pesticides show that all the pesticide treatments have a greater effect on the control of larvae of the chickpea pod borer than the control. all the six treatments have almost the same number of larval instar before the treatment of the first spray. the first larval population was observed in the table 3: pretreatment and post-treatment larval population of pod borer after 2nd spray. s. no larval population (pretreatment) larval population (post-treatment) treatments n m f total mean n m f total mean t1 0.9±0.36ab 0.2±0.18c 0±0b 1.1 0.36 0.4±0.2c 2.7±0.37c 0.6±0.35b 3.7 1.23 t2 0.7±0.3b 0.6±0.34c 0.2±0.18ab 1.5 0.5 3±0.6b 4.9±0.7b 1±0.7b 8.9 2.96 t3 1.6±0.6a 0.7±0.37bc 0.1±0.14b 2.8 0.9 0.2±0.1c 3.5±0.6bc 0.5±0.18b 4.2 1.4 t4 0.6±0.26b 1.4±0.31ab 0.3±0.16ab 2.3 0.76 2.1±0.33bc 4.2±0.31bc 1±0.62b 7.3 2.4 t5 0.8±0.29ab 0.7±0.28bc 0.1±0.1b 1.6 0.53 2.3±0.46bc 3.2±0.38bc 0.4±0.24b 5.9 1.96 t6 1.4±0.4ab 1.7±0.29a 0.6±0.3a 3.7 1.23 11.5±2.6a 15.5±1.6a 5.1±1.4a 32.1 10.7 where, n= neonate, m=middle and f=fully grown larval stages respectively comparative efficacy of bio-pesticide and synthetic agrochemicals on control of helicoverpa armigera larvae on chickpea vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 79 r a d s j . b i o l . r e s . a p p l . s c i 79 first week of april. the neonate, which is the first instar was present in all the observed subplots, and the middle instar which is formed by the conversion of neonate instar was observed only in the bifenthrin + emulsified neem oil combination and control treatments before the application and has a value of 0.2 each for both the treatments. the fully grown instar (f) was absent an all the pretreatments plots. after the application of the pesticides, it was found that emamactin has the highest effect on the control of the chickpea pod borer larvae. % reduction in the larval population was 63 after the first spray. bifenthrin + emulsified neem oil combination and emulsified neem oil treatments have an almost ttreatmenthe same effect on the control of pod borer larvae and % reduction in case of this two treatment was 60 in each. the chlorpyrifos has the larval % reduction of 56.4 while acetamiprid was the least effective treatment for the control of pod borer larvae after the application of the first spray. the % reduction in the larval population was more pronounced after the application of the 2nd spray. table 4 shows that emamactin has the lowest control of pod borer larvae and the % reduction in the larvae was 88. the chlorpyrifos showed the second best results after emamactin and the % reduction in the larval population, in this case, was reported to be 86.9. the bio-pesticide showed 81 % reduction in the larval population of pod borer. the bifenthrin + emulsified neem oil combination treatment which was almost the same as that of emulsified neem oil treatment after 1st spray showed a decreased efficacy as compared to emulsified neem oil treatment in lowering the larval population and showed a % reduction of 77. acetamiprid was the least effective in controlling the larval population after the 2nd spray and the total % reduction was noted to be 72. table 4: percent reduction of the chickpea pod borer larvae after each spray. treatments % reduction (1st spray) % reduction (2nd spray) t1 63 88 t2 42.3 72 t3 56.4 86.9 t4 60 77 t5 60 81 t6 ___ ___ percent pod damage and pod damage reduction the percent damage of pod for each treatment is shown in table 5. the highest pod damage was observed in control, while the lowest value was observed in case of emamactin with a pod damage % age of 13.33 the chlorpyrifos treatment showed a pod damage of 14 percent while the emulsified neem oil has a percent reduction of 14.66. in the case of pesticide treatments, acetamiprid has the highest pod damage after the control and the percent pod damage reported was 17.33 percent. similarly, the highest pod damage reduction over control was also noted in the case of emamactin application and the lowest value was observed in the case of acetamiprid having values of 44.4 % and 27.7 percent. the neem oil has 38.8 % damage reduction while bifenthrin + emulsified neem oil combination has a % age damage pod reduction of 30.5. table 5: percent pod damage and percent reduction of pod damage over control. treatments % damage pod % reduction in pod damage over control t1 13.33b 44.4 t2 17.33ab 27.7 t3 14b 41.66 t4 16.66ab 30.5 t5 14.66b 38.8 t6 24a __ grain yield the grain yield obtained after the harvest of the chickpea plant are shown in table 6. the highest grain yield was obtained for emamactin and the percent increase over the control was recorded to be 47.8 percent. the emulsified neem oil treatment yield was second after emamactin and was 22.5 kg and the percent increase was noted to be 40.6. the yield of grain was recorded for control and was 16 kg only. suneel kumar used emamactin against the chickpea pod borer and found that emamactin has 83.7 % control over the control. patel et al. found that emamectin showed a better activity against the chickpea pod borer15 and singh and yadav found the lowest pod damage 12.5 percent in chickpea in case of indoxacarb16. jawad et al. observed that neem oil possessed almost the same comparative efficacy of bio-pesticide and synthetic agrochemicals on control of helicoverpa armigera larvae on chickpea vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 80 r a d s j . b i o l . r e s . a p p l . s c i 80 activity against the h. armigera on tomato vegetables17. they noticed that mean larval of pod borer per plant were 0.40 and 0.46 for neem and emamectin treatments respectively. rajput et al. reported that synthetic insecticide was found to be the best treatment as compared to natural products18. gohokar et al. found that neem treatments gave best results than synthetic insecticides19. gilani noted that neem oil possesses repellent, insecticide and anti-feeding characteristics20. singh and kumar reported that emamectin was the effective insecticide in lowering the population of h. armigera in chickpea21. sontakke et al. found that emamectin benzoate was found effective in decreasing the population of fruit borer and fruit damage in okra as well as tomato crops22. neem oil showed a tremendous antifeedant, insecticidal and insect growth regulatory properties23. in one study, it was found that bollcure fraction and nske was highly effective and economical in lowering the h. armigera larvae in chickpea24. c o n c l u s i o n s the bio-pesticide of neem oil showed better results in controlling the pod borer larval population next to chlorpyrifos and emamectin after the 2nd spray. the highest grain yield was reported for emamactin followed by emulsified neem oil treatment. pakistan is the 3rd largest producer of chickpea and there is need to use the bio-pesticide due to wto constraints for the exportoriented agro-product and neem oil has proved to be a good bio-pesticide. this product is purely organic and a prominent candidate as a pesticide in organic farming. this product has been used effectively in controlling the aphid and potato leafhopper control. further study is recommended on further crops and a pest to study the effect of the biopesticide. acknowledgement the author is highly grateful to mr. alamzeb, deputy chief scientist of the plant protection division of nuclear institute for food and agriculture, peshawar for providing useful information for calculating the larval population of the chickpea pod borer. r e f e r e n c e s 1. ahmed k, awan ms. integrated management of insect pests of chickpea cicer arietinum (l.) walp. in south asian countries: present status and future strategies a review. pak j zool. 2013; 45: 1125-45. 2. ahmed s, zia k, shah n. validation of chemical control of gram pod borer, helicoverpa armigera (hubner) with new insecticides. intern j agri bio (pakistan). 2004; 6(6): 978-80. 3. sharma op, bhosle bb, kamble kr, bhede bv, seeras nr. management of pigeon pea pod borers with special reference to pod fly (melanagromyza obtusa). ind j agri sci. 2011; 81(6): 539-43. 4. yadav nk, singh ps. field evaluation of some new insecticide molecules against pod borers in mung bean. indian j entomol. 2013; 75(4): 360-1. 5. vinoth kb, kumaran n, kubendran d, kuttalam s. bio-efficacy of flubendiamide+thiacloprid 480 sc against insect pests of tomato. pestol. 2010; 34(1): 44-7. 6. pandey bm, tripathi mk, lakshmi v. seasonal incidence of gram pod borer, h. armigera (hub.) on chickpea in varanasi area. j exp zool 2012; 15(2):667-9. 7. sreekanth m, lakshmi msm, koteswar ry. bioefficacy and economics of certain new insecticides against gram pod borer, h. armigera (hubner) infesting pigeon pea (cajanus cajan l.). intern j plant ani environ sci. 2014; 4(1): 11-5. table 6: bio-mass and grain yield of chickpea for all treatments. treatments bio-mass (kg) grain yield (kg) % increase in grain over control t1 90.5a 23.66a 47.8 t2 79c 19.7ab 23 t3 81.5bc 20.33a 27 t4 85abc 21.8a 36.2 t5 88ab 22.5a 40.6 t6 67d 16b comparative efficacy of bio-pesticide and synthetic agrochemicals on control of helicoverpa armigera larvae on chickpea vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 81 r a d s j . b i o l . r e s . a p p l . s c i 81 8. singh p, singh r, kumar s, kumar v, kumar s. bioefficacy of certain new insecticides against the larval population of gram pod borer, h. armigera (hubner) in chickpea. the ecoscan. vii: 2015; 315-8. 9. kambrekar dn, somanagouda g, basavarajappa mp, halagalimath sp. effect of different dosage of emamectin benzoate 5 sg and indoxacarb 14.5 sc on pod borer h. armigera infesting chickpea. legume res 2012; 35(1): 13-7. 10. sarwar m. competency of natural and synthetic chemicals in controlling gram pod borer, h. armigera (hubner) on chickpea. inter j of agri sci. 2012; 2(4): 132-5. 11. meena lk, raju svs. bio-efficacy of newer insecticides against tomato fruit borer, h. armigera (hubner) on tomato, lycopersicon esculentum mill under field conditions. the bioscan 2014; 9(1): 34750. 12. hosamani ac, bheemana m, vinod sk, rajesh l. bio-efficacy of chlorantraniliprole 20 sc (rynaxypyr) against h. armigera of chickpea. ind j plant protec. 2013;41(2): 178-79. 13. kumar j. neem oil content and its chemical constituents in relation to the agro-ecological factors and regions in india. pestici res j. 2010; 9:216-25. 14. hossain a, haque a, ahmad m, prodhan mzh. development of an integrated management approach for pod borer, h. armigera (hubner) on chickpea bangladesh. j agril res. 2010; 35(2):201-6. 15. patil sk, ingle mb, jamadagni bm. bio-efficacy and economics of insecticides for management of h. armigera (hubner) in chickpea. ann plant protec sci. 2007; 15(2): 307-11. 16. singh ss, yadav sk. comparative efficacy of insecticides, bio-pesticides and neem formulations against h. armigera (hubner) on chickpea. ann plant protec sci. 2007; 15(2): 299-302. 17. shah ja, inayatullah m, sohail k, shah sf, shah s, iqbal t et al. efficacy of botanical extracts and a chemical pesticide against tomato fruit worm, helicoverpa armigera (lepidoptera: noctuidae). sarhad j of agri 2013; 29(1): 93-6. 18. rajput aa, sarwar m, bux m, toufiq m. evaluation of synthetic and some plant origin insecticides against helicoverpa armigera (hubner) on chickpea pak j biol sci. 2003; 6 (5): 496-9. 19. gohokar rt, thakre sm, borle mm. chemical control of gram pod borer (heliothis armigera hubner) by different synthetic pyrethroid and insecticides. pesticides 1987; 21 (11): 55-6. 20. gilani g. neem the wonder tree. in farming, octnov. ed: m.t. saleem, safe foundation 2001; 27-30. 21. singh ak, kumar a. evaluation of new molecules in sipm modules against helicoverpa armigera (hubner) in chickpea, ann pl protec sci. 2012; 20 (1): 19-23. 22. sontakke be, das n, panda pk, swain lk. bio efficacy of emamectin benzoate 5% sg against fruit and shoot borer in okra, ann pl protec sci. 2007; 4, 2: 30-3. 23. ramya s, jayakumararaj r. antifeedant activity of selected ethnobotanicals used by tribals of vattal hills on h. armigera (hübner). j pharmacol res 2009; 2:1414-8. 24. reghuraman m, birah a, gupta gp. management of h. armigera in chickpea with botanical formulations. ind j entomol 2008; 70 (2): 118-22. structural bioinformatics: computational software and databases for the evaluation of protein structure vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 94 r a d s j . b i o l . r e s . a p p l . s c i 94 op en ac ces s f u l l l e n g t h a r t i c l e structural bioinformatics: computational software and databases for the evaluation of protein structure ayisha amanullah* and suad naheed department of biotechnology, jinnah university for women, karachi, pakistan a b s t r a c t databases are the computerized platform where information is stored and can be retrieved easily by public users. biological databases are the repositories of biological data. these biological data libraries contain facts and figures related to various disciplines of research including genomics, proteomics, microarray technology, metabolomics and phylogenetics. by using biological databases, a broad collection of essential biological information can be exploited ranging from function, structure and localization of gene, clinical consequences of mutation to similarity index among biological sequences and structures. nowadays, different kinds of biological databases are available on the web. the present write up focuses on biological databases and bioinformatics tools for protein structure analysis. this review also aims to elaborate the searching schemes, available in different structural databases. the wide variety of different levels and types of information content related to 3d protein structures are available on web-based databases. regarding the biological functions and 3d structures of various proteins, these databases provide a huge range of useful links, schematic diagrams as well as strategies for detailed analysis of proteins and other macromolecules structures. 3d structural illustration of proteins stored in structural databases is determined and visualized by x-ray crystallography, electron microscopy and nmr spectroscopy. on regular basis, a large number of protein structures are submitted by structural biologists, updated and curated by subject experts. most familiar biological databases that store 3d protein and other macromolecules structures include, pdb, 3d genomics, cath, & scop. these databases contain valuable information of overall protein structures, domains and motif structures, protein-protein complex systems and complex of protein with other biomolecules. keywords database, pdb, cath, mmdb, scop, domain, protein-protein complex systems. *address of correspondence ayisha.aman24@gmail.com article info. received: april 02, 2018 accepted: september 24, 2018 cite this article: amanullah a, naheed s. structural bioinformatics: computational software and databases for the evaluation of protein structure. rads j. biol. res. appl. sci. 2018; 9(2): 94-101. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n a database is an organized and structured computational based repository that can be easily retrieved, analyzed, managed and updated. the biological database is an archival collection of biological data. biological databases fall into two categories i.e. primary database and secondary database. in the primary database, the information submitted is basically in its original form which is obtained experimentally. while in secondary databases, the information is highly curated after the evaluation of data, from primary databases. nowadays, gathering, processing and analyzing of data has become an important aspect of research, especially in the field of biological sciences. bioinformatics, a new and foremost sub-discipline of biotechnology, mainly focuses on data r e v i e w a r t i c l e structural bioinformatics: computational software and databases for the evaluation of protein structure vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 95 r a d s j . b i o l . r e s . a p p l . s c i 95 processing. through the various strategies of bioinformatics, biological databases are designed after data gathering and processing. these biological databases are incorporated with specific features1. among various databases, protein database is a collection of various sets of data about proteins. it is serving as a storehouse of information since 1971 and stores information about protein sequence, protein structure, its conformation and active sites. moreover, it also describes the physical and chemical nature and biological role of proteins as proteins are solely responsible for all the biological functions in the body2. protein structure databases elucidate the protein structure in normal as well as in diseased condition in the biological system. through these structure databases, the 3d structure of proteins and cell dimension can be studied. this review highlights the most common protein structure databases, that will be helpful to those individuals in the scientific community who would be interested to conduct research in bioinformatics specifically in protein structure studies. p r o t e i n d a t a b a n k ( p d b ) protein data bank is an online structural library of biological macromolecules, which is the only worldwide repository of macromolecular structure. the pdb was organized in 1971 at brookhaven national laboratories (bnl) as a platform of crystal structures of biomolecules. over the years, the data submitted to pdb was modified and approaches to access the pdb have changed, as a result of advancements in technology. in october 1998, research collaborator for structural bioinformatics (rcsb) has started to manage and maintain the activities of pdb. pdb data is curated and annotated by rcsb pdb. the major task of the rcsb is to generate such measures that allow the use and analysis of structural data3. pdb stores 3d structural information of biological molecules mainly nucleic acid and proteins. the structural information of biomolecules is commonly acquired experimentally by nmr spectroscopy, x-ray crystallography, electron microscopy etc. structural information of some chemical ligands and nucleotides are also available on pdb. pdb id is a fourcharacter identifier that is actually entitled as pdb entry. a user can access pdb at http://www.rcsb.org/pdb/ or http://www.pdb.org (fig. 1). fig. 1: a homepage of pdb. searching through pdb is done by a vast range of search engines ranges from pdb id and keywords to structural features of proteins and other biomolecules (fig. 2). fig. 2: searching strategies available at pdb. there are two formats that pdb uses to keep structural data: the pdb file format and macromolecular crystallographic information file format (mmcif). pdb file design is more commonly used in protein community as compared to mmcif. pdb offers various molecular structural visualization soft wares including jmol, pdb simple viewer, pdb protein workshop and rcsb-kiosk. structural confirmation of secondary structure is also provided by pdb4. the pdb depository is run by an association, named the worldwide protein data bank (wwpdb) which guarantees that the information is freely accessible to the public. structures for huge numbers of the proteins and nucleic acids required in the central procedures of life are available on pdb5. p d b s u m at present, more than 13000 3d structures of biomolecules have been exploited experimentally by using nmr spectroscopy and x-ray crystallography. most of these 3d structures mainly include proteins, dna and protein-ligand complexes (fig. 3 & 4). along with the structural bioinformatics: computational software and databases for the evaluation of protein structure vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 96 r a d s j . b i o l . r e s . a p p l . s c i 96 sequence, functional and physiochemical properties give a bundle of information, quite sufficient for developing knowledge regarding biochemical processes. these structures are submitted to pdb which can be obtained from rcsb's pdb web page. there are other structural archives that collect further information on the particular type of molecules or particular features of the molecules. pdbsum is one of the principal database. pdbsum was established in 1995 with the goal to make an available pictorial summary of biological macromolecules structures (dna, rna, protein, metal ions, water molecules and small molecule ligands), present in pdb, along with their major structural attributes. pdbsum keeps 3d images of the protein structure, protein secondary structure annotations, detailed structural statistics, developed from the promotif computer software, summary procheck consequences and flow diagrams that indicate connections between protein, ligands and dna molecules. rasmol focuses the crucial features of the protein structure including domains, protein-ligand correlations and prosite format for interactional 3d visualization. fig. 3: search engines available at pdbsum. fig. 4: pictorial summary of hydrolase/dna with pdb id: 1kfv at pdbsum. pdbsum is frequently upgraded, whenever any new structure is submitted to pdb. pdbsum is accessed freely through url: http://www.ebi.ac.uk/thorntonsrv/databases/cgi-bin/pdbsum 6. 3 d g e n o m i c s 3d-genomics is a structural database that gives information about the protein structure from the sequenced genome. the database stored information of 93 proteomes in august 2003. it stores information about homologous sequences from different sequence databases, patterns from prosite, protein domain information from pfam and scop, and characteristics of other predicted sequences such as transmembrane regions and coiled coils. the structure of the protein and its biochemical function can be annotated after the genome has been sequenced by this database. various searching policies are available on 3d-genomics database that allow a user to,  retrieve data directly for individual protein sequence by accession numbers or keywords.  investigate the desired sequence selected from summarized annotations for a specific proteome.  or approach pre-calculated frequency based cross proteome comparative study7. different methodologies are involved for retrieving the information such as the recognition of motifs which are responsible for structure and function, characterization of coiled and integral regions sequence identification and the identification of similar proteins whose function or the structure has already been reported8. c a t h d a t a b a s e protein evolution originates in the families of structurally closed protein, within which sequence identities can be very small. this can allow effective structure-based classification in distinguishing unpredicted associations in known structures and under ideal conditions, the function can also be allocated. the constant emerging variety of fully known protein structures is just so massive. hence it is impossible to classify all proteins manually. in order to overcome the situation, automated techniques are needed for quick assessment of these biological molecule structures9. structural bioinformatics: computational software and databases for the evaluation of protein structure vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 97 r a d s j . b i o l . r e s . a p p l . s c i 97 cath is a protein structure classification bank which stores hierarchical ranking (taxonomic classification) of protein domains based on their folding mechanisms. cath uses evolutionary information of proteins and classifies protein domains into superfamilies if they have emerged from same ancestors. cath database obtains domains (fig. 5) from protein structures, stored in pdb10. fig. 5: protein domains data, accessible through cath. almost, 114,215 domains are present in the latest version of cath, which was released in july 2008. these domains are graded in a hierarchical manner with four main divisions, named class (c), architecture (a), topology (t) and homologous superfamily (h), so the name coined "cath". cath can be browsed through url www.cathdb.info. the cath has four different visions when classifying protein domains, 1. class (c): at this level, domains are categorized on the basis of their secondary structure into four structural types: including alpha, beta, mixed alpha beta and forth type consists of domains comprised of few secondary structures. 2. architecture (a): this level classifies domains on the basis of the orientation of their secondary structures. 3. topology (t): this level mainly focuses on the topological connectivity of the secondary structures. 4. homologous superfamily (h): this level groups the domains according to their common evolutionary linkages; therefore, they can be demonstrated as homologous. the comparison among closely related domains is based on sequence and structural similarity, which is done by using ssapa dynamic programming algorithm11. construction of database the information in cath is received from pdb files submitted within the supermolecule information bank. the database encloses structures resolved with 4ao resolution or higher. the necessities of cath mainly include that domain should consist of at least 40 residues and side chains should be resolved with 70% or more12. as described in the introduction, the current edition of cath consists of 114,215 domains, treated from the proteins deposited in pdb. new domains structures are incorporated in cath on the basis of two fundamental principles, 1) deposited protein chains are chopped to acquire domains of those fragments. 2) afterward, these resulting domains are classified11. by analyzing structural families produced by cath database shows the outstanding options of macromolecule structure area. a well-defined info relevant to the structural families of macromolecules like cath can help the assigning of structure-function and phylogenetic relationships to each known and freshly determined structures of macromolecules. the cath database is effective for researcher and bioinformaticians. web surfing is made simple and convenient even for a single domain via a manageable user-friendly net interface for researchers having a particular task, whereas bioinformaticians with insight on comprehensive research can explore entire datasets provided for downloading. the database is frequently updated and supplemented with numerous approaching extensions with horizontal layers corresponding to the hierarchical data structure; cath is probably going to be an excellent useful protein taxonomic computational tool in the future. thus, operating with cath is remarkably uncomplicated13. p r o t e i n f o l d i n g d a t a b a s e the protein folding database (pfd) is freely available data house that is supplied with the data concerning to methodological, structural annotations, kinetic and thermodynamic folding patterns of a large number of proteins i.e. greater than 50 proteins belonging to 39 families14. this database collects overall protein folding structural bioinformatics: computational software and databases for the evaluation of protein structure vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 98 r a d s j . b i o l . r e s . a p p l . s c i 98 information into a solitary, effectively open asset. an easy to use web database has been produced that permits strong searching, browsing, information mining and data recovery while giving connections to other protein repositories. the protein folding database architecture represents the view of folding impressions in a helpful and innovative manner, with the everlasting goal that encourages data retrieval and bioinformatics procedures15. structural organization of the database a salient purpose of the database is to permit the examination of the exact and hypothetical connections between structural characteristics and folding frequencies of a protein, for instance, topology. consequently, new tables were included into databases in order to assemble information like sequence, expression tags, construct length, pdb identifier and disordered regions. additional tables also enable the accumulation of crude kinetic information and blunders for every single numerical data are presently recorded14. folding research and pfd the pfd deposits a stockpile of folding pattern information that can be examined with various factors and the results show or appear in the structural form. the repository allows a full information, spreadsheet-like rundown of outcomes allowing rapid exploration of common or general fashion in data. the inquiry data outcomes can be looked for on any caption, which is valuable, e.g. while reviewing the inconstancy of folding frequencies among proteins inside a family. information related to publications of each entry and url to access ncbi pubmed writing database is recorded15. futuristic approaches the pfd has been redesigned by the laws defined by the international fold omics consortium. latest approaches in this tool will motivate the further construction of the database, and new methods of presenting data information graphically will upgrade its utilization in protein science field. the main objective of future research will be on the advancement of further graphical portrayals of the folding data information. it is important to reveal here that this database is not only an information archive but rather turns into an intense analytical tool in folding research14. d a t a b a s e o f m a c r o m o l e c u l a r m o t i o n s macromolecular motion is crucial to understand the function. the macromolecular motion allows understanding the mode of catalysis, signaling and the mechanisms involved in the formation of complexes. database of macromolecular motion is a platform, to which structures are submitted and this database creates putative motion trajectories, that play a vital role in structural biology16. the database of macromolecular motions is used generally as a structural community. this database is freely available at http://bioinfo.mbb.yale.edu/molmovdb, it schematizes the protein and nucleic acid movement in order to obtain structural information. evidence about experimental information, structural similarity is available in this database. in order to implement a database, we can use the design of standard relation. in addition, the heterogeneity, as well as complexity regarding information, is available in the database thus promoting its link to an object-oriented approach. however, in order to keep complex data, the database is equipped with imaginable depictions for motion pathways, obtained from 3d interpolation among the known conformation17. s c o p d a t a b a s e there is another protein structural repository that stores information of those proteins that are similar at the structural level and possess common ancestors. scop (structural classification of proteins) was constructed for this purpose18. in 1994, scop was developed in the laboratory of molecular biology and center for protein. scop database consists of comprehensive details of evolutionary relatedness of known protein structures. the basic unit of classification is a protein domain, which is hierarchically classified into species, proteins, families, superfamilies, folds and classes. classification scop classifies the protein based on hierarchical levels, which are discussed below,  family, proteins are grouped into families according to two strategies, based on common evolutionary relationships. 1st all proteins with 30% or greater structural bioinformatics: computational software and databases for the evaluation of protein structure vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 99 r a d s j . b i o l . r e s . a p p l . s c i 99 residue identities, 2nd proteins having small sequence identities but they are similar at functional and structural level.  superfamily, this level occupies the families of proteins that hold least sequence identities, but their structural and in many conditions, functional characteristics share a same evolutionary origin.  common fold, it groups the proteins of families and superfamilies, have major similar secondary structures with the same order and topological connectivity.  class, this level groups different folds into classes. these protein folds are categorized into five classes, 1. all alpha, whose structure is composed of alpha helices. 2. all beta, whose structure consists of beta sheets. 3. α/β, whose structure is formed from alpha helices and beta sheets. 4. α+β, in these proteins, alpha helices and beta strands are massively sequestered. 5. multidomain, protein domains with different folds and for which homologs are unknown. searching facilities at scop for the browsing and searching of particular families of protein at scop, scop is provided with the variety of search engines and techniques for navigations including,  surfing through scop hierarchy  browsing by using amino acid sequence  search through a key  search through pdb identifier  browsing using history19. scop is freely available at url link http://scop.mrclmb.cam.ac.uk/scop/. scop 2 there was a closure of scop in 2010 and in january the prototype for a new scop2 database has become easily accessible, which reflects a novel approach for the classification of proteins that differs quietly from the previous version of scop, but best features have been introduced. in scop2 (fig. 6), proteins are also arranged according to the evolutionary and structural relationship but not like old version of scop which provides hierarchy in simple tress like structures, this database describes the classes fig. 6: a homepage of scop2. of proteins in a form of graph which is actually acyclic in shape with a network of complex nodes, which defines a relationship of protein of particular type with others20. m m d b ( m o l e c u l a r m o d e l i n g d a t a b a s e ) mmdb is the 3d structural database of biological macromolecules and their molecular interaction, developed by ncbi. mmdb can be accessed through ncbi entrez's search engine21. the main aim of mmdb is to circulate the information of the 3d structure of macromolecules mainly protein and functional annotations among molecular biologists 22. mmdb filters the contents of pdb. it links protein 3d structure information along with sequence information, sequence classification resources and pubcheman archive that keeps the chemical structural data of small molecule and their biological roles, thus making available various approaches to 3d structure data for molecular biologists, structural biologists, and chemists. mmdb gives a comprehensive detail on structural alignments and software for 3d structure visualization by graphical viewer cn3d. mmdb also focuses the quaternary structures and the molecular interactions between its components. mmdb is accessible through the world wide web at url http:// www.ncbi.nlm.nih.gov/structure 21. c o n c l u s i o n there are numbers of web-based protein structure databases that contain different extents of information on biological macromolecules structures on a different basis. generalized databases contain general information about protein structures; include those protein structures experimentally investigated by nmr spectroscopy and xray crystallography. they also give useful web resources and schematic diagrams related to the protein 3d structural bioinformatics: computational software and databases for the evaluation of protein structure vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 100 r a d s j . b i o l . r e s . a p p l . s c i 100 structure and their biological functions. there is another category of protein structure database which systemizes 3d structures by their folds as they display evolutionary relatedness which may be difficult to determine from sequence alignment alone. in addition, there are diverse databases and servers that contrast folds of protein structures are beneficial for newly determined protein structures, and especially for those proteins or protein structures which are still unknown. beyond these, there is a wide range of databases for the most specialized users that deal with particular families, different structural qualities, diseases and so on. pdb is the most widely used and popular among protein structure communities that provides excellent details of protein structure in 3d, allowing the user to study and visualize the 3d structures by different computational based software (jmol, ngl view etc.). pdb interprets the 3d structures to the user in an understandable way. on the other hand, scop and cath categorize protein 3d structures on the basis of their folds and evolutionary origin. mmdb is also structural archive, headed by ncbi that also stores an extensive number of macromolecular 3d structures. although the number of these structural databases is increasing day by day with great advancements and facilities but the databases that have been discussed in this article are the milestones in the field of bioinformatics1. abbreviations pdb: protein data bank cath: class architecture topology homologous superfamily scop: structural classification of proteins rcsb: research collaboratory for structural bioinformatics mmdb: molecular modeling database pfd: protein folding database r e f e r e n c e s 1. bagchi a. a brief overview of a few popular and important protein databases. computational molecular bioscience. 2012; 2:115-2. 2. zhang y, zhu y, he f. an overview of human protein databases and their application to functional proteomics in health and disease. sci china life sci. 2011; 54(11): 988-98. 3. berman hm, westbrook j, feng z, gilliland g, bhat tn, weissig h, et al. the protein data bank. nucleic acids res. 2000; 28(1): 235-42. 4. xu d, xu y. protein databases on the internet. curr protoc mol biol. 2004, ch. 19, unit 19.4. 5. about rcsb pdb: enabling breakthroughs in scientific and biomedical research and education. rcsb pdb; [cited 2018 march 19]. available from: http://www.rcsb.org/pages/about-us/index 6. laskowski ra. pdbsum: summaries and analyses of pdb structures. nucleic acids res. 2001; 29(1):221–2. 7. fleming k, muller a, maccallum rm, sternberg mj. 3d genomics: a database to compare structural and functional annotations of proteins between sequenced genomes. nucleic acids res. 2004; 32: d245–50. 8. li c, dong x, fan h., wang c, ding g, li y. the 3dgd: a database of genome 3d structure. bioinformatics. 2014:30(11): 1640-2. 9. orengo ca, michie ad, jones s, jones dt, swindells mb, thornton jm. cath: a hierarchic classification of protein domain structures. structure. 1997;5(8):1093108. 10. sillitoe i, lewis te, cuff a, das s, ashford p, dawson n.l, et al. cath: comprehensive structural and functional annotations for genome sequences. nucleic acids res.2015; 43: d376–81. 11. kundsen m, wiuf c. the cath database. human genomics. 2010; 4(3):207-12. 12. greene lh, lewis te, addou s, cuff a, dallman t, dibley m, et al. the cath domain structure database: new protocols and classification levels give a more comprehensive resource for exploring evolution. nucleic acids res. 2007;35: d291-7. 13. cuff a, redfern oc, greene l, sillitoe i. thecath hierarchy revisited – structural divergence in domain superfamilies and the continuity of fold space. structure. 2009;17(8):1051-62. 14. fulton k., bate m, faux n, mahmood k., betts c, buckle a. protein folding 8. database (pfd 2.0): an online environment for the international foldeomics consortium. nucleic acids res. 2007; 35(database issue): d304-7. 15. fulton kf, devlin gl, jodun ra, silvestri l, bottomley sp, fersht ar, buckleam. pfd: a database for the investigation of protein folding kinetics and stability. nucleic acids res. 2005; 33(database issue): d279-83. 16. flores s, echols n, milburn d, hespenheide b, keating k, lu j, et al. the database of macromolecular motions: new features added at the decade mark. nucleic acids res. 2006; 34: d296-301. 17. gerstein m, krebs w. a database of macromolecular motions. nucleic acids res.1998; 26(18):4280-90. 18. murzin ag, brenner se, hubbard t. and chothia c. scop: a structural classification of proteins database structural bioinformatics: computational software and databases for the evaluation of protein structure vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 101 r a d s j . b i o l . r e s . a p p l . s c i 101 for the investigation of sequences and structures. j. mol. biol.1995; 247:536-40. 19. lo conte l, ailey b, hubbard tj, brenner se, murzin ag. and chothia c. scop: a structural classification of proteins database. nucleic acids res.2000; 28: 257-9. 20. andreeva a, howorth d, chothia c, kulesha e & murzin ag. scop2 prototype: a new approach to protein structure mining. nucleic acids res.2014; 42: d310-14. 21. madej t, addess kj, fong jh, geer ly, geer rc, lanczycki cj, et al. mmdb: 3d structures and macromolecular interactions. nucleic acids res. 2012; 40: d461-4. 22. wang y, anderson jb, chen j, geer ly, he s, hurwitz di, et al. mmdb: entrez's 3d-structure database. nucleic acids res.2002; 30:249-52. rads journal of biological research & applied sciences vol 4 (2), july 2013 issn : 2305-8722 original articles study of evaluation of four varieties of onion (allium cepa l) in the environment of ust, bannu abdul malik, rehman ullah khan, saad ullah khan, syed aneel gilani, hidayat ullah khan, zeeshan ahmad......................................................................................................... 01 antimicrobial activity of shilajit aliya hayat, fatima sher ali .............................................................................................. 10 oral health status, the level of oral microbial flora in healthy girls sughra hasan, amna shafiq, aziz fatima, sayyada ghufrana nadeem........................13 detection of du antigen in rh negative blood group individuals naheed afshan, sarah tariq ............................................................................................... 16 bioethics education, awareness of ethics and dissemination of knowledge among teachers and students saima rasool, sumaira javed, sayyada ghufrana nadeem, shazia tabassum hakim ................20 effect of burn injury on the dissemination of candida albicans from the skin of mouse qudsia hussain, amna shafiq, shazia tabassum hakim, sayyada ghufrana nadeem ...............33 effect of cadmium levels on the growth curve of candida albicans aziz fatima, qudsia hussain, shazia tabassum hakim, sayyada ghufrana nadeem ................38 phosphatase production among candida species syeda hira batool, sayyada ghufrana nadeem ...............................................................42 effect of paneer booti during kinetics on candida tuba batool, sayyada ghufrana nadeem.......................................................................... 49 prevalence of transfusion transmitted infection in replacement and voluntary blood donor syeda kanwal zehra, saira bano, shazia tabassum hakim, sayyada ghufrana nadeem..........56 prevalence and risk factors for diabetes mellitus in urban population of karachi (a short study) sobia jawaid, sayyada ghufrana nadeem ........................................................................ 64 anti-proliferative effect of arsenic, cadmium and lead on human placental cells aftab ahmad, aamina shahm, abdul rauf shakoori ......................................................67 review articles expression of growth form factors during morphogenesis in candida albicans amna shafiq, aziz fatima, qudsia hussain, sayyada ghufrana nadeem, shazia tabassum hakim ...................................................................................................................................................................................................... 75 instruction to authors introduction onion (allium cepa l.) is most important cool season plant, it hold top position in area of cultivation and production amongst the bulb crops produced in the world (fao 2002). allium cepa is known only in c u l t i v a t i o n , g r u b b e n & d e n t o n ( 2 0 0 4 ) . onion is the chief food plant in which food is stored in a bulb. it is very old, its use going back over 4000 year beyond the beginning of authentic history. it is unknown in wild state the onion is probably a native of southern asia or mediterranean region. it has long been valued in china and india for its flavoring. onions are cultivated over large areas in temperate region and even in tropical climates. they prefer cool moist regions with a sandy soil. they are grown from seeds or small bulblets. onion should be dried prior to storage to develop the characteristic flavor and test, which are due to an acrid volatile oil, the allyl sulphide. they are both food plants and flavoring agents. over 250 species of allium are known. many occur as weeds. the commonest forms in cultivation are garlic, leeks, chives, shallots, and the true onion, hill (1951). onion is a shallow rooted crop a fairly high concentration of nutrient should normally be study of evaluation of four varieties of onion (allium cepa l) in the environment of ust, bannu. abdul malik1, rehman ullah khan1*, saad ullah khan1, syed aneel gilani2, hidayat ullah khan1, zeeshan ahmad 1 1department of botany, university of science and technology, bannu. 2 pakistan museum of natural history, islamabad, pakistan abstract a field experiment was conducted to evaluate the performance of different varieties, viz; bannu local, india, swat and quata of onion at awais ahmad ghani green house, department of botany, university of science and technology bannu, k.p.k pakistan. the trial was conducted in rcbd (randomized complete block design) and replicated 3 times to minimize the error. significant differences were recorded among varieties for the parameters like new leaf initiation, leaf length plant-1, leaves bulb-1. while non-significant results were recorded in case of fresh biomass plant-1, dry biomass plant-1, economic weight bulb-1, bulb diameter, yield kg hac-1. so for as the performance of quata variety is concerned it taken the highest number of days (24) for new leaf initiation after transplantation and longest leaf length (25.50cm) was observed in this variety. maximum fresh biomass (259.6 g plant-1), dry biomass (218.6 g plant-1), economic weight (210.1 g plant-1), bulb diameter (7.2cm) and yield (17149.850 kg h-1) was found in case of swat variety in the prevailing environment. as for as the potential and efficiency of the bannu local variety is concerned, it produced the higher number of leaves bulb-1 (26.7), while minimum value in all the parameters regarding yield components was observed for india variety. in the light of this effort, swat variety was recorded the best and most suitable for the environment like bannu. key words: biomass, leaf bulb, onion, swat. *corresponding author: rehman_g4u@yahoo.com 01 vol 4 (2), july 2013; 01-09 maintained at the surface of the soil for its optimum growth and yield. the importance of urea, phosphate fertilizer and potash fertilizer on the growth and yields of vegetable crops are well-known. onion genotypes vary in their nature of bulbing with wide range of production of yield parameters and yield, islam et al., (2007). there is no doubt that mineral fertilizers are essential in most cropping system if maximum yields are to be realized. however in longterm field experiments where mineral fertilizers have only been used, soil structure has been deteriorated and crop yield steadily decreased as reviewed by ristimaki et al., (2000). in pakistan the cultivation of onion is not uncommon, it is cultivated through out the pakistan, especially in irrigated area or where water is abundant. different cultivars are cultivated in different area of pakistan. the cultivars, which are in practice in bannu and nurang region, are naurang local, panyalla local, phulkara, shah alam local, swat, bannu local and india etc. one of the main problems in cultivation of onion is proper and adequate amount of water and use of proper amount of fertilizer, selection of fertilizer’s type and its method of application. our present work illustrate the evaluation of four (4) varieties of onion i.e. india, swat, local bannu, quata on the basis of various yield parameters by using urea and dap. materials and methods the small bulbelts or set of all the varieties of onion were sown in the green house area of main campus ust, bannu during the year 2010. the varieties are: v1 local bannu, v2 india, v3 swat, v4 quata the germplasm of all the varieties have been developed and maintain by private formers in their own fields. v1 and v4 were obtained from the field of sperka wazir fr bannu maintained by mir shahid ullah, v2 was obtained from the field of ghulam muhammad tatar khel karak, while v3 was maintained by mujeeb ur rehman zabbi karak. i transplanted all of these varieties as early as possible at 5 january 2010 to my experimental field of clay loam soil, due to effectiveness of early transplant. all the plots were irrigated thoroughly and adequately from well. similar and equal dose of np fertilizer was maintained in all the varieties to evaluate their actual performance against the prevailing condition. the fertilizer was used in three splits in top-dressing method. the experimental field was arranged in four replications with randomized complete block design. the measurement of experimental field was kept as 9x2.4 and was split out in to four rows with row to row distance 60 cm while plant to plant distance 50 cm. each replication was composed of four sub plots. each sub plot contained 80 plants, i.e. 20 plants in each row. in this way the total area along with 4 replications was 14 m x 11.1 m. total sub plots were 16 and each plot of 3 m x 2.4 m and thus the total number of plants were 1280,320 of each variety. statistical analysis: the data so collected for each parameter were averaged and was subjected to statistical analysis as proposed by steel and terrie (1980). when significant results were obtained for all parameters then l s d tests were applied for comparison among the means of treatment. all the results were compared at 5% probability level. the actual data was recorded in each treatment on the following parameters. data recorded on following parameters: 1:new leaf initiation, 2:leaf length, 3:fresh weight (biomass) of whole plant, 4:dry weight (biomass) of whole plant, 5:dry weight (economic weight) of bulb, 6:diameter of bulb, 7:leaves bulb-1, 8:yield kg ha-1: results and discussion analysis of variance: the results of the analysis of 02 vol 4 (2), july 2013; 01-09 variance indicate that the mean differences among the different varieties are highly significant for the parameters studied viz., days to new leaf initiation, leaf length and leaves bulb-1 (appendix 1, 2 and 7). in case of parameter like, fresh biomass, dry biomass plant-1, economic weight bulb-1, bulb diameter and yield kg/h-1 are non significant. the mean values for each parameter are shown in table i viii, while the graphic representations are highlighted in fig no. 01-08. the success of any crop lies in the constant vigilance and improvement through selection and evaluation for the individual plants, which is considered necessary for the synthesis of physiologically efficient and biologically superior strains showing promise of increased production per unit area per unit time under a given set of environmental conditions. to achieve these objectives, a comprehensive knowledge of the evaluation mechanism of the control of various parameters in different varieties of the onion crop under the prevailing environmental conditions have been greatly advocated by a large number of plant scientists like nourai (1992), rajcumar (1997), mohanty (2001), cheema (2003), jilani and ghafoor (2003), benkeblia (2005), nourai (2006), shaheen et al., (2007), halvorson et al., (2008), goussous et al., (2009) jotangee (2010), schroeder et al., (2010) and shaheen et al., (2011). 1. new leaf initiation: a reference to table no.i and fig. 01 with regard to leaf initiation reflects that the quata variety does extremely well then the other hybrids dyed with recital of 24 days, while the india variety and swat variety both ranked second by 21.5 days. moreover local bannu implemented most minuscule with 20 days. all the varieties were raised in akin environmental condition and were maintained with required agronomical practices. the local bannu variety responds maximally to the new situation after transplant and were not affected by new environment, so that starting growth and leading the other varieties. the quata variety does not established themselves to the new condition quickly so having minor range of adaptation or may slowly and gradually adopt itself to the new surroundings. the difference is not due to smaller nature of transplant and not differentiation in size all the plants were transferred at 4th leaves stage. new leaf initiation in the new field is not the only criteria in varietals evaluation, but it indicates quick and better adoptable characteristics. day taken to new leaf initiation depends on the genetic make up of variety and also on adaptation to the environment. the cultural practices also influence the growth of cultivar. 2. leaf length: data with reference to the leaf length is highlighted in the table no.ii and fig. 02. which depicts that leaf length is extensively pretentious by different sources of varieties. the data proved that quata variety presented the category first with set highest length of 25.50 inches followed by swat variety with leaf height of 22.25 inches. the least possible length of 16 inches is graded to india, in 03 vol 4 (2), july 2013; 01-09 figure 1. effect of different varieties of onion on leaf initiation. table i. new leaf initiation. varieties bannu local india swat quetta lsd value 20.0a 21.5b 21.5b 24.0a 1.131 mean in days the customary ecological conditions and crop growing procedures along with required maintenance. so for as the finding with reference to the research work is concerned is based on the highest leaf length found per plant including the neck. the leaf length shows the photosynthetic activity of plant. the plant having more leaf length is more photosynthetic in general but this is not always true, because the leaf width and nature of cells also play a vital role in photosynthesis. the results achieved have similarities to some extent with the finding of jilani and ghafoor (2003), while contradiction with mohanty, prusti (2001) and ishwari et al., (2006). such type of contradiction might be due to differences in genotype make up of the varieties but ishwari obtained non significant differences in height due to genotype and showed that transplanting date is effective in this regard. the differences and similarities may be due to e n v i r o n m e n t a n d a g r i c u l t u r a l p r a c t i c e s . 3. fresh biomass plant-1: an examination of data in table no. iii and fig. 03 confirms that fresh biomass is not radically affected by the varieties of onion. mean value revealed that swat variety acts best with denomination of 259.6 gm while plant-1 least weight of 125.8 gm is found in india variety. local bannu variety achieves second position by rank on the basis of performance in the prevailing e n v i r o n m e n t a n d a g r o n o m i c a l p r a c t i c e s . fresh biomass depends on all contents including water and other volatile substances. the fresh biomass may be greater for one variety while the dry biomass for another means that there is more water in the variety under study. the results so concluded confirm the finding of shaheen et al., (2007). the differences and similarities may be due to differences in germplasm sources and also due to different environmental conditions. irrigation of field also plays an important role in it but basically the water retention property of leaves of the variety is also significant. 4. dry biomass: a perusal of table no. iv and fig. 04 indicate the dry biomass bulb-1. numerical study shows non significant execution of genetically traits, 04 vol 4 (2), july 2013; 01-09 figure 2. effect of different varieties of onion in leaf length figure 3. effect of different varieties of onion in fresh biomass table ii. leaf length varieties bannu local india swat quetta lsd value 20.75b 16.0c 22.25b 25.50a 2.625 mean in inches table iii. fresh biomass varieties local bannu india swat quetta lsd value 225.8a 125.8a 259.6a 221.2a 106.5 mean in gm in contrast to the fundamental setting and analogous agricultural rehearsal. swat variety secured the top most status with weight of 218.6gm and local bannu variety go along with swat variety by merit of 175.7gm. the smallest value of weight is recorded as 138.5gm by india variety. the finding so for concluded by me contradicts the finding of cheema et al., (2003) who evaluated different varieties of onion in one trial under faisalabad condition and in another trial under different ecological zone of punjab. there are valuable differences in single plant weight in different varieties in same localities and even differences he recorded for same variety in different condition e.g. pulkara variety was noted with 68.50gm in one area while 86.67gm in another location by him. this type of contradiction might be due to different genetic materials and climatic conditions under which these experiments are performed. 5. economic weight/bulb: facts about the economic weight are expressed in table no. v and fig. 05. swat variety surpassed rest of the varieties tinted with utmost weight of 210.1 gm, while the india variety documented the bare minimum eight of 125.8 gm. in addition quata verity attained the subsequent attitude to swat variety with charge of 165.6 gm weight. so the swat variety secured the climax on the bases of functioning versus the existing environment and parallel crop growing condition. bulb weight influence the yield ha-1 higher the bulb weight more will be the yield ha-1. such types of findings are already reported by iqbal et al., (2000) who noted that bulb weight affect the yield ha-1 as the bulb has significant interaction with yield. the results so achieved here are in contradiction with coolong et al., (2008) who reported that yield is not increased by large size bulb as wala wala and ailsa craig two large bulb variety produce lower yield. similar results were also concluded by shimabuku et al., (1980) in their evaluation study of 15 varieties at different elevation. mr. max showed equivalency in bulb weight in both the elevation of 2000 and 1200 ft. the great effect of elevation on bulb weight was observed in yel-granex/rcs-1903, rcs-1004, rio bravo and savannah. the varieties mentioned in the previous line show maximum performance at 2000 elevation while minimum at 1200 elevation. cheema et al., (2003), also reported similar finding during varietals evaluation they found high bulb weight for 606 cal and ac-383-1. the deviation in present findings may be due to different germplasm of cultivars, environmental f a c t o r s a n d a l s o a g r o n o m i c a l p r a c t i c e s . 05 vol 4 (2), july 2013; 01-09 figure 4. effect of different varieties of onion in dry biomass table iv. dry biomass varieties local bannu india swat quetta lsd value 175.7a 138.5a 218.6a 149.2a 89.11 mean in gm table v. economic weight varieties local bannu india swat quetta lsd value 163.2b 125.8b 210.1a 165.6b 1.177 mean in gm 6. bulb diameter: analysis of the data revealed that bulb diameter is not significantly affected by the diversity of onion varieties as indicate in table no. vi and fig. 06. swat variety ranked first by diameter of 7.2 cm, while second position is maintained by both local bannu variety and quata variety with the diameter of 6.6 cm. the minimum diameter of 5.9cm is gained by india variety, in the prevailing environment and other required agricultural practices. the bulb diameter depends upon not only on the number of leaves bulb-1 but also on the thickness of rings of leaves and criteria of the researcher. many workers either do not mention their criteria or does not distinguish b/w bulb size and shape. onion bulbs are not completely spherical they are of two basic shapes either vertically elongated or horizontally elongated, for that reason the accurate diameter is equal to vertical diameter plus horizontal diameter divided by 2. diameter = vertical diameter + horizontal diameter 2 the work regarding the parameter of bulb diameter of this study is also supported by martinez et al., (2005). likewise the results are in contradiction to the finding of hasegawa et al., (2001) who worked out on the comparison of nine f1 hybrid varieties with eight local varieties. such type of fluctuation in research findings might be due to variation of germplasm of varieties of onion utilized and the difference in the ecological setup. the agronomic practices also influence the crops in various ways. 7. leaves bulb-1: the information regarding the parameter like leaves bulb-1 is exposed in table no. vii and fig. 07 which indicate that less number of leaves i.e.19.8 per bulb is noted in india variety and local bannu variety is noted for 26.7 leaves per bulb. at the other hand swat variety is recorded for 25 leaves per bulb and thus occupied the position next to the local bannu variety. in this way local bannu variety achieving the top most position in comparison to all the other varieties in the current study. it is necessary to mention that leaves per bulb is directly related to the diameter of the bulb and each variety differ from another in having rings bulb-1, no matter each variety has specific thickness of rings which contribute to the diameter also the rings of 06 vol 4 (2), july 2013; 01-09 figure 5. effect of different varieties of onion in economic weight figure 6. effect of different varieties of onion in bulb diameter table vi. bulb diameter varieties local bannu india swat quetta lsd value 6.6b 5.9c 7.2a 6.6b 1.177 mean in cm 07 vol 4 (2), july 2013; 01-09 leaves are of two types the outer tunica leaves which may be from 2-4 leaves and inner fleshy leaves in present study both type of leaves are estimated collectively in the leaves bulb-1. the finding regarding the leave bulb-1 is contradicting from the report of cheema et al., (2003) who stated that all the cultivars are not radically influenced by different location for leaves bulb-1. while the results published by anisuzzuman et al., (2009) are in full agreement to the finding of me who recorded differences in number of leaves at different plantation time. the differences may be due to different locality, soil and agricultural practices and temperature as anisuzzaman reported correlation of vegetables growth with temperature, he concluded that cool temperature and fully sunny day promote vegetative growth. 8. yield kg h-1: so far as the performance of the varieties is concerned on the basis of kg h-1 the variety swat excelled rest of the varieties with mean performance of 17149.85 kg h-1 while the india variety was recorded with minimum performance of 10264.75 kg h-1. further more the quata variety reflected the second position with value of 13517.10 kg h-1 and thus bannu local variety was ranked third on the basis of performance against the prevailing environment and similar agronomical practices. the results achieved so far for the parameter of yield (kg/h-1) is concerned are not in confirmation with the work of nelson (1986) who recorded 25.05 tons acre-1 for all the varieties in average, but to some extant in conformity with rajcumar (1997) who worked on the performance of different varieties, he recorded significant differentiation in yield in different cultivars, which range from 8.4-38.7 tons h-1. more over, the data of the current study is apposite to nourai (1992), who recorded significant differences due to seasonal changes and shows that long water intervals of irrigation reduce the yield. while similar with mohanty and prusti (2001) who observed highest yield of 21.06 ton h-1 for kalyan variety. shah and ishtiaq (2002) evaluated different verities in swat valley and reported highest yield for rio zorro (86.44 tons h-1) and minimum for contessa (23.57 tons h-1). the physical environment influence the yield h-1, but the gen pools are quite effective in this regard. the total yield is not affected by large size bulb as walla walla and ailsa craig two large bulb varieties studied for lower yield when planted in spring due to diseases, but are suitable for fall planting. so season is also effecting, some variety more then the other as reported in the above line by coolong et al., (2008). table vii. number of leaves bulb-1 varieties bannu local india swat quetta lsd value 26.7a 19.8c 25.8a 23.5b 1.852 means figure 7. effect of different varieties of onion in leaves\bulb table viii. yield kg h-1: varieties bannu local india swat quetta lsd value 13323.200b 10264.750c 17149.850a 13517.100b 6710 means kg h-1 08 vol 4 (2), july 2013; 01-09 conclusion and recommendation as a result of fore going discussion it is concluded that: quata variety take maximum days (24 days) in adaptation to the new surrounding after transplant. the maximum leaf length (25.50 inches) is recorded in quata variety while the swat variety produce maximum fresh biomass (259.6 gm), dry biomass (218.6 gm), economic weight (210.1 gm), bulb diameter (7.2 cm) and yield hector-1 (17149.850 kg ha-1). the bannu local variety has higher number of leaves (26.7) bulb-1. f r o m t h e a b o v e c o n c l u s i o n f o l l o w i n g recommendation may be offered for the guidance of future research worker; swat variety is suitable for higher yield and yield contributing parameters. the bannu local variety for more number of leaves bulb-1 and quata variety for longer leaf. so swat variety is suggested for cultivation in the local area of the bannu for higher yield and yield contributing parameters. references anisuzzaman m, m. ashrafuzzaman, m. r ismail, m. k. uddin and m. a. rahim. 2009. planting time and mulching effect on onion development and seed production.. department of horticulture, bangladesh agricultural university, mymensingh, bangladesh. benkeblia n., 2005. free-radical scavenging capacity and antioxidant properties of some selected onions (allium cepa l.) and garlic (allium sativum l.) extracts. graduate school of agriculture; hokkaido university. vol.48, n. 5: pp. 753-759. cheema k l, a saeed and m habib. 2003. bulb yield and other economic traits in eight onion cultivar under different ecological zones of punjab–pakistan. vegetable research institute, faisalabad-pakistan international journal of a g r i c u l t u r e & b i o l o g y. 0 5 – 2 – 1 8 8 – 1 9 0 coolong t. 2008. spring onion cultivar evaluation in central kentucky. department of horticulture, university of kentucky. fao (food and agricultural organization) 2002. fao production year book. rome 54:152-154. goussous s.j. and m.j. mohammad. 2009. comparative effect of two arbuscular mycorrhizae and n and p fertilizers on growth and nutrient uptake of onions. international journal of agriculture & biology 1560–8530. grubben, g.j.h. & o.a. denton 2004. plant resources of tropical africa 2. vegetables. prota foundation, wageningen; backhuys, leiden; cta, wageningen. halvorson .a. d., r.f. follett, m e. bartolo and frank c. 2002, nitrogen management nitrogen fertilizer use efficiency of furrow-irrigated onion a n d c o r n . a g r o n o m y j o u r n a l 9 4 : 4 4 2 4 4 9 hasegawa .a, h.yabuki,t. nabeura,h. fukui and t. iwata. 2001. evaluation of bulb shape and fresh w e i g h t o f d i f f e r e n t o n i o n cultivars.tech.bul.fac.agr.kagawa uni.,vol.71-77. hill a f. 1951. research fellow in economic botany harvard university. economic botany textbook of useful plants and plants products, ed.2nd, co mcgraw-hill book. iqbal, m. z., f. khan and s.a. khan, 2001. correlation and path coefficient analysis in potato. figure 8. effect of different varieties of onion in yield kg h-1 09 vol 4 (2), july 2013; 01-09 j. agric. res., 39: 301–306. ishwori p. g, b khatri and govinda p. paudel 2006. evaluation of different varieties of onion and their transplanting times for off-season production in mid hills of nepal. nepal agric. res. j. vol. 7, 21 islam. m.a., m.f.alam and a.k.m.r.islam. 2007. growth and yield response of onion (allium cepa l.) genotypes to different levels of fertilizers", bangladesh j. bot.36(1):33-38 jilani.m. s and a. ghafoor. 2003. screening of local varieties of onion for bulb formation. department of horticulture, faculty of agriculture, gomal university, dera ismail khan pakistan. international journal of agriculture & biology 1560-8530 jotangee l., 2010. effect of application methods of organic fertilizer on growth, soil chemical properties and microbial densities in organic bulb onion production. 124, n (3), p. 299-305 martínez.a..r., j. f. paz and j. l. andrés ares 2005. evaluation of local onion lines from northwest spain. spanish journal of agricultural research. 3(1), 9097. mohanty b. k. and a.. m. prusti. 2001. performance of common onion varieties in kharif seasons. journal of tropical agriculture 39 (2001): 21-23 nelson, j.l. 1986. onion variety tests at madras, oregon. in irrigated crops research in central oregon 1986. oregon agricultural experiment s t a t i o n . s p e c i a l r e p o r t 7 8 0 . p p . 11 1 8 . nourai. a. h 2006. effects of transplanting date, nitrogen nutrition and watering regime on yield, quality and storage of red onion. agricultural research corporation, shambat research station, khartoum north, sudan rajcumar.r. 1997. selection of onion cultivars for yield, early maturity and storage potential in mauritius. amas food and agricultural research council, reduit,mauritius ristimuki. l.m., i. papadopoulos, c. sannwel, and n.j. berhoyen. 2000. slow relese fertilizers on vegetables. acta hprt. 511: 125-131 schroeder. b.k., t.d. waters and franklin. 2010. evaluation of onion cultivars for resistance to enterobacter cloacae in storage. washington state university, mount vernon nwrec, mount vernon. 98273. shah, m.h.; ishtiaq, m 2002. evaluation of different exotic onion cultivars under the agro-climatic conditions of swat valley (nwfp agricultural univ., peshawar (pakistan) sarhad journal of agriculture shaheen.a.m., f.a. rizk, f.s. abdel-aal, h.a.m. habib. 2011. production of safe and economic vegetable. vegetable research department, national research centre, dokki, cairo (egypt). vol. 3. shaheen.a..m., a. mona, m. abdel-mouty, h. ali and f.a. rizk. 2007. natural and chemical phosphorus fertilizers as affected onion plant growth, bulbs yield and its some physical and chemical properties. australian journal of basic and applied sciences, 1(4): 519-524. steel.r.g.d and g.h. toreei, 1980. principles and procedure of statistics. mc graw hill book co. inc. new york. saliva a new horizon for estimating antioxidant profile of mobile phone user vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 23 r a d s j . b i o l . r e s . a p p l . s c i . 23 op en ac ces s f u l l l e n g t h a r t i c l e saliva a new horizon for estimating antioxidant profile of mobile phone user asra khan1, suad naheed2, *, mehtab alam3, saba salahuddin1 1department of biochemistry, jinnah university for women, karachi 74600, pakistan 2department of biotechnology, jinnah university for women, karachi 74600, pakistan 3department of biochemistry, dow international medical college, dow university of health sciences, ojha campus, karachi, pakistan a b s t r a c t this is the era of information and new technologies, among the brilliant inventions and advancement, global system of mobile phone communication (gsm) makes the life as approachable as one can think. it changes the social relation and social network of an individual. hence the mobile has two sides of a coin. advantages and disadvantages are associated with every feasibility or inventions. but some time blessings bring some harmful effects on the human body. mobile phone uses radio waves. some part of the radio wave is also absorbed in the body. the ear is the most exposed part of the body. the salivary gland is very close to the ear. hence, the affected part of the cellular phone radiations is a salivary gland. in the current study, we collected the unstimulated saliva sample from volunteers to estimate the antioxidant profile of the mobile phone user. the uric acids, catalase, c reactive protein (crp), reduced glutathione (gsh), and superoxide dismutase (sod) were included in the antioxidant profile. uric acid, gsh, sod was significantly decreased while crp and catalase were significantly increased. the result shows the increase the risk of inflammation and oxidative stress, which can predispose the cell phone user to a multitude of infectious & non-infectious oral diseases. keywords saliva, mobile phone, antioxidant profile, oxidative stress, oral disease. *address of correspondence suadnaheed@yahoo.com article info. received: august 13, 2018 accepted: january 8, 2019 cite this article: khan a, naheed s, alam m, salahuddin s. saliva a new horizon for estimating antioxidant profile of mobile phone user. rads j. biol. res. appl. sci. 2019; 10(1): 23-29. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n the 21st century is the century of technology; in this era, vast technologies are present around us that make human life as much comfortable as possible. among the brilliant inventions and advancements, cellular phones have made life as much approachable as one can think. mobile phones in present days have become a habit for each and every person. it shortens the distance; people get closer to each other. these devices use electromagnetic radiations, thereby producing possible harmful effects on the human body. advantages and disadvantages are associated with every facility or invention. global system of mobile communication (gsm) may play an important role in medical aspects or healthcare management; as they are associated with various ill effects1. mobile phones, being wireless communication devices use electromagnetic radiations of microwaves (radiofrequency ranges from 300 mhz to 300 ghz). technically, when you are speaking on phone, your sound wave is converted into a sine wave by using a transmitter. this transmitter uses the power of the highest 1 from shortest 0.75 watt, with 2w at hit the highest point. the antenna catches the signal from the transmitter and spreads it in all directions. the current of electric sine wave makes electromagnetic fields around the o r i g i n a l a r t i c l e saliva a new horizon for estimating antioxidant profile of mobile phone user vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 24 r a d s j . b i o l . r e s . a p p l . s c i . 24 transmitter. the electromagnetic radiations are formed by collapse and buildup of the fields due to the electric current moving continuously in back and forth motion. radio waves are launched by the antenna and are picked up by a receiver in the cell phone tower2. at present, there are 7 billion people of whom 6 billion are mobile users; surprisingly some of these mobile phone users are very young, i.e. less than 5-year-old3. the interaction between the radiations emitted and absorbed by the body can be calculated by sar (specific absorption rate), which may be defined as the velocity of energy at the point the radiation is absorbed by the human body. its unit is power by mass of tissue i.e. watt / kilogram4. there have been unpredictable increasing rates of using mobile phones in recent years. it is one of the fastest launched wireless communication devices among all. the mobile service is now playing a potential role in the advancement of education, health-care delivery and management5. but there is another side of the coin which shows its bad effects, such as when the mobile phone is vibrated, it poses harmful effects on the human body. scientists suggest that if there is long-term exposure of mobile phones, it may produce disturbances within the body6, 7. when humans are exposed to electromagnetic radiations emitted from mobile phones, the electromagnetic energy can pass through the skin. it is not necessary that the surface layer will always make a barrier against it. this radiation penetrates in the subcellular structures through barriers of the body. different epidemiological studies have shown that this radiation produces its adverse effect on the closed area of emission and leads to causing brain tumors, however, the mechanism is still unresolved3,8-9. almost 60% of the human body is made up of water. when the energy of the radio frequency range is absorbed in an aqueous medium, there is the production of chemically reactive species that can collaborate with solutes. this confirms the synthesis of hydrogen atoms and dioxin radical anion from the decomposition of water in the excited state. as all biomolecules exist in aqueous media, any change in the chemistry of water automatically changes the environment of biomolecules. for example, hydroxyl radical chemically interferes with deoxyribose nucleic acid (dna) and break its strands10. excessive synthesis of free oxygen radicals is known as an oxidative hassle, which may cause oxidation of protein, enzyme denaturation and peroxidation of lipids molecules beneath the plasma membrane internal structure thus resulting in structural and functional disturbances or abnormalities of the cell. free radicals damage dna and rna and increase chances of mutation11. a state that the interaction between magnetic surroundings and biological system entail the contribution oxidative stress particularly by the radical duo system. the use of low-level of the electromagnetic field for a minimal time period are not effective to alter the biological structures in human body12. in animal models, the electric field increased lipid peroxidation and change in antioxidant level13. however, the complaints of sleep disturbances, headache, dizziness, irritability, focused problems, and hypertension is also received from the nearby living persons to mobile base stations but the mechanism is still unknown14. salivary gland is situated near the ear. the major divisions of salivary gland area parotid gland, submandibular and sublingual glands from where human saliva is released. the major portion of saliva is constituted by water; saliva also has enzymes and proteins. when ear close with a mobile phone, parotid gland is nearest exposed site amongst all glands, hence it is the most affected part while using a mobile phone. the molecules present in saliva are gradually affected due to radiations emitted by mobile phones causing perturbation in their function, either they become reduced or increased in saliva15. there is a chance of activation and/or deactivation of hormones, enzymes or some functional proteins produced in that region. the contents present in salivary secretions other than hormones are salivary enzymes like salivary amylase, salivary lipase etc.; antioxidant enzymes like catalase, glutathione peroxidase, superoxide dismutase; and some proteins, e.g. crp (c-reactive protein) which may be significantly affected by mobile phone radiations. saliva also has antibacterial agents like iga, lysozyme, and lectoferrin16,17. the physiological action and scavenge capability of antioxidants can be impaired due to the presence of oxidative stress8,13. collectively inherent antioxidants in the immune system can be exclusively disturbed through amplification of oxidative stress caused by electromagnetic fields6. nature has created various biomolecules including glutathione peroxidase, saliva a new horizon for estimating antioxidant profile of mobile phone user vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 25 r a d s j . b i o l . r e s . a p p l . s c i . 25 superoxide dismutase, catalase, c-reactive protein, uric acid, ascorbic acid, etc. present in the human body as well as saliva18. these molecules are affected by heavy use of mobilephones9. among various causes of oxidative stress within the human body, some controversial studies have indicated electromagnetic radiation from cellular phones19. the aim of the current study was to find out the relationship between mobile phone use and oxidative markers in saliva. in pakistan, people are using a range of mobile phones from simple to smartphones. products of various companies are available in the market. the present study focused only on the duration of usage regardless of the quality of cellular phone to determine oxidative state/stress marker in saliva. m a t e r i a l s a n d m e t h o d s the study accomplished local population. volunteers were either in sex with different age groups. the saliva was collected by following the method of navazesh20. all participants were healthy and heavy user of global system of mobile communication (gsm). a questionnaire was filled with participants. the exclusion criteria of the study were, usage of drugs, smoking, pregnancy, any trauma, headaches, injuries, chronic systematic diseases, and the history of any chemotherapy and radiotherapy. the volunteer of non-mobile phone user was kept as control. the volunteers of the mobile phone user were further grouped into 3 groups (table 1). table 1. groups of mobile phone user. parameter group 1 group 2 group 3 calls per day (minutes) 5-10 5-10 10-25 time spent on each call (minutes) 5-15 30-60 5-15 s a m p l e c o l l e c t i o n saliva was collected by passive drool method without using any stimuli. the saliva collected in the sterile container between 9 am to 11 am. the unstimulated saliva was collected for at least 5 minutes after 60 seconds of intervals. after collection, all samples of saliva were centrifuged at 3500 rpm for 15 minutes to clear all unwanted substance. the supernatant was separated in eppendorf and stored at -20°c until tests were performed. b i o c h e m i c a l a n a l y s i s estimation of uric acid the assayed was based on enzymatic colorimetric method. the commercially prepared kit was supplied by randox in which uricase is responsible for the conversion of uric acid into allantoin and hydrogen peroxide, then under the catalytic influence of peroxidase, oxidizes 3,5dichloro-2-hydroxybenzene sulfonic acid and 4aminophenazone to form a red-violet quinoneimine derivative observe at 520 nm. estimation of c-reactive protein the assay based on the principle of agglutination. the commercially prepared kit was supplied by sbio in which semi-quantitative method was used by preparing the serial dilution of each sample. the test specimen was mix with sbio crp latex reagent to for the continuation of the reaction. if there was agglutination seen the crp is > 0.6 mg/ dl, then performed serial dilution and calculated the results. if there is no agglutination after mixing so the concentration of crp is less than 0.6 mg/dl. estimation of catalase in 0.1 ml of supernatant, the reaction mixture was made by adding up 1 ml of 0.01 m of phosphate buffer (ph 7.0), 0.4ml of 2 m h2o2. after the addition of 2ml dichromateacetic acid reagent (5% potassium dichromate and glacial acetic acid were mixed in a ratio of 1:3). checked it at 610 nm and calculate the values by using standard curve (0.2 molar-2 molars). the unit expressed as gm/dl 21. estimation of reduced glutathione (gsh) the 2% trichloro-acetic acid added in 0.5 ml of supernatant. at 3000rpm for 15minutes the mixture was centrifuged. then added 0.5 ml of ellman’s reagent and 0.2m phosphate buffer (ph 8.0). calculate percent inhibition after checking 412nm at absorbance22. estimation of superoxide dismutase (sod) in 0.1 ml of supernatant, 0.15 ml of ice chilled chloroform and 0.75 ml of ethanol mixed together. at 3000 rpm for 15 saliva a new horizon for estimating antioxidant profile of mobile phone user vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 26 r a d s j . b i o l . r e s . a p p l . s c i . 26 minutes place the tube for centrifugation. added 1.0 ml of 0.1 m of carbonate-bicarbonate buffer (ph 10.2) and 0.5ml of edta (0.6mm). the procedure was then continued after the addition of epinephrine (1.8 mm). at 480nm the readings were measured for 3 minutes to calculate percent inhibition23. s t a t i s t i c a l a n a l y s i s statistical analysis was done on the graph pad (www.graphpad.com), which is a freely available resource. the two tail p value was used to calculate significance (p< 0.05). r e s u l t s a n d d i s c u s s i o n in our study uric acid, reduced glutathione (gsh) and superoxide dismutase’s (sod), catalase and crp were measured. among these parameters uric acid, gsh, sod was significantly decreased than control as shown in figure 1. previous studies show that a significant decrease in antioxidant profile increases the risk of inflammatory diseases of the oral cavity such as gingivitis, periodontitis, and mucositis in individuals utilizing cell phones for longer durations24. reduced glutathione (gsh), superoxide dismutase (sod) and catalase contribute towards total antioxidant capacity (tac), that is an integral antioxidant defense mechanism that counteracts with reactive oxygen species (ros). saliva is not invasively collected, non-intrusive manner and acts as a first line scavenger of free radical species resulting in minimizing oxidative stress8. the previous study revealed the salivary uric acid is considered to be the most important antioxidant molecule in saliva as it contributes to almost 70% of the salivary tac. all the three groups in this study demonstrated significantly lower values of uric acid in a contrast to that of the control group, while group 2 shows a steep decline in salivary uric acid levels. thus, the oxidative stress of long-term cell phone usage results in a reduction of salivary uric acid and this directly possesses effector salivary antioxidants19. it was stated the significantly reduced capacity of the enzymatic profile of antioxidant with a concurrent rise in lipid peroxidation when exposed to even extremely low frequencies of electromagnetic radiation in albino wistar rats13. histochemical changing has been observed in rat’s paranoid gland when exposed from 2100 mhz of radiation that was emitted from the 3g mobile phone. these histopathological changes were observed in acinar cells, interstitial space, ductal system, vascular system25. these changes influence the rate of antioxidant level in saliva were proved19. so this it can be incurred that saliva is an indicator of oxidative stress when using mobile phones25, have demonstrated the antioxidant body clock in rats at the result of electromagnetic exposure26. however, on the other hand, the antioxidant, the short time contact of electromagnetic emission from mobile devices produce none of the significant effects13. it was reported experimental data which shows the short-term exposure of electromagnetic radiations may have a deleterious influence on blood serotonin and glutamate levels27. the reason for significantly lowered values of uric acid, gsh and sod was the alteration on the molecular level, the mobile phone emission of electromagnetic radiations may effect on the genetic expression which influences the oxidative stress28. the free radical’s activity detoxifies by the action of sod and gsh, the product of sod is hydrogen superoxide may inhibit sod activity. in our finding the gsh also decreased, both enzymes (gsh, sod) show a decrease in their activity may induce noxious metabolic outcome9. figure 1. estimation of uric acid, gsh, sod. results are expressed as mean ± sem (standard error of the mean). the value of n for control, group 1, 2 and 3 are 26, 38, 27, and 20 respectively. every single mean was run in triplicate. p (< 0.05) was calculated by two tail analysis. *significant when compared with control. in this study, catalase and crp was measured, in figure 2. both were significantly increased than control which saliva a new horizon for estimating antioxidant profile of mobile phone user vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 27 r a d s j . b i o l . r e s . a p p l . s c i . 27 shows that there is an activation induced by electromagnetic radiations. in accordance the creation of ros was scavenged through antioxidants, hence the catalase increases after the exposure of electromagnetic field29. in our study, only c reactive protein (crp) was determined through a semi-quantitative method. the relation between the mobile phone user of all three groups and crp was significantly increased than that of the control group. the levels of crp are in a linear relationship with the duration of cell phone usage. in addition, the physical activity will increase the value of crp and other inflammatory markers26. it was showed that the erythro-leukemic cell has increased their catalase, cytochrome p450, and nitric oxide synthase action when it is exposed to extremely low-frequency electromagnetic field (elf-emf)30. the findings of elevated salivary crp levels in this study are congruent with previous studies indicating the presence of gingival and periodontal inflammation in the absence of plaque and calculus31-33. our findings suggested that the long-term exposure of mobile phone generate oxidative stress, the reactive oxygen species are formed and the body increases the synthesis of catalase to convert the metabolic product of hydrogen peroxide in water19. the increment in c-reactive protein shows an inflammation response, also associated with oxidative stress. figure 2. estimation of crp and catalase. results are expressed as mean ± sem (standard error of the mean). the value of n for control, group 1, 2 and 3 are 26, 38, 27, and 20. every single mean was run in triplicate. p (< 0.05) was calculated by two tail analysis. *significant when compared with control. studies of antioxidants that comprise of the salivary tac are of utmost importance as a significant proportion of oral diseases have established associations with salivary tac18. imbalances between ros and tac in saliva perform a crucial role in causing of diseases and growth of infectious or non-infectious diseases of oral cavity3,28. the two most common non-communicable dental diseases namely dental caries and periodontitis are linked with alterations in tac of saliva34, it moderate and severe periodontitis has been associated with low levels of tac30. findings of this study implicate that cell phone usage for longer duration leads to significantly reduced levels of salivary antioxidants leading to a reduction in cumulative salivary tac and this is in congruence with previous studies16,30. reduced salivary tac increases the susceptibility of the oral cavity to a wide range of oral diseases from periodontitis to premalignant lesions and oral cancer35. c o n c l u s i o n the study provides the evidence implicating mobile phone usage and reduction in the antioxidant profile in human beings, as far as the effects of electromagnetic radiations on the modification of antioxidant markers. the undesirable things of electromagnetic radiations are existed in all three groups. these groups were divided according to the calls per day and the minutes spent on each call. although, this study shows clear evidence of electromagnetic radiation and the significantly decreased levels of salivary sod, gsh, uric acid activity while significantly increased levels of catalase and crp. we, therefore, conclude that the influence of radiation emitted by the mobile phone decrease the salivary levels of sod, gsh and uric acid. the increased activity of crp &catalase may implicate the risk of inflammation and oral pathologies from unabated actions of free radicals. r e f e r e n c e s 1. mohammadzadeh n, safdari r, rahimi a. cancer care management through a mobile phone health approach: key considerations. asian pac j cancer prev. 2013; 14(9):4961-4. 2. ahmaditameh a, ahmadi r, gohari a. long-term exposure to cell phone radiation and stress. eels. 2014; 43-5. 3. khadra kma, khalil am, samak ma, aljaberi a. antioxidant profile of saliva among young men using mobile phones. jjbs. 2014; 7(4):275-80. saliva a new horizon for estimating antioxidant profile of mobile phone user vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 28 r a d s j . b i o l . r e s . a p p l . s c i . 28 4. munshi a. cellular phones: to talk or not to talk. j can res ther. 2011; 7(4):476-7. 5. ahmed t, lucas h, khan as, islam r, bhuiya a, 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arbabi-kalati f, salimi s, vaziry-rabiee a, noraeei m. effect of mobile phone usage time on the total antioxidant capacity of saliva and salivary immunoglobulin a. iran j public health. 2014; 43(4):480-4. 17. mortavazi s, habib a, ganj-karami a, samimi-doost r, pour-abedi a, babaie a. alterations in tsh and thyroid hormones following mobile phone use. oman med j. 2009; 24(4):274-8. 18. battino m, ferreiro m, gallardo i, newman h, bullon p. the antioxidant capacity of saliva. j clin periodontol. 2002; 29(3):189-94. 19. abu khadra km, khalil am, abu samak m, aljaberi a. evaluation of selected biochemical parameters in the saliva of young males using mobile phones. electromagn biol med. 2015; 34(1):72-6. 20. navazesh m. methods for collecting saliva. annals of the new york academy of sciences. 1993; 694:72-7. 21. sinha a. k. colorimetric assay of catalase. anal biochem. 1972; 47(2):389-94. 22. ellman g.l. tissue sulfhydryl groups. arch biochem and biophys. 1959; 82(1): 70-7. 23. misra h.p., fridovich i. the role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. journal of bio chem. 1972; 247(10):3170-5. 24. dodwad r, betigeri av, preeti b. estimation of total antioxidant capacity levels in saliva of caries-free and caries-active children. contemp clin dent. 2011; 2(1):17-20. 25. aydogan f, unlu i̇, aydin e, yumusak n, devrim e, samim ee, et al. the effect of 2100 mhz radiofrequency radiation of a 3g mobile phone on the parotid gland of rats.american journal of otolaryngology. 2015; 36(1):39-46. 26. cao h, qin f, liu x, wang j, cao y, tong j, et al. circadian rhythmicity of antioxidant markers in rats exposed to 1.8 ghz radiofrequency fields. int j environ res public health. 2015; 12(2):2071-87. 27. eris a, kiziltan h, meral i, genc h, trabzon m, seyithanoglu h, et al. effect of short-term 900 mhz low-level electromagnetic radiation exposure on blood serotonin and glutamate levels. bratisl lek listy. 2014; 116(2):101-3. 28. khalil am, abu khadra km, aljaberi am, gagaa mh, issa hs.assessment of oxidant/antioxidant status in saliva a new horizon for estimating antioxidant profile of mobile phone user vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 29 r a d s j . b i o l . r e s . a p p l . s c i . 29 the saliva of cell phone users. electromagn biol med. 2014; 33(2) :92-7. 29. patruno a, tabrez s, pesce m, shakil s, kamal ma, reale m. effects of extremely low-frequency electromagnetic field (elf-emf) on catalase, cytochrome p450 and nitric oxide synthase in erythroleukemic cells. life sci. 2015; 121:117-23. 30. canakci v, yildirim a, canakci cf, eltas a, cicek y, canakci h. total antioxidant capacity and antioxidant enzymes in serum, saliva, and gingival crevicular fluid of preeclamptic women with and without periodontal disease. j periodontol. 2007; 78(8):1602-11. 31. vogel gl, carey cm, chow lc, tatevossian a. micro-analysis of plaque fluid from single-site fasted plaque. j dent res. 1990;69(6):1316-23. 32. rankine can, moreno ec, vogel gl, margolis hc. micro-analytical determination of ph, calcium, and phosphate in plaque fluid. j dent res. 1985; 64(11):1275-80. 33. silva-boghossian cm, orrico sr, gonçalves d, correa fo, colombo ap. microbiological changes after periodontal therapy in diabetic patients with inadequate metabolic control. braz oral res. 2018; 28:1-9. 34. hegde a, rai k, padmanabhan v. total antioxidant capacity of saliva and its relation with early childhood caries and rampant caries. j clin pediatr dent. 2009; 33(3):231-4. 35. kolanjiappan k, ramachandran c, manoharan s. biochemical changes in tumor tissues of oral cancer patients. clin biochem. 2003; 36(1): 61-5. pesticide exposure in female cotton pickers vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 76 op e n ac c e s s f u l l l e n g t h a r t i c l e assessment of pesticide exposure in female cotton pickers of district jamshoro, sindh muhammad abdullah avais1,*, hamida narijo2, shagufta kamal3, mike parker4, fareeha asghar ali5, umair aslam6 1rural education & economic development society (reeds), pakistan. 2department of sociology, university of sindh, jamshoro, pakistan. 3department of biochemistry, gc university, faisalabad, pakistan. 4freelance biostatistician, united kingdom. 5education department punjab, university of agriculture, faisalabad, pakistan. 6better cotton initiative (bci), pakistan. a b s t r a c t background: the butyrylcholinesterase (bche) is widely known chemical biomarkers to identify pesticide exposure. the presence of cholinesterase in the serum of female cotton pickers indicate the exposure to organophosphate and carbamate pesticides. assessment of the cholinesterase levels in agricultural workers provides the most important indicator for the initiation of medical treatment. objectives: the present study was designed to compare the activity of serum bche among female cotton pickers (exposed group) and non-cotton pickers (non-exposed group). methodology: the level of serum bche of 100 female cotton pickers was compared with that of 100 non-cotton picker females. a self-designed questionnaire was used for the collection of primary data after a review of related studies. the r computer program (a language and environment for statistical computing and graphics) was used for the analysis of primary data. results: the level of bche was recorded, and the means for the two groups were compared. the exposed mean was 5975.90 ± 541.85u/l, and the non-exposed mean was 6981.76 ± 782.92u/l and the difference was found to be statistically significant (p < 0.001). the result of the current study confirmed the negative association between decreased serum bche and pesticide exposure. it was also found that 76% of female cotton pickers did not wash their hands before eating during the cotton picking. similarly, 81% of pickers did not use gloves for their safety. conclusion: the inappropriate use of personal protective equipment (ppes) and the lack of awareness regarding pesticides' adverse the impact on female cotton pickers' health are significant factors of pesticide exposure. keywords butyrylcholinesterase, bche, female, occupational exposure, pesticides, sindh. *address of correspondence abdullahawais77@yahoo.com article info. received: february 22, 2021 accepted: july 01, 2022 cite this article avais ma, narijo h, kamal s, parker m, ali fa, aslam u. assessment of pesticide exposure in female cotton pickers of district jamshoro, sindh. 2022; 13(1):76-82. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n pesticides are commonly used to kill almost every crop's harmful pest1.it was estimated that all over the world, around 43% of the agriculture labor force is women2. in sub-sharan africa, 80% of women workers belonged to the agricultural sector compared to tanzania, where 70% of women were involved in the horticultural sector3. in developing countries such as pakistan, women play a vital role in the development of a nation, and their share is 67% in forestry, fishing, and agriculture4,5. o r i g i n a l a r t i c l e pesticide exposure in female cotton pickers vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 77 it has been gauged that 15,000 chemicals and 35,000 chemical formulations have been prepared and used in the agricultural sector since 19456. although chemical pesticides are a significant controlling mechanism over pests in the agricultural sector, they are also a threat to the environment and living organisms7. the cotton crop is more prone to pest attacks; therefore, due to a lack of knowledge and a lack of awareness regarding integrated pest management techniques (ipm), pakistani farmers widely use pesticides to control cotton crop pests. this intensive use of pesticides leads to pesticide exposure of female cotton pickers during the cotton harvesting, as well as to agricultural workers. pesticides impose two types of hazards (acute and chronic) on human health. the acute hazard results in headache, fatigue, rashes, feelings of weakness, vomiting, nausea, impaired vision, excessive sweating, panic attacks, tremors, cramps, skin discomfort, feelings of weakness, chronic bronchitis, and dizziness, along with coma and death in severe cases8,9. usually, acute poisoning is a result of accidental or intentional exposure to a high dose of pesticide. exposure to pesticides even in small quantities for longer intervals leads to acute illness. a study into the hazards of pesticides on farmworkers in colorado state identified that pesticide exposure is directly linked with different respiratory problems, i.e., wheeze, cough, allergy, and organic dust toxic syndrome (odts) among non-smokers10. the shared statistics may vary among other less developed countries, especially in pakistan, due to underreporting, lack of awareness, inaccessibility of health facilities, misdiagnosis and unavailability of the data management system, and non-availability of strict regulations for using or handling of pesticides11-13. in developing countries like pakistan, it is always challenging to assess occupational pesticide exposure of female cotton pickers. therefore, it is the easiest way to estimate the extent of exposure through biological monitoring. thus, the most common test for this purpose is the quantification of butyrylcholinesterase (bche) activity due to its inhabitation by carbamate (cm) and organophosphorus (of) pesticides14-16. both (pesticide exposure and level of bche) are inversely related to each other17. the statistical results of a research study showed that 80% of pesticides are used on cotton crops and the remainder on sugarcane, fruits, paddy, and vegetables in pakistan12. women are lynching excel in traditional sindhi families, and their sickness leads to crisis and chances of social disintegration. therefore, the present research aimed to determine the level of bche in the serum of female cotton pickers in district jamshoro, sindh, to evaluate the correlation between the level of bche and pesticide exposure. variables, such as the knowledge of female cotton pickers regarding symptoms of exposure, use of ppes during the cotton picking, socioeconomic background of pickers, and health-related issues of cotton pickers, were documented. it is a pioneer study on female cotton pickers in district jamshoro, and there is an urgent need for such studies on a mass level in developing countries such as pakistan. this data may be used to better understand the extent of pesticide exposure among female cotton pickers. m a t e r i a l s a n d m e t h o d s the ongoing study examined the lethal effects of frequently used pesticides on the female cotton pickers' health in district jamshoro. it is primarily a chemical biomarker study based on female cotton pickers' health concerns. one district of sindh-pakistan; jamshoro was selected for the study. the district consists of the indus plain's hilly and plain (cultivated) areas. jamshoro district is spread over 11,517 square kilometers and is administratively subdivided into 4 talukas (kotri, sehwan sharif, thana bulla khan, and manjhand). agriculture is the district's primary income source involving cotton, rice, sugarcane, maize, bajra, wheat, barley, and pulses. five villages of district jamshoro (bhutto, lashari, sobho hajana, sobho lashari & umeed ali), taluka manjand, were selected randomly from 1st october 2019 to 30th november 2019. an attempt was made to select respondents from the same socioeconomic and demographics. after the selection, respondents were segregated into two groups: exposed and non-exposed. both groups contained 100 respondents. thus, face-to-face interviews of 200 females (female cotton pickers-100 & non-cotton pickers-100) were conducted to complete a structured questionnaire. this can be regarded as an expensive method for data collection, but it provided the rich and complex information required, along with the highest response rate. the pesticide exposure in female cotton pickers vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 78 questionnaire was developed after the review of the literature and was pre-tested before the collection of data. the respondents were selected through a purposive sampling technique, and written consent was obtained from participants after sharing the aims and objectives of the study in the local language (sindhi). participation was voluntary, and all the respondents were informed of their right to withdraw their consent at any time. analyses have been performed using the computer program r18. sample collection in addition to the face-to-face interview, 3ml of blood was collected from all participants (200) through venipuncture to determine butyrylcholinesterase. the blood samples were preserved in specialized glass tubes without any coagulant. each tube was assigned a unique number for identification for confidentiality. the serum was obtained from blood and stored at -20ºc for analysis. working principle of bche serum bche is a plasma cholinesterase, a serine hydrolase in all mammals, and can be detected in the liver and plasma19-21. bche is the front defense line against toxic agents/compounds reaching the bloodstream. thus, the exposure to pesticides, especially (organophosphate) or nerve agents, can be assessed by measuring the decreased level of bche22. there are various methods of detecting the pesticide residue, including mass spectrometer (ms)23, high-performance liquid chromatography (hplc)24, colorimetric assay25, and liquid chromatography (lc)26 and biosensors27. cholinesterase hydrolyses butyryl thiocholine into thiocholine and butyric acid. further, thiocholine decreases yellow potassium hexacyanoferrate (r+) to colorless potassium hexacyanoferrate. the decrease of absorbance was measured at 405nm on merck micro lab-300. the said method is already used in a study on female cotton workers in pakistan with the help of merck micro lab 20017. analysis procedure the modified spectrophotometric method28 was adopted to determine ache activity based on the rate of hydrolysis of acetylthiocholine by ache. the yellow color liberated by the reaction of thiocholine with dtnb (5,5′ dithio-2nitrobenzoic acid) was quantified spectrophotometrically at λmax 412nm. the variation in optical density (od)/min for four minutes is directly proportional to ache activity that is determined by the following formula: ache activity (u) = ∆a×132.35 where, δa is the mean absorbance. considering that 1 ku.l−1 = 1000 u.l−1, ache activity was expressed as u.l−1, 25, 29. a commercial laboratory kit (bche a8k7p8) was applied to measure the serum bche level. the change in the intensity of the yellow-colored compound (hexacyanoferrate-iii) to colorless (hexacyanoferrate-ii) was monitored spectrophotometrically at λmax 412nm. inclusion and exclusion criterion only married female cotton pickers having more than 3 years of cotton-picking experience and 100 married females from the same area who were not involved in cotton picking (as a control group) were interviewed. all the respondents were thoroughly briefed regarding the aims and objectives of the research. they were informed of their right to withdraw from the study without giving any reason. r e s u l t s the percentage of adopted precautionary measures by female cotton pickers during the picking and the health status of female cotton pickers is indicated in table 1. only 26% of females washed their hands before eating during cotton picking or after cotton picking. the data shows that 51% of cotton pickers take a bath before engaging in domestic work. only 23% cover their heads, and 20% cover their faces or use a mask during cotton picking. only 16% of females cover their feet, and 19% use gloves for their protection. out of all, 51% of respondents were lactating mothers, and 15% of respondents were pregnant. the level of bche was recorded, and the means for the two groups were compared. the exposed mean was 5975.90 ± 541.85u/l, and the non-exposed mean was 6981.76 ± 782.92u/l, and the difference was found to be statistically significant (p < 0.001). pesticide exposure in female cotton pickers vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 79 table 1. precautionary measures taken by female cotton pickers during the picking of cotton and their health status. precautions percentage wash hands during or after picking before eating anything? 26% taking a bath before involving in domestic work 51% cover head 23% use mask/cover face 20% use gloves/cover hands 19% used closed shoes/ covered feet 16% lactating mothers 49% pregnancy 15% table 2. summaries of continuous variables by exposure group. variable exposure group mean difference n e p-value e = exposed (n = 100) n = non-exposed (n = 100) standard error 95 % confidence limits mean <median> sd (iqr) [range] mean <median> sd (iqr) [range] lower upper age of 38.06 8.360 35.95 9.460 -2.110 -4.579 0.338 0.095 respondent <40.00> (30 to 45) <36.50> (28 to 45) (1.254) (years) n = 100 [20 to 55] n = 100 [19 to 55] height 149.44 7.740 155.33 7.380 5.889 3.765 7.915 < 0.001 (cm) <149.35> (143.3 to 155.4) <152.40> (152.4 to 161.5) (1.0593) n = 100 [134.1 to 176.8] n = 100 [125.3 to 167.6] weight (kg) 52.22 12.390 54.82 7.190 2.600 -0.199 5.341 0.075 <50.00> (43.0 to 58.0) <53.00> (50.0 to 59.0) (1.4151) n = 100 [30.0 to 91.0] n = 100 [39.0 to 78.0] standard deviations are based on within-group data (not on pooled estimates). standard errors of differences between means and confidence limits have been obtained using 9999 bootstrap samples. p-values for the comparison of the group means have been obtained using 10000 permutation samples. table 3. number of abortions by exposure group. presence of abortion exposure groups overall exposed non-exposed count % count % count % none 43 43.0 89 89.0 132 66.0 1 or 2 57 57.0 11 11.0 68 34.0 overall 100 100 100 100 200 100 fisher's exact test p-value for association between presence of abortion and exposure group is p < 0.001. the quantitative variables have been summarized by the exposure group, using numerical statistical summaries and graphical displays. the means for the two exposure groups have also been compared. it has been found that the difference between the means for the groups of the heights of the respondents (non-exposed – exposed) is 5.889cm (95% cl 3.765 to 7.915), and this difference is statistically significant (p < 0.001) (table 2). the differences between pesticide exposure in female cotton pickers vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 80 the group mean for age and weight have not been found to be statistically significant. abortion counts have been tabulated and compared by the exposure group (table 3). the difference between the percentages of mothers having any abortions for the two groups (exposed – non-exposed) is 46% (95% cl: 33.6 to 56.4), and the difference is statistically significant p < 0.001). d i s c u s s i o n various studies suggest that cotton pickers and agriculture workers are prone to pesticide exposures, even though they are not directly involved in the use of pesticides30-32. the serum bche activity as a biological indicator of pesticide exposure was evaluated in both exposed and non-exposed groups of females33, 34. serum bche is a wellrecognized and reliable indicator of pesticide exposure35, 36. decreased levels of (bche) in the exposed group compared to the non-exposed group in this study confirm the adverse impacts of pesticide exposure in female cotton pickers. various research studies propounded the decrease in cholinesterase levels due to exposure to a pesticide in the human population36-41 and strengthened the ongoing research findings. the primary cause of pesticide exposure among female cotton pickers is the lack of, or less use of personal protective equipment's (ppes) due to the unavailability of guidelines for cotton pickers from the provincial as well as federal government in pakistan. the study revealed that 15% of pregnant women were involved in cotton picking, and only 26% pickers were washing their hands before eating. the results suggest that the risk of pesticide toxicity is directly linked to the extent of exposure, inappropriate use of ppes, and exposure to pesticide residue on cotton crops. the misuse of pesticides on the cotton crop is a severe threat to the environment and farmworkers of the cotton crop. in pakistan, cotton picking is a role of women; therefore, they are more prone to pesticide exposure. they can be exposed to pesticides during picking through the dislodging of pesticide dust from the boll stem, and leave settled and fluffy bolls on their naked parts of the body, i.e., arms, hands, face, or through inhalation42. in jamshoro, women are involved in cotton picking for 2-3 months. therefore, continuous and prolonged pesticide exposure through different entry points (eyes, nose, skin, and mouth) poses greater health-related issues. most female cotton pickers belong to needy families, and they are forced to work even in pregnancy to cope with financial upheavals without knowing the consequences of pesticide exposure on their bodies and the fetus. it was estimated that approximately 200,000 people out of 1-5 million cases around the world die every year due to pesticides among agricultural workers, and the majority of exposed people belonged to developing countries43, 44. policymakers, non-governmental organizations, and civil societies must provide training and technical support along with ppe for female cotton pickers to protect them from hazardous pesticide residue. c o n c l u s i o n lack of awareness, illiteracy, careless attitudes, traditional cotton picking methods, and the unavailability of ppes are the leading causes of pesticide exposure among female cotton pickers. the result of this study suggests that the bche biomarker helps study the adverse effects of pesticides on female cotton pickers. moreover, the results may be generalized to other crops due to the increasing involvement of females in different phases, from sowing to picking. e t h i c a l a p p r o v a l ethical approval for the study was obtained from the research & education development department of rural education & economic development society (reeds), pakistan. c o n f l i c t o f i n t e r e s t the authors declare no conflict of interest. f u n d i n g s o u r c e rural education & economic development society (reeds) financially supported the study. a c k n o w l e d g e m e n t s the author is grateful to the concerned institution for providing facilities to conduct this study. moreover, the authors are thankful to cotton pickers’ women for their voluntary participation in the study. pesticide exposure in female cotton pickers vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 81 l i s t o f a b b r e v i a t i o n s ipm integrated pest management odts organic dust toxic syndrome ppes personal protective equipment’s serum bche serum butyrylcholinesterase r e f e r e n c e s 1. adu p, forkuo ek, issah a, asumadu io, cadmansackey e, quarshie aa, et al. high incidence of moderately reduced renal function and lead bioaccumulation in agricultural workers in 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8573 r a d s j . b i o l . r e s . a p p l . s c i . 82 chromatography with ultraviolet detection. j chrom. 1992; 607(1):37-45. 25. ellman gl, courtney kd, andres jr v, featherstone rm. a new and rapid colorimetric determination of acetylcholinesterase activity. biochem pharmacol. 1961; 7(2):88-95. 26. hogendoorn e, van zoonen p. recent and future developments of liquid chromatography in pesticide trace analysis. journal of chromatography. 2000; 892(1-2):435-53. 27. xu yl, li fy, ndikuryayo f, yang wc, wang hm. cholinesterases and engineered mutants for the detection of organophosphorus pesticide residues. sensors. 2018; 18:4281-9. 28. ellman gl, courtney kd, andres jr v, featherstone rm. a new and rapid colorimetric determination of acetylcholinesterase activity. biochem pharmacol. 1961; 7(2):88-95. 29. harlin ks, ross pf. enzymatic-spectrophotometric method for determination of cholinesterase activity in whole blood: collaborative study. j assoc official analy chem (usa). 1990; 1-8. 30. bradman a, salvatore al, boeniger m, castorina r, snyder j, barr db, et al. community-based intervention to reduce pesticide exposure to farmworkers and potential take-home exposure to their families. 2009; 19(1):79-89. 31. coronado gd, thompson b, strong l, griffith wc, islas i. agricultural task and exposure to organophosphate pesticides among farmworkers. environmental health perspectives. 2004; 112(2):1427. 32. bakhsh k, ahmad n, kamran ma, hassan s, abbas q, saeed r, et al. occupational hazards and health cost of women cotton pickers in pakistani punjab. bmc public health. 2016; 16(1):1-1. 33. ghafouri-khosrowshahi a, ranjbar a, mousavi l, niliahmadabadi h, ghaffari f, zeinvand-lorestani h, et al. chronic exposure to organophosphate pesticides as an important challenge in promoting reproductive health: a comparative study. j educat health prom. 2019; 8-13. 34. nili-ahmadabadi a, pourkhalili n, fouladdel s, pakzad m, mostafalou s, hassani s, et al. on the biochemical and 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338(8761):223-7. 44. bunggush ra, anwar t, chaudhry g, noor n, qazi a, elsebae a, et al. public health impact of pesticide used in agricultural. pak j bio sci. 1992; 3(11):25-33. prevalence and antibiotic susceptibility profiling of mdr p. aeruginosa vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 1 op e n ac c e s s f u l l l e n g t h a r t i c l e prevalence and antibiotic susceptibility profiling of mdr pseudomonas aeruginosa from uti patients of southern punjab, pakistan iqra arooj1, ⃰, aysha asghar1, mehvish javed1, amina elahi2, asghar javaid3 1department of microbiology & molecular genetics, the women university, multan, pakistan. 2university institute of medical laboratory technology, university of lahore, lahore, pakistan. 3department of pathology, nishtar medical university, multan, pakistan. a b s t r a c t background: urinary tract infections (utis) are of major concern in health care settings. an increasing percentage of organisms causing nosocomial infections have now become multidrug resistant. hence, constant surveillance of etiology and antimicrobial susceptibility of uropathogens including pseudomonas aeruginosa is compulsory. objective: main purpose of this study was to assess the prevalence rate of various pathogens, and to evaluate the multidrug resistance pattern of pseudomonas aeruginosa in uti patients of southern punjab, pakistan. methodology: a cross-sectional study was performed to obtain quantitative data of uti patients, for which one hundred and fifty (150) urine samples were obtained from uti patients admitted to nishtar hospital, multan. samples were processed for culture, bacterial isolation and identification. antibiotic sensitivity of p. aeruginosa isolates was determined in agreement with clinical laboratory standard institute guidelines. results: prevalence of uti was greater in females (59%) when compared to males (41%). among 150 urine samples, uropathogens isolated were escherichia coli-50(33.33%), followed by klebsiella pneumoniae-27(18%), proteus spp.-15(10), staphylococcus saprophyticus-13(8.66%), staphylococcus aureus-11(7.33%), pseudomonas aeruginosa-10(6.66%), enterobacter spp.-9(6%), candida albicans-8(5.33%) and citrobacter spp.-7(4.66%). antibiotic sensitivity profiling of pseudomonas aeruginosa revealed highest sensitivity to cefixime (80%), followed by gentamicin (70%), piperacillin/tazobactam (60%) and nitrofurantoin (40%). conversely, maximum resistance was observed against amikacin (80%), followed by ciprofloxacin (60%), imipenem (50%) and amoxicillin (50%). conclusion: knowledge of dynamic etiology and ever-changing drug resistance patterns is essential for appropriate management and treatment of utis necessitating their exploration on a regular basis. keywords antibiotic sensitivity, multidrug resistance, nosocomial infections, pseudomonas aeruginosa, susceptibility, urinary tract infection. *address of correspondence iqra.6051@wum.edu.pk article info. received: april 04, 2021 accepted: february 04, 2022 cite this article arooj i, asghar a, javed m, elahi a, javaid a. prevalence and antibiotic susceptibility profiling of mdr pseudomonas aeruginosa from uti patients of southern punjab, pakistan. 2022; 13(1):1-9. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. o r i g i n a l a r t i c l e prevalence and antibiotic susceptibility profiling of mdr p. aeruginosa vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 2 i n t r o d u c t i o n urinary tract infections (utis) have been reported as one of the most predominant microbial infections globally, that can be diagnosed both in the outdoor and in the clinics1. these infections are generally categorized into hospital and community-acquired infections2. patients develop a high risk of acquiring hospital-borne utis when admitted to hospital for 48hrs or longer3. calculated percentage of utis that are acquired from hospitals is 35% in admitted patients4. in uti patients, microorganisms can affect any part of urinary system by weakening natural body defense mechanisms which can be diagnosed depending on the infection site as pyelonephritis (inflammation of kidneys), cystitis (inflammation of bladder), and urethritis (inflammation of urethra), respectively5. on average, 95% of uti cases are reported because of the presence of single microbial species, while more than one specie is usually present in urine culture due to contamination6. out of all, 80-85% of the uti cases are attributed towards gram negative bacteria including escherichia coli, klebsiella pneumoniae, enterobacter spp. and pseudomonas aeruginosa, however the most frequently encountered pathogen among these is e. coli7. specifically, among catheterized patients, the major causative agent is p. aeruginosa, a gram-negative pathogen considered as superbug in numerous human infections8. its opportunistic behavior and ubiquitous existence enable it to become associated with pathogenesis particularly in immunocompromised patients9. p. aeruginosa is held responsible for 11% of nosocomial infections and it produces a multitude of virulence factors that enable its attachment to host cell surface, facilitating entry into the host tissues and suppression of host immunity10. in addition to virulence factors, p. aeruginosa produces biofilms and chemical signals for quorum sensing on the surface of urinary catheters leading towards the development of infections11. antimicrobial resistance is one of the most significant and challenging issues emerging with regard to hospital as well as community-acquired utis12. continuously rising drug resistance rate is creating complications with reference to treatment, morbidity rate, duration of stay in hospital, etc.13. rate of utis varies between developing and developed countries depending on random usage of antibiotics. antibiotic resistance against common bacteria has become a huge trial for clinicians in our part of world in terms of devising therapeutic strategies against utis14. the leading cause of increasing resistance rate is mutation and resistance gene transfer among pathogens. plasmids and transposons are the carriers of resistance genes making single type of organism resistant to several drugs precipitating in the emergence of multiple drug resistant (mdr) pathogens15. recently, continuous increase in antibiotic resistance has been observed particularly against ampicillin and trimethoprim which is adversely affecting the management and treatment of utis16. furthermore, encountering pathogenic infections that are contagious on routine basis is aggravating the problems while pathogens are becoming resistant to first-line antimicrobial agents17. it is hereby critical for physicians to recommend appropriate treatment in accordance with the susceptibility pattern of causative agents of utis18. when infection is in a specific area, an organized empirical therapy plan is required to reduce spread of resistance among pathogens, for which knowledge of prevalence rate of particular pathogens and their most recent antimicrobial susceptibility profile is compulsory19. keeping this in view, we aimed to isolate, purify, and identify p. aeruginosa which is the predominant cause of utis among catheterized patients. furthermore, we evaluated the antibiotic susceptibility profile of p. aeruginosa isolates in order to estimate emerging sensitivity pattern in the local population of southern punjab, pakistan where 10% of population is speculated to suffer from uti at some stage of their life20. m a t e r i a l s & m e t h o d s sample collection one hundred and fifty (150) samples of midstream urine were collected in sterilized containers from the uti patients bedded in nishtar hospital, multan for this hospital-based study after gaining ethical approval from the ethical committee of nishtar hospital. the main inclusion criterion was referral and admission to nephrology, urology, gynecology, neurology, & medicine wards of nishtar hospital in addition to catheterization and noncatheterization as well as both genders. further, inclusion prevalence and antibiotic susceptibility profiling of mdr p. aeruginosa vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 3 was based on appearance of symptoms within two days of stay in the hospital and diagnosis by physicians. all those cases showing symptoms of infection prior to 48hrs stay in the hospital, and/or diagnosed with infections other than uti were excluded from the study. personal information including demography and geography as well as medical information encompassing symptoms of patients was documented on a pre-designed questionnaire proforma signed by patients to ascertain informed consent. the samples were processed in microbiology section, pathology department of nishtar medical university, multan, under standardized procedures to reduce the chances of contamination. isolation and identification of p. aeruginosa for the purpose of microscopic examination, centrifugation of urine samples was performed at 5000rpm for 5mins and sediment was considered for standard operating procedures. cases with considerable pus cells (≥10 wbcs/mm) were administered for further analysis. for isolating and identifying uropathogens, urine samples were streaked onto cysteine lactose electrolyte deficient (cled) agar plates maintaining sterility and incubated at 37ºc for 18-24hrs under ventilated conditions as cled media is strongly recommended for isolation, identification, and quantification of the uropathogens. subsequent to incubation, colony characteristics were observed by using hand lens. p. aeruginosa colonies on cled medium were light green with typical intertwined surface, while colonies of e. coli were yellow due to its ability to ferment lactose. colonies of klebsiella spp. were also yellow and enormously mucoid in terms of appearance. colonies of proteus spp. were radiant blue and those of s. aureus were deep yellow. gram staining was done subsequently to observe gram-negative rods of p. aeruginosa. various biochemical tests comprising of triple sugar iron, oxidase, motility, citrate utilization, methyl red / voges-proskauer and indole tests were performed for species identification. antimicrobial susceptibility profiling antimicrobial susceptibility pattern of p. aeruginosa isolates was determined by performing kirby-bauer method (disc diffusion assay) using mueller hinton (mh) agar. about 3-5 colonies from pure cultures of p. aeruginosa isolates were transferred to a sterilized test tube containing 5ml normal saline, with the help of sterile wire loop and this suspension of colonies was compared with 0.5 mcfarland standard. with the help of sterile cotton swab dipped in microbial suspension, each mh agar plate was inoculated and antibiotic discs were applied to it at specific and appropriate distance. the plates were then incubated at 37ºc for 24hrs subsequent to which antibiotic sensitivity zones were measured in mm by ruler and recorded as sensitive, intermediate sensitive or resistant following the clinical and laboratory standard institute (clsi) guidelines. isolates were evaluated for their susceptibility or resistance towards amikacin (ak), ciprofloxacin (cip), imipenem (ipm), amoxicillin (amx), cefixime (cfm), gentamicin (cn), piperacillin/tazobactam (tzp), and nitrofurantoin (nf). r e s u l t s baseline characteristics of study population of the 150 urine samples of patients admitted to different wards of nishtar hospital collected and investigated for the presence of uropathogens, 88 were females whereas 62 were males. out of all the females, 58 were associated with catheter while 30 were not associated with catheter. out of all the males, 35 were associated with catheter and 27 were not associated with catheter. patients belonged to different regions of multan and nearby localities (fig. 1). this highlights the fact that patients from all over south punjab become admitted to nishtar hospital, multan, thereby making our sample population representative of entire southern punjab region. symptomatic frequency was observed to be greater in females when compared to males as evidenced by the fact that frequency of urination, burning sensation during urination, flanking pain and dysuria were all more frequently experienced by females in comparison with males (fig. 2). information regarding prevalence of various risk factors associated with uti was also documented and it was observed that indwelling catheter was the major risk factor associated with utis followed by pregnancy, presence of stone, enlarged prostate, diabetes, injured spinal cord and presence of tumor in kidney, respectively (fig. 3). prevalence and antibiotic susceptibility profiling of mdr p. aeruginosa vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 4 figure 1. geographical distribution of study population. figure 2. gender-based prevalence of symptoms associated with uti in study population. figure 3. prevalence of risk factors associated with uti among study participants. prevalence and antibiotic susceptibility profiling of mdr p. aeruginosa vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 5 age and gender based prevalence of utis utis were recorded as being more prevalent among females (59%) as compared to males (41%) in the study population highlighting the substantial gender-based differences in prevalence of uti. furthermore, uti frequency was observed as being higher among younger females and males as compared to older males and females, in general (table 1). in both genders, utis were the most prevalent among individuals that were 20 to 30 years old, and its frequency constantly decreased while females grew older to 70 years and males grew older to 60 years. a slight increase in uti frequency was observed among females older than 70 years and males older than 60 years. prevalence of microbes in uti patients based on the urine cultures, it was confirmed that in all urine samples, at least one kind of urinary microbe was present. isolates from various samples included p. aeruginosa, e. coli, enterobacter spp., klebsiella spp., proteus spp., s. aureus, s. saprophyticus, candida albicans and citrobacter spp. it was observed that e. coli was the most predominant uropathogen among males as well as females followed by k. pneumoniae, proteus spp., s. saprophyticus, s. aureus, p. aeruginosa, enterobacter spp., c. albicans and citrobacter spp., respectively (table 2). table 1. age and gender based prevalence of uti in the study population. age in years males (n=62) females (n=88) 20-30, n (%) 29 (46.77) 44 (50) 30-40, n (%) 12 (19.35) 20 (22.72) 40-50, n (%) 8 (12.90) 11 (12.50) 50-60, n (%) 3 (4.83) 3 (3.40) 60-70, n (%) 4 (6.45) 3 (3.40) above 70, n (%) 6 (9.67) 7 (7.95) n = number of individuals table 2. gender based prevalence of uropathogens isolated from uti patients included in the study. uropathogen total (n=150) females (n=88) males (n=62) e. coli, n (%) 50 (33.33) 31 (35.22) 19 (30.64) k. pneumoniae, n (%) 27 (18) 17 (19.31) 10 (16.12) proteus spp., n (%) 15 (10) 8 (9.09) 7 (11.29) s. saprophyticus, n (%) 13(8.66) 7 (7.95) 6 (9.67) s. aureus, n (%) 11 (7.33) 6 (6.81) 5 (8.06) p. aeruginosa, n (%) 10 (6.66) 6 (6.81) 4 (6.45) enterobacter spp., n (%) 9 (6) 5 (5.68) 4 (6.45) candida spp., n (%) 8 (5.33) 4 (4.54) 4 (6.45) citrobacter spp., n (%) 7 (4.66) 4 (4.54) 3 (4.83) n = number of individuals prevalence and antibiotic susceptibility profiling of mdr p. aeruginosa vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 6 table 3. antibiotics used against isolates of p. aeruginosa with their mechanism of action and prevalence in study participants. antibiotic group antibiotic mechanism of action code prevalence ( ⃰ s or ⃰ r) aminoglycoside amikacin inhibitor of protein synthesis ak 80% (*r) fluoroquinolone ciprofloxacin inhibitor of nucleic acid synthesis cip 60% (*r) carbapenem (betalectamase inhibitor) imipenem inhibitor of cell wall synthesis ipm 50% (*r) beta-lactamase inhibitor amoxicillin inhibitor of cell wall synthesis amx 50% (*r) cephalosporin cefixime inhibitor of cell wall synthesis cfm 80% (*s) aminoglycoside gentamicin inhibitor of protein synthesis cn 70% (*s) beta-lactamase inhibitor piperacillin/ tazobactam inhibitor of cell wall synthesis tzp 60% (*s) urinary tract antiseptic nitrofurantoin inhibitor of cell wall and nucleic acid synthesis nf 40% (*s) *r=resistant; s=sensitive antibiotic susceptibility profile of p. aeruginosa antibiotic susceptibility pattern was determined specifically for p. aeruginosa isolates and the results have been summarized in table 3. cefixime, which is a cephalosporin antibiotic, was demonstrated to be the most effective of all the drugs tested against p. aeruginosa isolates whereas amikacin, an aminoglycoside, was recorded as being the least effective drug in this regard. in terms of effectiveness against p. aeruginosa uropathogen, cefixime was followed by gentamicin, piperacillin/tazobactam and nitrofurantoin, respectively. with reference to ineffectiveness, amikacin was followed by ciprofloxacin, imipenem and amoxicillin, respectively. furthermore, majority (90%) of the isolates had become resistant to two or more antibiotics classifying them as mdr pathogens. d i s c u s s i o n p. aeruginosa was conferred for the first time in 1882 by gessard when he isolated it from green pus8. urinary tract is the most prevalent site of healthcare-associated infections, with p. aeruginosa accounting for 7-10% of utis diagnosed in patients admitted to hospitals21. because of the dynamic nature and recent shift in the etiology and antimicrobial susceptibility pattern of utis, it has become essential for the clinicians to keep abreast of the etiological agents and their antimicrobial susceptibility profiles, so as to direct and improvise the preliminary empirical treatment22. in the light of these observations, this study was conducted to evaluate etiology of utis in a population of southern punjab, pakistan and antibiotic susceptibility profiling of mdr p. aeruginosa was evaluated. it was observed that uti was more prevalent in females as compared to males, is due to the differences in the anatomy of their urogenital organs; as short urethra in females allows microbes to gain relatively easy access to bladder so as to cause infection23. in addition, females are inclined towards several circumstances like vaginitis as well as decay and prolapse of vagina or womb which leads towards the development of uti24. the symptoms of uti were also more prevalent among females which reinforces the previous observations in this regard25. it has been reported that uti is the most significant cause of hospital-acquired infections. among patients who are hospitalized, urinary catheter has been revealed as the major predisposing factor for utis26. accordingly, the most prevalence and antibiotic susceptibility profiling of mdr p. aeruginosa vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 7 substantial risk factor for uti detected in the present study was presence of indwelling catheter. previously, this has been reported that the duration of catheterization is directly proportional to the risk of developing uti27. another important risk factor observed here was pregnancy which has been commonly reported as a factor responsible for making females vulnerable to the development of utis28. we found that majority of the patients were 20 to 30 years old which has been scarcely reported previously and mostly, old age has been documented as a risk factor for utis29. this could possibly be explained based on the sexual behavior and use of contraceptive agents30. conceivably due to the rise in rate of sexual action, as well as associated rise in the number of pregnancies and utilization of various kinds of contraceptive agents, younger individuals were the most affected ones31. however, study on a larger sample population is required to confirm this speculation. e. coli was the most commonly isolated uropathogen in individuals belonging to both genders, and p. aeruginosa was responsible for 7% of the overall cases, approximately. similar observations have been reported previously in another population of pakistan32. a vast majority of the p. aeruginosa isolates were revealed as being mdr here which is in line with the emerging data regarding prevalence of mdr uropathogens33. cefixime was reported here as being the most effective of all the drugs tested against p. aeruginosa whereas amikacin was the least effective owing to high degree of resistance against it. previously, in various populations the drug resistance patterns have been similar as well as dissimilar implying that antibiotic resistance profile of uropathogens varies across the globe34,35. over-enthusiastic use of antibiotics has led to the global emergence of resistance among various bacteria represented by recommendation of antibiotics without susceptibility reports or prolonged usage of drugs36. antibiotics resistance has been declared as global human health issue by who and over-ambitious usage of broad-spectrum antibiotics has elevated the rate of drug resistance across the world37. c o n c l u s i o n utis account for a considerable burden of antibiotic prescriptions and gradual rise in resistance against commonly used antibiotics needs to be deliberated immediately. such growing resistance in uti patients by p. aeruginosa can be addressed by adopting essential management options by physicians as well as medical professionals based on the knowledge of etiology and antibiotic susceptibility of pathogens. quest for alternative treatment options which may be combined with antimicrobial drugs is the need of the hour. e t h i c a l a p r o v a l ethical approval for the study was obtained from ethical review board of nishtar hospital, multan, pakistan. c o n f l i c t o f i n t e r e s t the authors declare no conflict of interest. f u n d i n g s o u r c e the study received no external funding. a c k n o w l e d g e m e n t s we acknowledge the cooperation of study participants who facilitated our work by providing their personal information and samples. l i s t o f a b b r e v i a t i o n s ak amikacin amx amoxicillin cfm cefixime cip ciprofloxacin cled cysteine lactose electrolyte deficient clsi clinical and laboratory standard institute cn gentamicin ipm imipenem mdr multiple drug resistant mh mueller hinton nf nitrofurantoin tzp piperacillin/tazobactam utis urinary tract infections r e f e r e n c e s 1. abubakar emm. antimicrobial susceptibility pattern of pathogenic bacteria causing urinary tract infections at the specialist hospital, yola, adamawa state, nigeria. j clin med res. 2009; 1(1):1-8. prevalence and antibiotic susceptibility profiling of mdr p. aeruginosa vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 8 2. öztürk r, murt a. epidemiology of urological infections: a global burden. world j urol. 2020; 38:2669-79. 3. siegman-igra y, levin r, weinberger m, golan y, schwartz d, samra z, et al. listeria monocytogenes infection in israel and review of cases worldwide. emerg infect dis. 2002; 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antimicrob agents chemother. 2000; 44(4):1089-92. 13. hooton tm, besser r, foxman b, fritsche tr, nicolle le. acute uncomplicated cystitis in an era of increasing antibiotic resistance: a proposed approach to empirical therapy. clin infect dis. 2004; 39(1):75-80. 14. savas l, guvel s, onlen y, savas n, duran n. nosocomial urinary tract infections: micro-organisms, antibiotic sensitivities and risk factors. west indian med j. 2006; 55(3):188. 15. chen q, lin y, li z, lu l, li p, wang k, et al. characterization of a new transposon, tn6696, on a blandm–1-carrying plasmid from multidrug-resistant enterobacter cloacae ssp. dissolvens in china. front microbiol. 2020; 11:525479-85. 16. ny s, edquist p, dumpis u, grondahl-yli-hannuksela k, hermes j, kling am, et al. antimicrobial resistance of escherichia coli isolates from outpatient urinary tract infections in women in six european countries including russia. j glob antimicrob resist. 2019; 17:25-34. 17. schito am, piatti g, stauder m, bisio a, giacomelli e, romussi g, et al. effects of demethylfruticuline a and fruticuline a from salvia corrugata vahl. on biofilm production in vitro by multiresistant strains of staphylococcus aureus, staphylococcus epidermidis and enterococcus faecalis. int j antimicrob agents. 2011; 37(2):129-34. 18. sorlózano-puerto a, gómez-luque jm, luna-delcastillo jd, navarro-marí jm, gutiérrez-fernández j. etiological and resistance profile of bacteria involved in urinary tract infections in young children. biomed res int. 2017; 4909452-7. 19. sugianli ak, ginting f, parwati i, de jong md, van leth f, schultsz c. antimicrobial resistance among uropathogens in the asia-pacific region: a systematic review. jac-amr. 2021; 3(1):1-11. 20. manzoor a, ishaq n, kanwal a. incidence, distribution and management of community acquired urinary tract infections among patients in hospitals of lahore, pakistan. int j biotech trends technol. 2020; 10(1):15-21. 21. tumbarello m, raffaelli f, peghin m, losito ar, chirico l, giuliano g, et al. characterization and risk factor profiling of pseudomonas aeruginosa urinary tract infections: pinpointing those likely to be caused prevalence and antibiotic susceptibility profiling of mdr p. aeruginosa vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 9 by multidrug-resistant strains. int j antimicrob agents. 2020; 55(4):105900-9. 22. balkhi b, mansy w, alghadeer s, alnuaim a, alshehri a, somily a. antimicrobial susceptibility of microorganisms causing urinary tract infections in saudi arabia. the j infec dev ctries. 2018; 12(04):220-7. 23. odongo i, ssemambo r, kungu jm. prevalence of escherichia coli and its antimicrobial susceptibility profiles among patients with uti at mulago hospital, kampala, uganda. interdiscip perspect infect dis. 2020; 8042540-7. 24. shah da, wasim s, abdullah fe. antibiotic resistance pattern of pseudomonas aeruginosa isolated from urine samples of urinary tract infections patients in karachi, pakistan. pak j med sci. 2015; 31(2):341-5. 25. bent s, nallamothu bk, simel dl, fihn sd, saint s. does this woman have an acute uncomplicated urinary tract infection? jama. 2002; 287(20):2701-10. 26. tenke p, mezei t, bőde i, köves b. catheterassociated urinary tract infections. eur urol suppl. 2017; 16(4):138-43. 27. letica-kriegel as, salmasian h, vawdrey dk, youngerman be, green ra, furuya ey, et al. identifying the risk factors for catheter-associated urinary tract infections: a large cross-sectional study of six hospitals. bmj open. 2019; 9(2):022137-42. 28. ranjan a, sridhar st, matta n, chokkakula s, ansari rk. prevalence of uti among pregnant women and its complications in newborns. indian j pharm pract. 2017; 10(1):45-9. 29. tenney j, hudson n, alnifaidy h, li jt, fung kh. risk factors for aquiring multidrug-resistant organisms in urinary tract infections: a systematic literature review. saudi pharm j. 2018; 26(5):678-84. 30. gebremariam g, legese h, woldu y, araya t, hagos k, gebreyesuswasihun a. bacteriological profile, risk factors and antimicrobial susceptibility patterns of symptomatic urinary tract infection among students of mekelle university, northern ethiopia. bmc infect dis. 2019; 19(1):1-12. 31. behzadi p, behzadi e, pawlak-adamska ea. urinary tract infections (utis) or genital tract infections (gtis)? it's the diagnostics that count. gms hyg infect control. 2019; 14:1-7. 32. ullah a, shah sr, almugadam bs, sadiqui s. prevalence of symptomatic urinary tract infections and antimicrobial susceptibility patterns of isolated uropathogens in kohat region of pakistan. moj biol med. 2018; 3(4):85-9. 33. mohsenzadeh m, abtahi-eivary sh, pirouzi a, khaledi a, rahimi m. a systematic review and meta-analysis of urinary tract infection, frequency of is elements and mdr isolates retrieved from adult patients. gene rep. 2020; 20:100707-15. 34. hossain a, hossain sa, fatema an, wahab a, alam mm, islam mn, et al. age and gender-specific antibiotic resistance patterns among bangladeshi patients with urinary tract infection caused by escherichia coli. heliyon. 2020; 6(6):4161-9. 35. hussain m. bacteriological spectrum and sensitivity pattern in culture proven urinary tract infection in children. j rawalpindi med coll. 2017; 21(3):290-2. 36. aypak c, altunsoy a, düzgün n. empiric antibiotic therapy in acute uncomplicated urinary tract infections and fluoroquinolone resistance: a prospective observational study. ann clin microbiol antimicrob. 2009; 8(1):1-7. 37. muraki y, kitamura m, maeda y, kitahara t, mori t, ikeue h, et al. nationwide surveillance of antimicrobial consumption and resistance to pseudomonas aeruginosa isolates at 203 japanese hospitals in 2010. infection. 2013; 41(2):415-23. geospatial analysis of indus river meandering and flow pattern from chachran to guddu barrage, pakistan vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 67 r a d s j . b i o l . r e s . a p p l . s c i 67 op en ac ces s f u l l l e n g t h a r t i c l e geospatial analysis of indus river meandering and flow pattern from chachran to guddu barrage, pakistan danish raza* and aqeel ahmed kidwai department of meteorology-comsats university islamabad, islamabad, pakistan a b s t r a c t natural and anthropogenic influence affects directly ecologic equilibrium and hydro morphologic symmetry of riverine surroundings. the current research intends to study the hydro morphologic features (meanders, shape, and size) of indus river, pakistan by using remote sensing (rs) and geographical information science (gis) techniques to calculate the temporal changes. landsat satellite imagery was used for qualitative and analytical study. satellite imagery was acquired from landsat thematic mapper (tm), enhanced thematic mapper plus (etm+) and operational land imager (oli). temporal satellite imagery of study area was used to identify the variations of river morphology for the years 1988,1995,2002,2009 and 2017. research was based upon the spatial and temporal change of river pattern with respect to meandering and flow pattern observations for 30 years’ temporal data with almost 7 years’ interval. image preprocessing was applied on the imagery of the study area for the better visualization and identification of variations among the objects. object-based image analysis technique was performed for better results of a feature on the earth surface. model builder (arc gis) was used for calculation of temporal variation of the river. in observation many natural factor involves for pattern changes such as; floods and rain fall. the object-oriented classification was applied for land use/land cover (lulc) features of the study area for the years 1988 and 2017 and abrupt change observed. overall, 1988 to 2017 the indus river in the study area has changed its path and pattern. keywords indus river, remote sensing, gis, objectbased image analysis, lulc. *address of correspondence danish_raza52@yahoo.com article info. received: august 15, 2018 accepted: october 19, 2018 cite this article: raza d, kidwai aa. geospatial analysis of indus river meandering and flow pattern from chachran to guddu barrage, pakistan. rads j. biol. res. appl. sci. 2018; 9(2): 6774. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n water is the renewable natural resource because it can be replenished after used. the rivers flow the water from one place to another. the river channel normally contains a single stream of water but some river flow as multiple streams that are interconnected 1. river water flows from upstream to downstream area with respect to elevation or topographical change. indus river (ir) lies in northwestern (nw) part of southern asia within the boundaries of china, pakistan, india and afghanistan 2. northern area of pakistan contains pamir mountain ranges and the karakoram which make enough substantial topography of indus river 3. indus river is a main tributary of pakistan its starts from the northern area of pakistan and end in arabian sea 4,5. at the downstream of panjnad upper side of mithankot five major rivers from east (jehlum, chenab, ravi, beas and sutlej) meets with indus river. these five rivers fall into the arabian sea with the flux of sediments 5. these rivers are flowing through the punjab province densely populate and agricultural areas. at the downstream of mithankot, indus o r i g i n a l a r t i c l e geospatial analysis of indus river meandering and flow pattern from chachran to guddu barrage, pakistan vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 68 r a d s j . b i o l . r e s . a p p l . s c i 68 river has no dam and water flow with high magnitude without any interruption. river pattern changes take place at above the delta, especially near kashmore and sehwan and old paths can be recognized towards the indus delta in lower sindh 6. due to the climate change heavy floods occurred in indus river. the rise in temperature is the major cause of snow melting as well as change in rainfall cycle on the watershed area of indus river cause of floods. in indus river thirteen super floods and high floods occurred in the years 1955, 1956, 1957, 1958, 1959, 1964, 1973, 1975, 1976, 1978, 1983, 1986, 1988, 1989, 1992, 1995, 1997 and 2010 7. remote sensing and gis (geographic information system) approach is very useful in river morphological changes detection. gis and remote sensing are used to calculate scattering of unusual and difficult areas to monitor in a cost-effective way with a high accuracy level 8. rivers are the natural feature on the earth surfaces that change the shape and size either by increasing or decreasing flow with the passage of time due to erosion, sedimentation and deposition. the gis-based analysis allows the river management and decision-making process 9. monitoring of the temporal morphological changes is not easy with the traditional methods. remote sensing and gis provides a possible solution to evaluate these changes in less time with quality work. accurate, fast and costeffective information can be achieved easily 10. remote sensing and gis are essential to identify these changes in a cost-effective way. satellite imagery of the land surface with the passive sensors allows the detection and perception of ground objects 11. gis technique used for the observation of spatial and temporal trend of river and its association with other features 12. in object-oriented classification, exported data in vector format in form of polygons that contain all the attribute information 13. land use planning do not possible without the satellite imagery. gis and remote sensing method used for change detection of land 14. remote sensing is a tool to help in detecting the degree of land use land cover change that has taken place on river watershed 15. on the other hand, statistical analysis performed by using arc gis: model builder that is a graphical user interface that represented by diagrams to create, edit and execute the geospatial work 16. the purpose of this research is to observe the indus river (chahcran-guddu) temporal flow pattern and meandering changes because the variations in river flow pattern occur due to flow of water with passage of time. temporal pattern of river flow identification is the main purpose of the study. land cover changes will be identified after the object-oriented classification of an area of interest to establish the scenario of land cover changes. m a t e r i a l a n d m e t h o d study area geographically study area is located at 28.31°-28.99° n, 69.54° 70.49° e (see fig. 1). the length of the study area is around 88 km from chachran to guddu barrage. this study area was chosen because of the water flow is very high due to all river of punjab meet at the point that near to study area. table 1 is showing the major settlements around the study area. guddu barrage was constructed to distribute the flow of water. indus river is the biggest river in pakistan that fall into the arabian sea 4, 17. the study area was chosen for flow pattern observation by using landsat satellite images. guddu barrage is located near kashmore alongside of indus river. the construction of guddu barrage was completed in 1962. the maximum discharge capacity of guddu barrage is 1200000 cusecs (m3 / s-1) 18. main purpose of guddu barrage is to distribute the water flow for irrigation 19. fig. 1: location map of the study area, indus river (chachran to guddu barrage). geospatial analysis of indus river meandering and flow pattern from chachran to guddu barrage, pakistan vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 69 r a d s j . b i o l . r e s . a p p l . s c i 69 table 1: major settlement around study area (indus river). northern west southern east mithankot chachran sharif rojhan dilkusha shahwali data and analysis multispectral satellite images of 151 paths and 40 rows were downloaded from the free available website of united states geological survey (usgs). satellite images from 1988 till 2017 with the gap of almost 7 years were downloaded. landsat-5 tm (thematic mapper) images were acquired for the years of 1988, 1995, 2009 and landsat-7 etm+ (enhanced thematic mapper plus) for the year 2002 was downloaded. meanwhile, landsat-8 oli (operational land imager) data was used for the year 2017 (see table 2). the landsat data used in the process were already geo-referenced with the world geodetic system (wgs-84) and universal transverse mercator (utm), zone 42, north projection. the satellite images were obtained of only for the summer season to avoid seasonal water variations. because in monsoon season river is under flooding consequently variations and changes of the river may not be easy observed. in the summer season, there is a controlled water flow in the river and flow pattern changes are easily identified using satellite images. image preprocessing performed on the acquired satellite imagery. atmospheric corrections and subset of satellite imagery of the area of interest were done by using the shape file of the study area. table 2: landsat sensors specification. satellite sensor resolution date landsat 5 tm 30 meters 06/12/1988 15/05/1995 22/06/2009 landsat 7 etm+ 26/05/2002 landsat 8 oli 27/05/2017 object based image analysis object-oriented classification technique has a higher accuracy level to classify the satellite imagery as opposed to old traditional classification techniques 20. the objectoriented classification was performed on pixel’s values of landsat data of 1988, 1995, 2002, 2009 and 2017 only on the study area. overall observation based upon the temporal changes reliable due to the accuracy of results to enhance calculations results which are also the main objective of the research. the resultant image detected the river water, vegetation, barren and built up areas. segmentation based classification segmentation of the objects depends upon shape parameters that indicate how objects are created. it will depend upon the size of the segments, the larger the segments smoothing is also required, compactness value and shape size have an important role to create appropriate segments. compactness is basically the procedure of merging features segment. the image displays clear segmentations with the targets (features). this may be realized that water has differences in reflectance because it could be clear, muddy and or intrusions. the segments are generated when the objectoriented process has been run, and they based on the target clustering (pixels). the image is divided into many objects and it is based upon the standard of nearest neighbor. every image of landsat has a number of pixels of different intensity but in object-based classification, objects are generated on the base of pixel values of same target or the pixels that have the almost similar intensity and increase the feature deficiency. two algorithms used in the object-oriented method; one is a multi-resolution segmentation and the second is knowledge-based classification. in the first step of segmentation, the desired segmentation of that object is made. fig. 2: hierarchy of raster dataset in object-oriented classification (source: benz et al. 21). geospatial analysis of indus river meandering and flow pattern from chachran to guddu barrage, pakistan vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 70 r a d s j . b i o l . r e s . a p p l . s c i 70 later using the merging technique, the neighboring similar objects are merged to form bigger object (fig. 2). then, these bigger objects are merged on the basis of color (intensity), shapes and digital numbers of the objects. knowledge-based (shape, size, texture and dn values) classification on the generated segments. yu describes that object-based classification is based upon the object segments. he describes that the classes of features and segments for the classification of remotely sensed data generated according to the objects 22. the scale parameter directly depends upon the segments, when the number of segments increase than the scale parameter is small because objects creation depends upon the value of scale parameter 23. in this research, the multi-resolution segmentation algorithm is used to generate the segments according to the dataset. to generate accurate segments, the value of the scale parameter is set at five on the basis of the targets (features) in imagery. according to requirement, the scale parameter is necessary to achieve the goal that is used to increase or decrease the segments. for the composition of homogeneous criterion, the values that are used for shape and compactness is 0.1 and 0.3 respectively. algorithms that are applied on the data are nearest neighbor; geometry and layer value standard applied. for the purpose of classification; algorithm is named as classification which is used to execute the classification results. to obtain reliable results 0.3 factor value is applied to avoid any mixed pixel effect. segmentation of the objects is depending upon shape parameters that indicate how objects are created. it will depend upon the size of the segments, the larger the segments smoothing is also required, if the segments are smaller in size, it is necessary to apply the smoothness. in this research compactness and shape size value have been used 0.3 and 0.1 respectively. the smoothing was required for balance the homogenously of segments shape. the classes were labeled as water, vegetation, built-up, barren land after the segmentation process. sampling of the objects was carried to separate the classes. the nearest neighbor algorithm was applied based on the layer (band) value and the geometry of the object. after the sample selection; algorithm of classification is used to execute classification results when all the sampling has been done. later, the result could be exported into shapefile to use in gis software. modal builder (arc gis) a detailed analysis has been carried out for the change of course of the river i.e. meandering, change of flow pattern etc. for the observation of change in pattern for the years 1988 and 2017 in the study area has noticeable. the study was carried out to observe the temporal changes according to its meandering and flow pattern. the study region has tremendous changes due to flow in form of its path to the area of the river in this research divided into several zones with respect to its meandering changes in multiple directions as observed in the results selection. division of study region in multiple zones is useful to observed multi-variations in the large study area 24. these changes were studied and analyzed using the overlay operations such as intersect, erase, and union to calculate the changed area. the influence of various factors over the river sinuosity was studied using the overlay analysis in the gis. three overlay operations, intersect, erase and the union was applied simultaneously. r e s u l t s & d i s c u s s i o n s in this research, the flow pattern of indus river (chachran guddu) for the period of 30 years from 1988 to 2017 was observed. the object-oriented classification was applied on landsat satellite for extraction of the results according to the shape of river body. the only reason for using object-oriented classification is that segments are generated according to the targets (features) specified. object-oriented classification technique is applied to the imagery of landsat 1988. the area of water body in 1988 was 195.9 km2. fig. 3 shows the indus river flow pattern from northeast to southwest and the area of the river in 1995 was 193.3 km2. from the above results in 1988, the area of water body was large as compared from the year of 1995. in 1995 the area of water body decreased with the difference of 2.6 km2. the area of water body in 2002 was 248.3 km2 and the area of the water body increased to 55 km2 according to the comparison of 1995 imagery. the flow pattern of water body from chachran to guddu barrage in 2009 has variations and the area of water body in 2009 was 285.2 km2. the difference between the water bodies of 2002 and 2009 was 36.9 km2 that observed. fig. 2 also shows the flow pattern of water body in 2017 that indicates the river in 2017 was 207.4 km2. geospatial analysis of indus river meandering and flow pattern from chachran to guddu barrage, pakistan vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 71 r a d s j . b i o l . r e s . a p p l . s c i 71 fig. 3: resultant map of object oriented classification for change detection. summarizing the results of all the maps that are generated from object oriented classification technique, table 3 showing change in specified time frame. table 3: results of temporal variations that occurred in observed period. observed period overlapped area (%) spatially changed (%) 1988-1995 39.81 60.19 1995-2002 40.62 59.38 2002-2009 47.62 52.38 2009-2017 52.31 47.69 meandering and flow patten observation for the years 1988 and 2017 area of the river increased up to 11.5 km2 in 2017 which makes the total area of water body 207.4 km2. the modal that builds (fig. 4) to observe the change in river pattern indicates the abrupt variations such as; an area of 25.49 % of the river in 2017 overlapped with the old position of the river 1988 however, abrupt changes could be observed in fig. 5 where it can be seen clearly that 74.51% area of the river in 2017 has changed its flow pattern due to the temporal variations as compared to 1988. overall scenarios in results show the temporal variation in river flow pattern and meandering within 30 years’ gap. an observed variation indicates that there are many factors involved in the changed in the condition of water level and path change processes such as rainfall and floods. according to memon flash flood is the main reason that involves in erosion on the river bed, because of the (catchment area of province baluchistan) flow of water towards indus river, eventually spatial and temporal changes occur caused by the floods. mainly, jacobabad, kashmore and shahdadkot areas get affected in floods and eventually the agricultural land, roads, forest, and other damages occur 25. also, he indicates the factors that may involve in the water flow in this region so, the results of the research show the abrupt change in the resultant map (fig. 5). consistency flow of the river in its channel is the source of energy which removes every object that falls in its way as the resulting shape of the river changed 26. fig. 4: model builder process for identification of river pattern changes in between the years 1988 and 2017. fig. 5: river flow pattern in 2017, model builder result shows the red color that indicate the remain of river 1988 and blue color indicate river path that changed till 2017. salma divided the study area into five zones according to the means and median of climate to observe the climate change 24. in this research, the study area is divided into 5 zones based on a meandering, flow pattern and direction of temporal variations in the path of the river. in this research, the study area is divided into 5 zones based on a meandering, flow pattern and direction of temporal geospatial analysis of indus river meandering and flow pattern from chachran to guddu barrage, pakistan vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 72 r a d s j . b i o l . r e s . a p p l . s c i 72 change path of the river. zone 1 in fig. 6 shows the temporal trend of the river in zone 1 where the river shifted up to 2.7 km, the trend of river pattern/meandering changed towards the south east direction. it is clearly observed in zone 2 that the river changes the trend of flow in nw direction and changed the path to almost 2.8 km away from the original path. moreover, in zone 3 and zone 4 respectively river flow pattern changed in se and in nw that also has an abrupt change in form of meandering of the river. the river changed the path up to 7.7 km that is a large deviation from the original path. in zone 5 it is observed that the river shifted its path slightly in nw direction again. river pattern and path of the river changed up to 2.5 km in this region. fig. 6: positional variation in flow of indus river (chachran-guddu) from 1988 to 2017. the floods are caused by the fluvial hazards in the current study area. he also describes that the unpredictable floods were observed at guddu barrage from 1973 to 1995 and also in 2010. it also faced ten super floods from 1973 to 2010 (an average of five years’ gap) and seven very high floods (an average of seven years gap) from 1981 to 2015. superfloods peak discharge varies from 26,740 to 33,951 (m3 / s) while a very high flood discharge varied from 20,077 to 23,524 (m3 / s). eventually, these variations in flooding cause severe meandering and changes in the flow pattern, trend, and directions. the floods at guddu barrage receive heavy rainfall in the northern areas of pakistan is the major cause of rising water level in indus river 7. graph (fig. 7) shows the temporal trend of rainfall that derived from annual average rainfall (mm) of punjab province observatories for the years 1988 to 2017. rainfall data acquired from the pakistan meteorological department (pmd). rainfall data based on the measurement of bahawalpur, bahawalnagar, islamabad, faisalabad, rawalpindi, jehlum, khanpur, lahore, mianwali, multan, murree, sargodha, shorkot and sialkot stations. change in rainfall has effects on channel movement and these variations are the main cause of river positional change. fig. 7: graph showing punjab annual average rainfall (mm) for observation of rainfall changes from 1988 to 2017 (pmd 27). satellite based land use / land cover change detection due to the temporal variations in the flow pattern of the river; land use and land cover changes occur. fig. 8 displays the map of land use/land cover classification for the years 1988 and 2017. temporal changes can be observed in the land cover where the vegetation has decreased while the built up, barren land and water body have increased with the passage of time. table 4: descriptions of land use/ land cover classes. serial number classes name class color 1 built up dark brown 2 barren land olive 3 vegetation green 4 water blue gis is an important technology for temporal analysis otherwise change detection is very difficult. change detection is possible with this technology in less time and cost 10, 28. in this research four lulc classes were used to observe the change (see table 4). fig. 9 shows the bar graph of the year 1988 and 2017 where it can be visualized increasing and decreasing trend of targets which are classified from landsat satellite imagery by geospatial analysis of indus river meandering and flow pattern from chachran to guddu barrage, pakistan vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 73 r a d s j . b i o l . r e s . a p p l . s c i 73 using the object-oriented classification. in 1988 vegetation can be seen in classified results, vegetation exists along the river. abrupt changes occurred in 2017 where the vegetation became as barren land and converted into built-up areas. temporal changes occurred in the downstream area (study area) after panjnad barrage due to the high flow of water 5. the research also established a loss of vegetation has occurred where the river changed its flow path and pattern. fig. 8: object oriented classified results of land use/ land cover change for the years 1988 and 2017. fig. 9: graph of temporal changes in land use/ land cover change in km2 for the years 1988 and 2017. the research work also establishes that the ratio of barren land, built up and vegetation in 1988 was 39.08 %, 0.49 %, and 49.14 % respectively. barren land, built up and vegetation covered area as; 48.31 %, 1.35 % and 38.74 % in 2017 the research shows that the built up and barren land increased but the vegetation deceased in the observed time period. built up and barren land have been also increased 0.86 % and 9.23 % respectively. vegetation cover in 2017 has decreased up to 10.4 % with respect to the vegetation in 1988. the water of the river and built up area increased during the 30 years’ gap. c o n c l u s i o n s in this research, indus river (chachran to guddu barrage) flow pattern and land use / land cover changes are analyzed. the results show that the water body has increased based upon the object-oriented classification. water flow pattern changes (shape, size and path) occur over the years according to its flow as analyzed using the landsat imagery of 1988 to 2017. indus river (chachran to guddu barrage) has variations in flow pattern with respect to the time. large variations are observed in flow pattern and meandering since last 30 years in the between zones 3 and 4 of the study area yet starting and ending points of path of river encountered less changes with respect to middle of the study area. object-oriented classification technique is best fit to detect the river morphology and results are satisfactory because image classified on the bases of their targets (objects) segment. the results show variations have occurred about 2.7 to 7.7 km in the river flow pattern. study area fall at the border of punjab province and receives a huge flux of water flows towards the guddu barrage and eventually, variation occurs due to erosion or deposition. the research established that the vegetation has decreased and the water, built up and barren land have increased in observed time period. r e f e r e n c e s 1. walther jv. earth's natural resources. jones & bartlett publishers; 2013. 2. kravtsova vi, mikhailov vn, efremova na. variations of the hydrological regime, morphological structure, and landscapes of the indus river delta (pakistan) under the effect of large-scale water management measures. water resources.2009; 36(4): 365-79. 3. hewitt k, wake cp, young gj, david c. hydrological investigations at biafo glacier, karakoram range, himalaya; an important source of water for the indus river. ann glaciol.1989; 13: 103-8. 4. hill d. the politics of water in south asia. transforming cultures ejournal. 2006; 1(2): 135-158. geospatial analysis of indus river meandering and flow pattern from chachran to guddu barrage, pakistan vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 74 r a d s j . b i o l . r e s . a p p l . s c i 74 5. akhtar s. the south asiatic monsoon and flood hazards in the indus river basin, pakistan. j basic applied sci. 2011; 7(2): 101-15. 6. hancock gs, anderson rs, whipple kx. beyond power: bedrock river incision process and form. rivers over rock: fluvial processes in bedrock channels. 1998; 35-60. 7. mahessar a, qureshi a, dars g, solangi m. climate change impacts on vulnerable guddu and sukkur barrages in indus river, sindh. surj)2017; 49(1):137-42. 8. alam ms, khan ja, kushwaha sp, agrawal r, pathak bj, kumar s. assessment of suitable habitat of near threatened striped hyena (hyaena hyaena linnaeus, 1758) using remote sensing and geographic information system. asian j geoinform. 2014; 14(2): 1-10. 9. alam jb, uddin m, ahmed u j, cacovean h, rahman hm, banik bk, et al. study of morphological change of river old brahmaputra and its social impacts by remote sensing. geographia technica. 2007; 2, 1-11. 10. seker dz, goksel c, kabdasli s, musaoglu n, kaya s. investigation of coastal morphological changes due to river basin characteristics by means of remote sensing and gis techniques. water sci technol. 2003; 48(10): 135-42. 11. ijaz mw, siyal aa, mahar rb, ahmed w, anjum mn. detection of hydromorphologic characteristics of indus river estuary, pakistan, using satellite and field data. arab j sci engineer. 2017; 42(6): 253958. 12. porter md, massong tm. analyzing changes in river channel morphology using gis for rio grande silvery minnow habitat assessment. gis/spatial analyses in fishery and aquatic sciences.2004; 505: 433-446. 13. zhou w, troy a. an object oriented approach for analysing and characterizing urban landscape at the parcel level. international j rem sens. 2008; 29(11): 3119-3135. 14. bato va, paningbatan jr ep, bartolome bj. high resolution satellite data for comprehensive land-use planning. j environ sci manag. 2011; 14(1):12-23. 15. hua ak. land use land cover changes in detection of water quality: a study based on remote sensing and multivariate statistics. j environ pub health. 2017; 2017:1-12. 16. esri. model builder for arcview spatial analyst 2. redlands, ca: esri press; 2000. 17. khan b, iqbal mj, yosufzai ma. flood risk assessment of river indus of pakistan. arab j geosci. 2011; 4(1-2): 115-22. 18. inam a, clift pd, giosan l, tabrez ar, tahir m, rabbani mm, et al. the geographic, geological and oceanographic setting of the indus river. large rivers: geomorphology and management. 2007; 333345. 19. chaudhry sa. pakistan: indus basin water strategypast, present and future. lahore j econom. 2010; 15: 187-211. 20. akar ö, güngör o. classification of multispectral images using random forest algorithm. j geodesy geoinform. 2013; 1(2). 21. benz uc, hofmann p, willhauck g, lingenfelder i, heynen m. multi-resolution, object-oriented fuzzy analysis of remote sensing data for gis-ready information. isprs j photogramm rem sens. 2004; 58(3-4), 239-58. 22. yu q, gong p, clinton n, biging g, kelly m, schirokauer d. object-based detailed vegetation classification with airborne high spatial resolution remote sensing imagery. photogramm engineer rem sens. 2006; 72(7): 799-811. 23. matinfar hr, sarmadian f, alavi panah sk, heck rj. comparisons of object-oriented and pixel-based classification of land use/land cover types based on lansadsat7, etm+ spectral bands (case study: arid region of iran). american-eura j agri environ sci. 2007; 448-56. 24. salma s, shah ma, rehman s. rainfall trends in different climate zones of pakistan. pak j meteorol. 2012; 9: 37-47. 25. memon aa, muhammad s, rahman s, haq m. flood monitoring and damage assessment using water indices: a case study of pakistan flood-2012. egyp j rem sens space sci. 2015; 18(1):99-106. 26. garde rj. history of fluvial hydraulics. new age international; 1965. 27. pmd. pakistan meteorological department, pmd headquarters office, sector h-8/2 islamabadpakistan; 2017. 28. rawat js, kumar m. monitoring land use/cover change using remote sensing and gis techniques: a case study of hawalbagh block, district almora, uttarakhand, india. egyp j rem sens space sci. 2015; 18(1): 77-84. effect of drying-dehydration techniques on organoleptic quality of ml vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 66 op e n ac c e s s f u l l l e n g t h a r t i c l e effect of various drying and dehydration techniques on the organoleptic quality of mango leathers aamir azeem1, aasia panhwar1, parkash meghwar1,*, asif irshad1, umaid ali soomro2, syeda mahvish zahra2,3 1institute of food sciences and technology, sindh agriculture university, tandojam, pakistan. 2food technology and nutrition section, pcsir labs complex, karachi, pakistan. 3department of environmental design, health and nutritional sciences, allama iqbal open university, islamabad, pakistan. a b s t r a c t background: mango is known as the king of fruits. it has several varieties however, some are more popular in the country including sindhri. each variety has its significance when used in different food products. the pulp of mango is used for developing jams, beverages, chutney (dips) etc., and sometimes preserved with some additives to be used for the production of mango leather. in general, the application of drying techniques to fruit puree yields a stable shelf-life product called fruit leather, which is of soft rubbery texture with a sweet taste and especially dehydrated. objectives: to analyze the effect of various drying techniques (i.e. sun drying, oven drying, and dehydration) on the organoleptic quality (i.e. quality that affects how a consumer experiences the food via their senses e.g. look, taste, smell, and touch) of mango leather. methodology: a drying experiment was performed like sun drying, hot air oven drying, and commercial dehydrator to determine the effect of drying times on the quality of mango leathers using these techniques. the effect of the storage period was also studied for the quality of mango leathers. the dried mango leathers were sensory analyzed by the trained panelists. results: a minimum drying time of 8-10hours was achieved in a commercial dehydrator for mango fruit leather at 70°c. it was also observed that despite the longer dehydration time, the mango leathers produced remained equally acceptable compared to other dehydration techniques. the mango leathers dried in dehydrater at 70°c ± 2°c gave the highest score of color (6.20), texture (6.13), flavour/taste (5.88), appearance (5.88), and overall acceptability (5.85), while mango leather dried in an oven at 70°c ± 2°c has recorded a score of color (5.93), texture (5.87), flavour/taste (5.77), appearance (5.53), and overall acceptability (5.70). the storage of mango leathers had also a significant effect (p < 0.05) in the storage period of mango leathers as compared to control. a non-significant difference was also observed in all the organoleptic parameters after 10-12 weeks of storage. conclusion: it is obtained from the current study that for the production of mango leather, the commercial dehydrator based drying techniques results in acceptable sensory characteristics as well as longer storage period by comparing other drying techniques, as it has a controlled environment. keywords mango leathers, commercial dehydrator, hot oven drying, sun drying, sensory characteristics. *address of correspondence kparkash707@gmail.com article info. received: april 18, 2021 accepted: june 11, 2021 cite this article azeem a, panhwar aa, meghwar p, irshad a, soomro ua, zahra sm. effect of various drying and dehydration techniques on the organoleptic quality of mango leathers. rads j biol res appl sci. 2021; 12(1):66-74. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o r i g n a l a r t i c l e effect of drying-dehydration techniques on organoleptic quality of ml vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 67 i n t r o d u c t i o n mango (mangifera indica l.) is known as the king of all fruits. pakistan is ranked as the 5th largest country in terms of world mango production. it is 2nd prime fruit of the country followed by citrus. in pakistan, major mango growing provinces are punjab and sindh. it shows abundant market presence in the months of may, june, and july which is also the peak harvesting period. greater than 200 types of mangoes are cultured in pakistan. chaunsa and sindhri are the most well-known varieties in the country. however, its production is low due to insufficient and unstable fertilization, high water table, salinity, and deficiency of acceptable plant safety actions. the pulp of mango is used as a basic ingredient to develop jams, juices, sauces, chutneys (dips), and preserved with just intent of raw material for the development of delicacies like pastries fillings or cake toppings instead of its sole utilization as fruit leather. this is the reason that in pakistan, more industries focus on the preservation of mango pulp as compared to its processing for other purposes1. using chemicals either alone or in combination for preservation is mainstreamed in pakistan, which helps to prevent microbiologically safe and rancidity or oxidation-free shelf-stable fruit preserves2. at times, chemical preservation is accompanied by storage at very low temperatures, which in case of prolonged duration calls for surface decay by discolouration and texture deterioration named as chilling injury3. the drying process involves the elimination of water from food either naturally or artificially with measured situations so that the attack of numerous spoilage microbes can be prohibited thus, preserving the food4. drying is nothing but ensuring the minimization of free water in the fruit tissues which the microbes use for growth and survival. application of drying techniques to fruit puree yields a stable shelf-life product fruit leather, which is of soft rubbery texture with a sweet taste and especially dehydrated5. the appetizing fruit slices (one or more varieties) are smashed, combined with other additives to enhance their nutritional and functional properties, molded, cooked, and processed for moisture evaporation on a smooth tray until consistent fruit leather is obtained6. fruit leathers are appealing, nutritionally recommended and satiety providing fruit products that can be eaten for healthy snacking itself or in the processing of other fruit product development. several drying methods are being used for fruit leather preparations namely: commercial dehydrator drying, cabinet drying or hot-air oven drying, sun drying, and vacuum oven drying5, 7. the composition of the final fruit product may vary depending on the processing conditions. the finished fruit product's composition can differ based on the operating conditions. conventionally, solar drying has been used to produce fruit leather from mature fruit. however, sun-dried items can discolour and the procedure can be unsanitary and time-consuming8. sun drying is also a matter of nature's mercy and can be problematic due to rain or prevailing dust in the air. hot air drying, on the other hand, is an efficient process that takes less time and increases the dried fruit's consistency9, 10. however, it has been shown that hot air drying can promote a decrease in the antioxidant capacity of fruit (such as oranges) at hightemperature drying or long-time drying at about 40°c11. remarkably, the drying technique i.e. commercial dehydrator for the production of mango leather results in a product with better-quality flavour and colour associated with old-style sun-drying way; thus seems to be an ideal drying process for the production of mango fruit leathers12. sun-drying allows the product to have a luminous presence, a typical colour, and a sticky texture. though, there are drawbacks like drying process exposure of the products to ecological pollution, reliance on climate circumstances, and hand labour necessities. so, another drying method was settled to stun the difficulties of cleanliness and time, as these methods are safe, fast, and manageable13. modern techniques-based dryers, such as forced air circulation, cabinet dryers and tunnel dryers, are being used for manufacturing organoleptically appealing fruit leathers especially focusing on flavour and colour. the product deviations during drying contain puffing and shrinkage due to the use of hot air occasionally, coupled with chemical reactions that cause changes in colour, texture, odor, and other properties in the final product. drying occurs from the vaporization of liquid by the provision of heat to the wet material. in various methods, improper drying results in irrevocable harm to the quality of the product which makes the product non-saleable14, 15. with modern dehydrators and well-designed drying methods, fruit leather can be dried at any time of the year to reach the requirements of customers. effect of drying-dehydration techniques on organoleptic quality of ml vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 68 the appropriate selection of mango variety and postharvest maturation phase is very important to maintain the product superiority and sensory properties after processing. raw mangoes are acidic and ironic in vitamin c whereas, ripe mangoes comprise moderate levels of vitamin c. the content of β-carotene, carotenoids, and xanthophyll esters in fruit upsurges exponentially during ripening16. it has been stated that air-drying marks in substantial fluctuations in the color though freeze-drying results in goods with higher color17. the elevated temperature of drying amplified the blackening of mango fruits. observed signs expressed that drying could either rise arytenoids content or decrease it. extreme sufferers in the vitamin c contents of mango fruits after drying have also been informed. despite wide study on mango slices of many varieties, there is limited data accessible on the effect of convectional air drying on mango leather18. therefore, this research was conducted to develop the mango fruit leather under various drying methods and to evaluate the best suitable drying method for the development of mango leathers from the sindhri mango variety. m a t e r i a l & m e t h o d s the study was carried out to examine the effect of several drying methods on the progress of the quality of mango leathers at the laboratories of the institute of food sciences and technology, faculty of crop production, sindh agriculture university, tandojam, during the year 2019. the development of mango fruit leather was conducted as the method applied by maskan, et al. 2002 with slight modification13. the mature ripe mangoes (sindhri variety) were procured from the mango orchard nearby tandojam. the mangoes were peeled off and pulp was extracted using the lab scale pulper, mixed with the different additives as given in table 1. table 1. preparation of mango leather. ingredients quantity pulp 3 kg sugar 1 kg citric acid 2g ascorbic acid 0.8g the pulp with additives was cooked to achieve the 50% total soluble solids and poured into the moulding trays6. the material was subjected to three different treatments (i.e., sun drying, hot air oven drying at 70°c ± 2°c and drying in a commercial dehydrator at 70°c ± 2°c). inclusion criteria with the development of technologies, several drying techniques have been introduced throughout the world and are still in progress to make the process more efficient and reliable. some common and widely used techniques for the quality assessment of mango leathers are given here. sun drying the puree mixture was spread on a grease-free paper (butter paper) and was dried on a perforated tray at a temperature of 35ºc ± 2ºc for 4 days in sunlight, continuously as to maintain the content of moisture up to 15-20%. the dried leather was transferred to the cutting table, rolled, and desired shaped product was obtained after cutting. the final product was then packed in wrappers and stored in an airtight container at ambient temperature for further analysis19, 20. commercial dehydrator the cooked pulp was transferred to parchment paper and dried in preheated tray dryer at 70ºc ± 2ºc for 8-10hours to maintain the moisture content of 15-20%. after obtaining dried leather it was moved to the tray and rolled to spread uniformly, and cut into the desired size. the final fruit leather product was then packed in packages impermeable to moisture and stored in an airtight container at shelfstable temperature for further analysis20, 21. oven drying the puree was spread on a grease butter paper and dried in preheated oven at 70°c ± 2°c for 24hours to maintain the moisture content of 15-20%. the dried leather was removed from the tray and cut into the desired size or rolled. the final product was then stored at ambient conditions after packing in wrappers to facilitate further analysis20. sensory analysis the sensory characteristics (color, appearance, texture, flavor/taste, and overall acceptability) were assessed by a panel of trained judges following a 7-point hedonic scale (larmound, 1977). the acceptability criteria was defined effect of drying-dehydration techniques on organoleptic quality of ml vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 69 as each characteristic will remain acceptable in the obtained mean score of 5 or above. statistical analysis three different drying treatments were performed and samples were obtained from each treatment. the data were subjected to statistical analysis using spss 23 version, two and the one-way analysis of variance (anova) was performed to know the significant results of different drying techniques to formulate the method of drying significantly (p < 0.05) influenced the sensory qualities followed by steel et al. (1996) and storage period. r e s u l t s the mango fruit is used widely in the preparation of various food products including mango leather. the food industries preserve mango pulp with the addition of preservatives to enhance the storage period along with to retain the sensory quality. later, they use the preserved pulp for the production of juices, jams, mango leather and so on. our study focused on the drying methods and storage duration of mango leather with the observation of sensory characteristics as discussed here. colour scores the results for a colour score of mango leather are presented in fig. 1. it was observed that the score of colour was slightly decreased (p < 0.05) in the drying score of mango leather dried by the different drying methods during storage time. mango leather dried by commercial dehydrator at 70ºc ± 2ºc gave the highest score of colour (6.20), hot air oven drying at 70ºc ± 2ºc was recorded (5.93) colour score, whereas the mango leather dried under sun-drying at 35ºc ± 5ºc gave the lowest score (5.88) of mango leather colour. in the results regarding the storage of the product prepared, there was a significant decrease (p < 0.05) in the storage period as compared to the initial stage (control). the mango leathers stored after 4 weeks gave the highest (6.33) score of colour, while those stored after 06, 08, and 10 weeks were observed as 6.23, 6.20, and 5.43 scores of mango leather colour, respectively. the mango leather stored after 12 weeks gave the lowest (5.33) colour score. there was no significant difference observed in colour after 10 and 12 weeks of storage. texture scores the results for the texture score of mango leather are presented in fig. 2. it was observed that the score of texture was slightly decreased (p < 0.05) in the drying score of mango leather texture dried by the different drying methods. mango leather dried by commercial dehydrator at 70ºc ± 2ºc gave the highest score of texture (6.13), while those dried by hot air oven drying at 70ºc ± 2ºc was recorded (5.87) texture score and, those dried under sundrying at 35ºc ± 5ºc gave the lowest score (5.65) of texture. in the results regarding the storage of mango leather, there was a significant decrease (p < 0.05) in the storage period as compared to control. the mango leather stored after 4 weeks gave the highest (6.13) score of texture, while those stored after 06, 08, and 10 weeks were observed 5.93, 5.87, and 5.53 score of texture, respectively and after 12 weeks of storage, gave the lowest (5.47) texture score. there was no significant difference observed in texture after 10 and 12-weeks storage. figure 1. effect of different drying methods on the color of mango leather. effect of drying-dehydration techniques on organoleptic quality of ml vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 70 figure 2. effect of different drying methods on the texture of mango leather. flavor / taste the results for the flavour/taste score of mango leather are presented in fig. 3. it was observed that the score of flavour/taste was slightly decreased (p < 0.05) in the drying score of mango leather flavour/taste dried by the different dehydration methods used.the mango leather dried by the commercial dehydrator at 70ºc ± 2ºc gave the highest score of flavour/taste (5.88), while mango leather dried by hot air oven drying at 70ºc ± 2ºc was recorded (5.77) flavour/taste score, whereas under sun-drying at 35ºc ± 5ºc gave the lowest score (5.65) of mango leather flavour/taste. in the results regarding the storage of mango leather, there was a significant decrease (p < 0.05) in the storage period of mango leather as compared to control. mango leather stored after 4 weeks gave the highest (6.13) score of flavour/taste, while those stored after 06, 08, and 10 weeks were observed 5.77, 5.73, and 5.33 scores of flavor/taste, respectively. the mango leather stored after 12 weeks gave the lowest (5.23) flavour/taste score. there was a non-significant difference observed in flavor/taste after 10and 12-weeks storage. figure 3. effect of different drying methods on flavor/taste of mango leather. effect of drying-dehydration techniques on organoleptic quality of ml vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 71 figure 4. effect of different drying methods on the appearance of mango leather. appearance the results for the appearance score of mango leather are presented in fig. 4. it was observed that the score of appearance was slightly decreased (p < 0.05) in the drying score of mango leather appearance dried by the different drying methods. mango leather dried by commercial dehydrator at 70ºc ± 2ºc gave the highest score of appearance (5.88), while mango leather dried by hot air oven drying at 70ºc ± 2ºc was recorded (5.53) appearance score, whereas mango leather dried under sun-drying at 35ºc ± 5ºc gave the lowest score of (5.28) appearance. in the results regarding the storage of mango leather, there was a significant decrease (p < 0.05) in the storage period of mango leather as compared to control. mango leather stored after 4 weeks gave the highest (5.75) score of appearance, and those stored after 06, 08, and 10 weeks were observed at 5.60, 5.50, and 5.43 scores appearance, respectively. the mango leather stored after 12 weeks gave the lowest (5.27) appearance score. there was a non-significant difference observed in appearance after 10 and 12-weeks storage. overall acceptability the results for the overall acceptability score of mango leather are presented in fig. 5. it was observed that the score of overall acceptability was slightly decreased (p < 0.05) in the drying score of appearance dried by the different drying methods. mango leather dried by a commercial dehydrator at 70ºc ± 2ºc gave the highest score of overall acceptability (5.85), while those dried by hot air oven drying at 70ºc ± 2ºc was recorded (5.70) overall acceptability score, whereas the mango leather dried under sun-drying at 35ºc ± 5ºc gave the lowest score (5.62) of overall acceptability. in the results regarding the storage of mango leather, there was a significant decrease (p<0.05) in the storage period of mango leather as compared to control. mango leather stored after 4 weeks gave the highest (6.10) score of overall acceptability, while mango leather stored after 06, 08, and 10 weeks were observed 5.87, 5.83, and 5.20 scores of overall acceptability, respectively. the mango leather stored after 12 weeks gave the lowest (5.03) overall acceptability score. there was a non-significant difference observed in overall acceptability after 10 and 12-weeks storage. effect of drying-dehydration techniques on organoleptic quality of ml vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 72 figure 5. effect of different drying methods on overall acceptability of mango leather. d i s c u s s i o n fruit leather can be obtained using drying processes of fruit puree into a leathery sheet from dried sheets of fruit pulp that have a soft, rubbery texture along with a sweet taste (raab and oehler, 2011)22. it has been obtained from the results that the mango leather dried by commercial dehydrator at 70ºc ± 2ºc gave the highest score of colour (6.20), texture (6.13), flavour/taste and appearance (5.88), and overall acceptability (5.85), while mango leather dried by hot air oven drying at 70ºc ± 2ºc was recorded with the score of colour (5.93), texture (5.87), flavour/taste (5.77), appearance (5.53) and overall acceptability (5.70), whereas the mango leather dried under sun-drying at 35ºc ± 5ºc gave the lowest score of colour (5.88), texture (5.65), flavour/taste (5.65), appearance (5.28) with overall acceptability (5.62) and results are significant among all the drying methods. the multi-origin fruit pulps at times with edible peels are converted to puree, mixed with few compositional additives to enhance organoleptic and physicochemical properties, cooked, poured to moulding trays, cut into desired shapes, and then dried to form cohesive fruit leather catering especially the product appeal (phimpharian et al., 2011)6. fruit leathers can be eaten as snack foods or added to a variety of food preparations and textural properties can be modified by using different drying techniques and varied equipment (raab and oehler, 201122; irwandi et al., 20187). the results further showed that the storage of mango leather had also a significant effect (p < 0.05) in the storage period as compared to control. mango leather stored after 4 weeks gave the highest score of colour (6.33), texture (6.13), flavour/taste (6.13), appearance (5.75), and overall acceptability (6.10). the results further revealed that the mango leather stored after 12 weeks gave the lowest mango leather score of colour (5.33), texture (5.47), flavour/taste (5.23), appearance (5.27), and overall acceptability (5.03). there was a non-significant difference was observed in all the sensorial parameters after 10 and 12-weeks storage. fruit product composition changes with modifications in processing conditions. the most conventional technique used for the development of fruit leather is sun drying, although the process of sun-drying is time-consuming and gives less hygienic product with a streak of discolouration, and susceptibility towards the microbial load (teshome et al., 2012)8. high-temperature treatment at above 80ºc or even treatment at 40ºc for a longer duration decreases the antioxidant potential of fruit leathers (heikal et al., 2016)11. drying of fruit puree in hot air oven is comparatively a better solution for improved organoleptic and compositional quality of the end product developed from fruits as it utilizes less time (maskan et al., 20139; garau et al., 201710); however, it has been shown that hot air drying can promote a decrease in the effect of drying-dehydration techniques on organoleptic quality of ml vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 73 antioxidant capacity of fruit (such as oranges). there are limited studies found about the influence of conventional drying techniques for mango leather development from the sindhri variety. c o n c l u s i o n it is concluded from the above study that the sun-drying method is found time-consuming as compared to hot air oven drying and commercial dehydrator techniques. additionally, the traditional drying technique results in poor sensory properties of mango leather. furthermore, a commercial dehydrator is a more suitable and controlled method, showing the highest scores of sensory qualities than sun-drying and hot air oven drying of mango leather. moreover, storage condition results showed that the product remains shelf-stable for at least 6-month, without the need for chemical preservatives due to the reduction in moisture content of the products. s u g g e s t i o n s / f u t u r e p e r s p e c t i v e it is suggested that further studies are required to study nutritional facts and the microbiological quality of mango leather in the storage period. also, the texture of mango leather can be improved by adding hydrocolloid, guar gum/pectin etc. the mango properties during processing must be monitored and measured so that if there is any problem developed it can be quickly detected, and the process will be adjusted to compensate for it. this helps to improve the overall quality of the mango leather and reduce the amount of material and time wasted. the final mango fruit leather product has to be also analyzed and characterized to ensure that it retains its desirable properties up to the time when it is consumed, meets the appropriate high-quality requirements, and that it is safe for consumption. c o n f l i c t s o f i n t e r e s t none. f u n d i n g s o u r c e none. a c k n o w l e d g m e n t s the author is grateful to the concerned institution for providing facilities to conduct this study. l i s t o f a b b r e v i a t i o n s anova analysis of variance kg kilogram r e f e r e n c e s 1. hussain i, zeb a, shakir i, shah as. combined effect of potassium sorbate and sodium benzoate on individual and blended juices of apricot and apple fruits grown in azad jammu and kashmir. pak j nut. 2008; 7(1):181-5. 2. hashmi ms, alam s, riaz a, shah as. studies on microbial and sensory quality of mango pulp storage with chemical preservatives. pak j nut. 2007; 6(1):858. 3. wills rbh, lee th, mcglasson dg, hall eg. postharvest – an introduction to the physiology and handling of fruit and vegetables. avi pub. co. inc, westport, conn. alternaria alternate in mango fruits. postharvest biol technol. 2016; 15: 165-74. 4. mathooko fm, okoth em, sila dn, onyango ca, owino wo, musyimi sm. evaluation of chemical and nutritional quality attributes of selected mango varieties at three stages of ripeness, grown in lower eastern province of kenya. j anim plant sci. 2013; 17(3):2619-30. 5. ofoedu ce, ubbaonu cn, agunwah i, obi cd, odimegwu ne, okeke fk. production and comparative evaluation of leather products from pawpaw (carica papaya) and banana (musa acuminata) fruit pulp. croatian j food sci technol. 2020; 12(2):218-28. 6. phimpharian c, jangchud a, jangchud k, therdthai n, prinyawiwatkul w, no hk. physicochemical characteristics and sensory optimisation of pineapple leather snack as affected by glucose syrup and pectin concentrations. int j food sci technol. 2011; 46(5):972-81. 7. irwandi j, man yc, yusof s, jinap s, sugisawa h. effects of type of packaging materials on physicochemical, microbiological and sensory characteristics of durian fruit leather during storage. j sci food agricul. 1998; 76(3):427-34. effect of drying-dehydration techniques on organoleptic quality of ml vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 74 8. teshome b. effect of processing on some quality attributes of mango (mangifera indica) fruit leather. j food sci. 2012; 35(4):554-63. 9. maskan a, kaya s, maskan m. effect of concentration and drying processes on color change of grape juice and leather (pestil). j food eng. 2002; 54(1):75-80. 10. garau mc, simal s, rossello c, femenia a. effect of air-drying temperature on physico-chemical properties of dietary fibre and antioxidant capacity of orange (citrus aurantium v. canoneta) by-products. food chem. 2007; 104(3):1014-24. 11. dhiman ak, thakur p, attri s, kathuria d, ramachandran p. utilization of ripe pumpkin (cucurbita moschata) for the development of fruit bar. curr j appl sci technol. 2020; 63-73. 12. islam f, sujan mh. rationality of hog plum cultivation in jhalokathi district, an empirical analysis in the socioeconomic context of bangladesh. j adv soc sci human. 2016; 2(1):8-18. 13. maskan a, kaya s, maskan m. hot air and sun drying of grape leather (pestil). j food engg. 2002; 54(1):818. 14. huang x, hsieh fh. physical properties, sensory attributes, and consumer preference of pear fruit leather. j food sci. 2005; 70(3):177-86. 15. singh gh, singh bs. effect of hydrocolloids on the dehydration kinetics, color, and texture of mango leather. int j food prop. 2003; 6(2):269-79. 16. pott i, marx m, neidhart s, mühlbauer w, carle r. quantitative determination of β-carotene stereoisomers in fresh, dried, and solar-dried mangoes (mangifera indica l.). j agricul food chem. 2003; 51(16):4527-31. 17. azeredo hm, brito es, moreira ge, farias vl, bruno lm. effect of drying and storage time on the physicochemical properties of mango leathers. int j food sci technol. 2006; 41(6):635-8. 18. karabulut i, topcu a, duran a, turan s, ozturk b. effect of hot air drying and sun drying on color values and β-carotene content of apricot (prunus armenica l.). lwt-food sci technol. 2007; 40(5):753-8. 19. yılmaz fm, yüksekkaya s, vardin h, karaaslan m. the effects of drying conditions on moisture transfer and quality of pomegranate fruit leather (pestil). j saudi soc agricul sci. 2017; 16(1):33-40. 20. sarkar t, chakraborty r. formulation, physicochemical analysis, sustainable packagingstorage provision, environment friendly drying techniques and energy consumption characteristics of mango leather production: a review. asian j water environ pollut. 2018; 15(3):79-92. 21. azeredo hm, brito es, moreira ge, farias vl, bruno lm. effect of drying and storage time on the physico‐ chemical properties of mango leathers. int j food sci technol. 2006; 41(6):635-8. 22. raab c, oehler n. making dried fruit leather. oregon state university extension service, usa. 2011; 1-4. investigating age dependent diversification of bone biomarkers vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 53 op e n ac c e s s f u l l l e n g t h a r t i c l e investigating age dependent diversification of bone biomarkers in females farah jabeen*, s. ayesha sehrish, afshan zeeshan wasti, farha aziz department of biochemistry, jinnah university for women, karachi 74600, pakistan a b s t r a c t background: bone is responsible to perform vital functions to provide support and maintain the structure of the body. due to certain reasons, the bone encounters certain disorders which affect the bone functionality, especially aging in females. objectives: the study was designed to establish a relationship between biomarkers of bone metabolism with age & to analyze the occurrence of bone disorders with increasing age, specifically in females. methodology: random samples of females were collected from the population of karachi spliced as control with less than 30 years of age and as tests with late 30 years of age. the analysis was done exclusively with the detection of biochemical markers of bone turnover, including calcium, phosphorus, alkaline phosphatase, rheumatoid factor (rf) and c-reactive protein (crp). the relationship between bone health, lipid profile, glycemic index and blood profile was also analyzed. results: results depicted that alterations in normal serum concentrations of all biomarkers were frequent in elder females as compared to the younger ones. conclusion: in conclusion, the biomarkers of bone metabolism are closely related to age, evaluating that older females are more prone to the risk of developing bone diseases. the variables portrayed were useful in understanding their role concerning age in the development of bone. keywords bone, biomarkers, calcium, rheumatoid factor (rf), crp, blood profile *address of correspondence farah786star@yahoo.com article info. received: may 18, 2018 accepted: october 28, 2019 cite this article: jabeen f, sehrish a, wasti az, aziz f. investigating age dependent diversification of bone biomarkers in females. rads j biol res appl sci. 2020; 11(1): 53-60. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n bone is a living, hard and metabolically active connective tissue that builds up the vertebral skeleton. bone is mainly composed of minerals, organic matrix, water and lipids in decreasing proportions. hydroxyapatite [ca10 (po4)6 (oh)2] a key component of bone is insoluble salt of calcium and phosphorus1. the principal function of bone is to provide mechanical support to the body, protection to the vital organs, maintaining structure, acid-base balance, mineral homeostasis and hematopoiesis. several clinical conditions are associated with bones including osteoporosis, fractures, tumors and bone cancer. the process of bone formation is termed osteogenesis that happens on soft tissues during developmental stages or the healing processes. bone modeling is the formation of bones on the existing bone tissues for proper morphological development, involving the changes in shape, curvature or thickness of bone. bone remodeling is the coupled process of bone resorption followed by ossification on specific sites in the adult skeleton for the o r i g i n a l a r t i c l e investigating age dependent diversification of bone biomarkers vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 54 maintenance of bone turnover and bone mass. the events of bone formation and resorption are strictly regulated by cytokines, growth factors and different systemic hormones2. bone biomarkers biomarkers are the quality features whose quantification and evaluation elucidate the altered physiological conditions. as indicated by the name, “biological markers” are the reflection of biological processes in an individual. bone disease analysis is done exclusively with the detection of biological markers of bone along with imaging techniques and radiographic analysis. some important bone biomarkers are: • biochemical constituents and enzymes as bone biomarkers including calcium, phosphorus, magnesium, alkaline phosphatase (alp), calcitonin, osteocalcin, parathyroid hormone, vitamin d, creactive protein (crp), rheumatoid factor (rf), type 1 collagen etc. • hematological indices as a bone marker • glycemic index as a bone marker • lipid profile as a bone marker biochemical constituents and enzymes as bone biomarkers among biochemical constituents and enzymes as bone biomarkers, calcium is very important as it is the most abundant mineral in the body, of which 99% is stored in bones and teeth and the rest in other parts of the body. its normal range and requirement depending upon age and gender. as in elderly people especially women, the risk of osteoporosis increases with a decrease in body calcium. the dietary requirement for an adult in a day range from 1000-1200mg. moreover, phosphorus is the second most plenteous mineral of the body. it is among the main bone component i.e. hydroxyapatite making bone strong. its daily requirement for an adult is 700mg. alteration in amounts of phosphorus in the body can lead to health conditions like joint pain and muscle fatigue. excessive intake may also ruin bone health. magnesium being the third important bone biomarker is a necessary mineral that maintains the functioning of the heart, bones and blood pressure. its recommended dietary allowance for adult women is 320mg and plays a vital role in maintaining bone health by coordinating calcium absorption and metabolism. alkaline phosphatase (alp) is found in many tissues of the body including the liver, bone, intestine, and placenta. its altered level in serum is normally indicative of two main courses of diseases: either hepatobiliary obstruction or bone disorder usually caused by an increase in osteoblastic activity. calcitonin is a hormone synthesized mainly in thyroid glands along with a variety of other tissues. its reported main function is the involvement in phosphorus and calcium metabolism. it particularly drops calcium levels in the blood by acting upon parathyroid hormone and decreasing bone resorption. besides calcitonin, another important mediator osteocalcin is also synthesized by osteoblasts exclusively plays a key role in bone mineralization as it contains gia proteins, whose geometry completely fits the calcium structure to be incorporated in hydroxyapatite. however, the functions of osteocalcin associated with bone mineralization remain controversial3. parathyroid hormone (pth) is released from parathyroid glands and is known to increase blood calcium level. its release is triggered by the magnesium levels in the blood. it increases the calcium concentration in the blood by either activating osteoclasts, reabsorbing calcium excreting from kidneys or enhancing vitamin d synthesis. pth has also been reported for having anabolic treatment opportunities in bone diseases as in the case of osteoporosis4. vitamin d is a fat-soluble vitamin (also considered a hormone), sometimes plays an important role in calcium metabolism and homeostasis. the normal serum concentrations of 25-(oh)-vitamin d is ≤10ng/ml, contributing to bone diseases. its deficiency can cause hyperparathyroidism, increased bone turnover, bone loss, and in extreme cases can also lead to osteomalacia5. c-reactive protein (crp) is an inflammatory marker usually associated with diseases like diabetes, cardiovascular diseases, lupus and rheumatoid arthritis. it is synthesized in the liver and increases in response to the extent of inflammation in the body. high sensitivity c-reactive protein (hs-crp) is allied with a low mineral density of bones. another inflammatory biomarker is the rheumatoid factor, an autoantibody against igg in humans usually detected for the diagnosis of rheumatoid arthritis i.e. an autoimmune disorder characterized by chronic investigating age dependent diversification of bone biomarkers vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 55 inflammation of joints. rf can also be checked in people with other autoimmune disorders6. type 1 collagen is chiefly found in bones along with soft tissues providing a matrix for mineralization. procollagen type 1 c-terminal pepeptide (pcip) is secreted into the circulation by osteoblasts during early proliferative stages of bone formation which is followed by bone resorption being identified by the presence of cross-linked telopeptide of type 1 collagen7. hematological indices as bone marker hematological indices are also considered as a bone marker. some studies demonstrated the positive association between bone health and blood profile regarding the influence of hematopoiesis and reported it to be a presumed indicator of bone mineral density (bmd)8. studies further represent that in rheumatoid arthritis the haemoglobin and erythrocyte count decreases that stimulate the risk of anaemia, whereas no considerable change was observed in total wbcs. in another study, neutrophils-lymphocyte and platelet-lymphocyte ratios were reported as biomarkers of rheumatoid arthritis9. glycemic index as bone marker glycemic index is also considered as a bone marker e.g.: in diabetes type 1, the insulin deficiency results in altered bone metabolism because insulin has anabolic effects on bone metabolism that can preserve bone quality10. however, in type 2 diabetes, the body does not respond to the insulin produced with reduced risk of fractures and higher bone mineral density (bmd). anti-diabetic medicines have also been found associated with decreased bone quality11. lipid profile as bone marker while considering the lipid profile as a bone marker, recent studies based on epidemiological characteristics and clinical trials reported that high-fat mass contributes significantly to bone fragility and an extent of osteoporosis. increased percentage of body fat is significantly associated with the increased risk of bone diseases, particularly osteoporosis, osteopenia, and non-spine fractures. total cholesterol and low-density lipoprotein cholesterol had a negative correlation with bone mass density at the femur and spine. decreased hdl levels can affect bone turnover as osteoblastic functions are disturbed due to the inflammatory environment resulting in ultimately decreased bone mass12. other bone health predictors are obesity, bmi, and physical inactivity in which obesity can cause deleterious effects on bone as it can lead to fat deposition in bone marrow tissues. several factors like ageing, diabetes, metabolic syndrome, and hormone impairment may lead to bone loss which could be overcome by maintaining a balance between diet and exercise13. women with high abdominal fat have been reported to have the worst bone quality. adipose tissues are associated with the secretion of certain hormones like estrogens in postmenopausal females and other adipokines which interferes with bone formation and resorption. bone formation is enhanced as the result of increased weight and mechanical load although this can be inversed as lipotoxic effects of adipocytes14. bmi is considered a more approachable index when correlated with conditions like diabetes and hypertension. sharmin et al. reported that bone loss was associated significantly with low bmi and ageing process15. although age, weight, and bmi reflect bmd, bone loss can also be influenced by other factors such as lifestyle and nutritional habits15. physical exercises that focus on bone density are shown to enhance bone strength and cause a decline in bone loss and bone pain. the preservation of bmd in pre and postmenopausal women was reported in response to physical activity. for young adults despite sex variation, bmd is directly associated with physical exercise16. therefore; the current study was designed to establish a relationship between biomarkers of bone metabolism with age; to analyze the occurrence of bone disorders with increasing age specifically in females. m a t e r i a l s a n d m e t h o d s healthy young female participants were included in the study. blood samples of women were collected randomly from the population in karachi. there was no specification considered rather than gender. in total, 55 samples were collected out of which 30 were considered as control and 25 were considered as a test, depending upon age i.e. females with <30 years of age were selected as control whereas females with >30 years of age were included in tests. women who had amenorrhea due to hysterectomy or any other known causes like those taking any hormone investigating age dependent diversification of bone biomarkers vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 56 replacement therapy or on any bone-related medications, those with a history of thyroid disorders, jaundice and liver diseases were excluded from the study. a questionnaire regarding anthropometry was conducted along with all controls and tests models. data including age, height, weight, habits, lifestyle and medical history was collected. yellow capped clot activating gel vacutainer was used to collect serum; whereas purple capped ethylenediaminetetraacetic acid, (edta) vacutainer was used for plasma. collected samples were centrifuged to separate serum /plasma which was then stored at 2-8ºc. all samples were thawed before tests execution. parameters estimated calorimetrically including fasting blood glucose by following glucose oxidase method (randox-glucose-gluc-pap)17, cholesterol by cholesterol oxidase/peroxidase method (biosystems)18, triglyceride by using glycerol phosphate oxidase/peroxidase method (biosystem)19, hdl via phosphotungstate/mg-cholesterol oxidase/peroxidase method (biosystems)20, ldl cholesterol was calculated using friedewalds formula (estimation of the concentration of low-density lipoprotein cholesterol in plasma) without using the preparative ultracentrifuge21. calcium was estimated by calcium monor by diacon systems22,23. enzyme activity of alkaline phosphatase was estimated via the kinetic method by using p-nitrophenylphosphate as substrate (lab kit)24,25. phosphorus-phosphomolybdate (spinreact) was used for the estimation of phosphorus on uv-visible spectrophotometer (jenway 6305)26. the crp level was determined by a quantitative turbidimetric method using atlas medical–crp latex kit27,28 while, rheumatoid factor (rf) was determined by atlas medical – rf latex kit29,30. r e s u l t s total 55 females (30 control & 25 test subjects) fulfilling the selection criteria were allocated. the mean age of control is 23.35 ± 0.27 years and 58.13 ± 2.41 years for test subjects anthropometric data are given in table 1. the biochemical profiling of this study included the evaluation of fasting glucose levels, hba1c, lipid profiling, alkaline phosphatase activity, calcium and phosphorus serum levels as well as analysis of crp and ra factor in the subjects. the results of biochemical parameters including enzymatic assay and determination of crp and ra were compared in test and control subjects. no significant difference in fbg and hba1c were found in a test as compared to the control group. whereas a significant increase in lipid profile including cholesterol, triglycerides, ldl was found in test subjects while comparing with control (table 2). the study demonstrated that calcium levels drop-off with the increase in age (p = 0.0227), whereas phosphorus levels rise with the increasing age (p < 0.0001); showing the inverse relationship between calcium and phosphate levels i.e. the decrease in serum concentration of calcium causes the increase in phosphorus concentration in serum and vice versa (fig. 1). figure 1. comparison of calcium and phosphorus levels in control and test subjects table 1. statistical results of anthropometric data. s. no. parameter age height weight bmi 01 control (n=30) 23.35 ± 0.27 52.37 ± 1.58 158.044 ± 1.980 21.140 ± 0.733 02 test (n=25) 58.13 ± 2.41 66.67 ± 1.58 162.983 ± 2.014 25.190 ± 0.682 n = no of subjects.. student t-test was done to calculate the p-value, the significance level was assumed to be < 0.05. values are represented as mean ± sem. investigating age dependent diversification of bone biomarkers vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 57 table 2. statistical results of quantitative biochemical parameters. s. no. parameters control (n = 30) test (n = 25) p-value 01 glucose (75-115mg/dl)* 91.349 ± 4.151 104.208 ± 7.574 p = 0.1149 02 hba1c (4-5.6%)* 4.811 ± 0.140 5.266 ± 0.251 p = 0.0953 03 cholesterol (200-239mg/dl)* 185.217 ± 7.877 225.318 ± 10.729 p = 0.0036 04 triglycerides (150-199mg/dl)* 138.975 ± 9.332 191.253 ± 10.628 p = 0.0007 05 hdl (> 60mg/dl)* 67.956 ± 2.958 81.877 ± 7.583 p = 0.0581 06 ldl (130-160mg/dl)* 89.4`65 ± 10.035 105.189 ± 9.571 p = 0.2869 07 alp (60-170u/l)* 42.33 ± 2.693 96.125 ± 3.395 p = 0.0001 08 calcium (8.5-12.0mg/dl)* 9.284 ± 0.323 7.896 ± 0.532 p = 0.0227 09 phosphorus (2.5-5.0mg/dl)* 3.732 ± 0.298 5.720 ± 0.340 p < 0.0001 10 crp (mg/dl) *(1-3mg/l) 1.85 ± 0.23 4.38 ± 0.25 p < 0.0001 11 rf + (less than 5%) +++ (approx 80%) n = no of subjects. student t-test was done to calculate the p-value, the significance level was assumed to be < 0.05. values are represented as mean ± sem. therefore, it has been evidenced that the increasing age has negative effects on bone biomarkers resulting in poor bone quality and requiring the increased aid of health assistance as the life proceeds. alp and phosphorus levels were found to increase in test subjects while comparing with control. a significant decrease was found in hdl and calcium level in a test as compared with control subjects. crp and ra factor analysis revealed that creactive protein was found to be significantly increased in test subjects as compared to the control group. while the ra found to be positive (approx 80%) only in test subjects in comparison to control subjects (less than 5%) indicating the presence of bone-related inflammation in these subjects. d i s c u s s i o n biomarkers of bone metabolism are closely related to age. this study evaluated that elder female are more prone to the risk of developing bone diseases when compared to younger females. this indicates the hazard of bone deterioration with the growing age. blood glucose level and bone health diabetes and osteoporosis are among the most prevalent diseases of the era. altered levels of blood glucose levels are found to be associated with altered bone metabolism, which results in decreased bone quality. diabetes type 1 is associated with insulin deficiency and results in altered bone metabolism because insulin has anabolic effects on bone metabolism that can preserve bone quality10. however, in diabetes type 2, the body does not respond to the insulin produced with reduced risk of fractures and higher bmd. this study demonstrated an increase in blood glucose levels and hba1c scores in elder females when compared with the control group, illustrating that aged women are more prone to the risk of diabetes as well as bone disease. lipid profile as a bone marker this study showed a significant increase in cholesterol and triglycerides levels in elderly women, exhibiting the risk of decreased bone quality. previous studies also reported the risk of low bmd with increased cholesterol and triglycerides levels31. another study reported the positive association between lipid profile and osteoporosis in postmenopausal investigating age dependent diversification of bone biomarkers vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 58 women however no significant relation between bmd and osteoporosis was reported32. alkaline phosphatase as a bone marker high levels of alp are usually associated with higher age regardless of the fact of presence of isozymes which can indicate several clinical conditions. although in this study, no significant relationship between elderly and young females was observed, an increase in mean serum concentrations was observed for elder females. another study reports a significant increase in alp in postmenopausal women with an inverse relation to serum calcium levels33. calcium as a bone marker the calcium level of the test group was significantly decreased pointing to overage as the dominating reason for the risk of bone health disruption, as low calcium levels cause an increase in bone resorption34. the reason behind low calcium levels in elder people can be malabsorption, one study reported that calcium absorption decreases significantly as age increases especially after 60 years of age35. that could be related to the declined levels of estrogen and other hormones at menopause35. phosphorus as a bone marker in this study, phosphorus was significantly increased in the test group. increased phosphorus has deleterious effects on bone and can also relate to heart disease. it has been reported that low blood pressure was associated with high phosphorus36. hypophosphatemia is also associated with osteomalacia as it will cause the impaired formation of hydroxyapatite. a finding showed that long term exposure to high phosphorus can lead to bone impairment37. association between calcium and phosphorus this research depicts the inverse relation between calcium and phosphorus levels i.e. high phosphorus and low calcium in the test group and vice versa. a negative relation between skeletal health and low calcium and phosphorus ratio was reported. thus, the risk of development of bone disease in older females was higher than the younger ones. calcium and phosphorus are dependent on parathyroid hormone and vitamin d levels; increased phosphorus has disastrous effects on bone whereas increased calcium levels have protective effects38. c-reactive protein (crp) as a bone biomarker c-reactive protein is an inflammatory marker produced by the liver, indicating tissue damage. bone health is closely associated with crp, as high crp levels indicate poor bone health and increased fracture risk39. several studies do not report crp as an indicator of low bmd, but relate it as the cause of bone loss, poorer bone quality, and increased fracture risk and joint destruction40. rheumatoid factor (rf) as a bone biomarker rheumatoid factor is an antibody targeted against persons own tissues, resulting in synovial inflammation, leading to rheumatoid arthritis. the solitary study showed 16.66% positive results for the test group that included elder females thus, indicating the risk of rheumatoid arthritis in aged women. another study reported that anti-citrullinated protein antibodies (acpa) were also associated with the risk of ra41. treatment and prevention of bone diseases antiresorptive drugs are effective in preventing bone fractures in combination with anabolic therapy. specific treatments are also available for specific bone conditions. bone diseases can be prevented by meeting the basic nutritional needs to maintain bone health. avoiding alcohol and smoking can also be beneficial. an appropriate combination of physical activity and weight should also be managed in a routine to maintain bone health. maintaining adequate exposure to sunlight and dairy intake can also aid in maintaining bone health. elderly people should also manage to see doctor and routine checkup to reduce the risk of fractures. awareness regarding health state can also be considered. c o n c l u s i o n in conclusion, the alteration of biochemical markers of bone in association to increasing age is intact enough to be considered as the strong predictor of bone health that regards older females at higher risk of developing bone diseases. the parameters analyzed, discussed and described in this study coordinated well in analyzing the role of age in the occurrence of bone disorders, particularly in females. results exposed that elderly women are at risk to encounter bone disorder at the late ages of their life. therefore, women should maintain compatibility among their dietary habits and exercise routine to avoid ending up investigating age dependent diversification of bone biomarkers vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 59 in a diseased state. medical assistance should be taken regularly to monitor bone health and maintaining bone quality. a c k n o w l e d g e m e n t s this work was supported by institutional funds of jinnah university for women, karachi, pakistan. l i s t o f a b b r e v i a t i o n s acpa anti-citrullinated protein antibodies alp alkaline phosphatase bmi body mass index bmd bone mineral density edta ethylenediaminetetraacetic acid fbg fasting blood glucose hdl high-density lipoprotein hs-crp high sensitivity c-reactive protein igg immunoglobulin-g pth parathyroid hormone pcip procollagen type 1 c-terminal pepeptide ra rheumatoid arthritis rf rheumatoid factor r e f e r e n c e s 1. boskey al. bone composition: relationship to bone fragility and antiosteoporotic drug effects. bonekey rep. 2013; 2:447. 2. seibel mj. biochemical markers of bone turnover part i: biochemistry and variability. clin biochem rev. 2005; 26(4):97-122. 3. zoch ml, clemens tl, riddle rc. new insights into the biology of osteocalcin. bone. 2016; 82:42-9. 4. martin tj. bone biology and anabolic therapies for bone: current status and future prospects. j bone metab. 2014; 21(1):8-20. 5. javed r, ghafoor f. a review of vitamin d in 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exercise associated with improved bmd independently of sex and vitamin d levels in young adults. eur j appl physiol. 2016; 116(7):1297-304. 17. barham d, trinder p. an improved colour reagent for the determination of blood glucose by the oxidase system. analyst. 1972; 97(1151):142-5. 18. roeschlau p, bernt e, gruber w. enzymatic determination of total cholesterol in serum. z klin chem klin biochem. 1974; 12(9):403-7. 19. jacobs nj, vandenmark pj. enzymatic determination of serum triglyceride. biochem biophys. 1960; 88:2505. 20. grove th. effect of reagent ph on determination of high-density lipoprotein cholesterol by precipitation investigating age dependent diversification of bone biomarkers vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 60 with sodium phosphotungstate-magnesium. clin chem. 1979; 25(4):560-4. 21. friedewald wt, levy ri, fredrickson ds. estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. clin chem. 1972; 18(6):499-502. 22. farell ec. calcium. kaplan a et al. clin chem the cv mosby co. st louis. toronto. princeton. 1984;1051255. 23. kessler g, wolfman m. an automated procedure for the simultaneous determination of calcium and phosphorus. clin chem. 1964; 10:686-703. 24. wenger c, kaplan a, rubaltelli ff, hammerman c. alkaline phosphatase. clin chem. 1984;1094-8. 25. tietz nw. clinical guide to laboratory tests, 3rd edition. 1995. 26. chaube a, gupta vk. spectrophotometric determination of phosphate in polluted waters by solvent extraction of molybdenum blue. analyst. 1983; 108(1290):1141-4. 27. pepys mb. c-reactive protein fifty years on. lancet. 1981; 317(8221): 653-7. 28. werner m. serum protein changes during the acute phase reaction. clin chim acta. 1969; 25(2):299-305. 29. taborn jd, walker se. rheumatoid factor: a review. lab med. 1979; 10(7):392-5. 30. dorner rw, alexander jr rl, moore tl. critical review: rheumatoid factors. clin chim acta. 1987; 167(1):1-21. 31. brownbill ra, ilich jz. lipid profile and bone paradox: higher serum lipids are associated with higher bone mineral density in postmenopausal women. j women’s health. 2006; 15(3):261-70. 32. bijelic r, balaban j, milicevic s. correlation of the lipid profile, bmi and bone mineral density in postmenopausal women. materia socio-medica. 2016; 28(6):412-5. 33. bhattarai t, bhattacharya k, chaudhuri p, sengupta p. correlation of common biochemical markers for bone turnover, serum calcium, and alkaline phosphatase in post-menopausal women. malays j med sci. 2014; 21(1):58-61. 34. hosking sm, pasco ja, hyde nk, williams lj, brennan sl. recommendations for dietary calcium intake and bone health: the role of health literacy. j nut food sci. 2016; 6(1):1-3. 35. beto ja. the role of calcium in human aging. clin nutr res. 2015; 4(1):1-8. 36. takeda e, yamamoto h, yamanaka-okumura h, taketani y. dietary phosphorus in bone health and quality of life. nutr rev. 2012; 70(6):311-21. 37. penido mg, alon us. phosphate homeostasis and its role in bone health. pediatr nephrol. 2012; 27(11):2039-48. 38. lamberg-allardt c, kemi v. interaction between calcium and phosphorus and the relationship to bone health. in clinical aspects of natural and added phosphorus in foods. springer, new york. 2017; 14557. 39. ishii s, cauley ja, greendale ga, crandall cj, danielson me, ouchi y, et al. c‐reactive protein, bone strength, and nine‐year fracture risk: data from the study of women's health across the nation (swan). j bone miner res. 2013; 28(7):1688-98. 40. danila mi, westfall ao, raman k, chen l, reynolds rj, hughes lb, et al. the role of genetic variants in crp in radiographic severity in african americans with early and established rheumatoid arthritis. genes immun. 2015; 16(7):446-51. 41. tedeschi sk, cui j, arkema ev, robinson wh, sokolove j, lingampalli n, et al. elevated bmi and antibodies to citrullinated proteins interact to increase rheumatoid arthritis risk and shorten time to diagnosis: a nested case–control study of women in the nurseʼs health studies. semin arthritis rheu. 2017; 46(6):6928. biology journal.indd biology journal.indd rads journal of biological research & applied sciences vol 5 (1), january 2014 issn : 2305-8722 original articles prevalence of single and multiple parasitic infection among school, pre-school children in karachi by sex and age group rukhsana talat, yasmeen a.qadir, talat syeda, farzana ibrahim ......................................01 keratitis causing micro-organisms isolated from ophthalmic contact lens solutions mohniyal abbasi, aliya hayat .................................................................................................. 05 carbapenem resistant acinetobacter: restricted the therapeutic alternatives naheed afshan, huda fatima, darakhshan jabeen ...............................................................09 antibiotic resistance pattern and detection of newly emerging resistant gene new delhi metallo-b-lactamase-1 (blandm-1) in escherechia coli and klebsiella pneumoniae erum mazhar, abdul basit khan, sayyada ghufrana nadeem, shazia tabassum hakim ........14 effect of zinc on lentil (lens culinaris l.) metabolites and antioxidant enzyme activities. saima ibrahim, sidra shabbir.................................................................................................. 20 pathogens identification and evaluation of nigella sativa’s (kalonji) antibacterial activity urooj arif, sumaira javed, sayyeda ghufrana nadeem, shazia tabassum hakim ...........25 bacteriological analysis of nile tilapia fish (oreochoromis niloticus) rouqia fatima, qudsia hussain, sayyada ghufrana nadeem, shazia tabassum hakim .........31 isolation and characterization of lactobacillus from fruits & vegetables syesa anum zehra, sumaira javed, sayyada ghufrana nadeem, shazia tabassum hakim .....36 instruction to authors prevalence of single and multiple parasitic infection among school and preschool children in karachi in pakistan by gender and age group rukhsana talat1*, yasmeen a.qadir1, talat syeda2, farzana ibrahim1 1department of zoology, jinnah university for women 5-c nazimabad karachi, pakistan 2department of biochemistry, jinnah university for women 5-c nazimabad karachi, pakistan abstract a total of 770 students have been observered from 5 different schools of karachi in pakistan during january 2013 to december. their stool samples were collected and observed under research microscope (40 xs) and found ascaris lumbricoides (round worm) and ancylostoma duodenale (hook worm). the prevalance of single parasitics infection was found 83.9% of which 39.5% in boys and 44.4% in girls. multiple parasitic infection was observed 42.1%, with 23.7% in boys and 18.4% in girls. keywords: ascariasis, acylostomiasis, prevalence, health, stool introduction the most important infection in asia which is contributing the biggest share of clinical disease burden is write out (sth) infection. there is indication to recommend that the occurrence of ascariasis, predominantly of ascaris lumbricoides, is increasing due to water resources development projects, population increase or displacement, human migration and competing priorities in the health sectar. the adult ancylostoma duodenale (hook worm) dwells in the human intestine and infection is acquired when third stage larvae present in soil penetrates through the skin while ascaris lumbricoides (round worm) infection enters through the oral route by taking contaminated food or water. sth infections are caused by ascaris lumbricoides (round worm) and ancylostoma duodenale (hook worm); it is the third leading parasitic cause of death after malaria and schistosomiasis. (sebastiaan, 2007 ). it is also a big problem and serious health threat in tropical and subtropical developing areas (ohnishi et al 2004). siddiqui et al. (2002) observed the prevalence of parasites infection in a rural area of karachi pakistan. waqar et al. (2003) studied the intestinal parasites in children from northern pakistan. chaudhry et al. (2004) observed the epidermiological factors affecting prevalence of intestinal parasites in children of muzaffarabad pakistan. rana and shivanda (2005) worked on prevalence and distribution of intestinal parasites among school children in kaski district, western nepal. shaukat et al. (2006) studied a record of intestinal parasitic infection from ehsanullah diagnostic laboratory, nazimabad karachi. wani et al 2007 studied the intestinal helminthesis in a population of children from the kashmir valley india. seghal et al. (2010) observed the prevalence of intestinal parasitic infections among school children and pregnant women in socio-economic area, chandigarh, north india. khan et al 2011 investigated intestinal parasite under above 15 years age in farmers of swat .talat et al. (2012) studied the prevelance of helminth infection in different areas of karachi. intestinal infections are more common in developing countries of the world, and cause high mortality rate. pakistan is one of the developing countries where*corresponding author: rukhsanatalat4@gmail.com 01 vol 5 (1), january 2014; 01-04 the incidence of intestinal parasitic infecticion is more common in the province of sindh at karachi city. large numbers of people are suffering with polyparasitism. material and methods the present investigation was carried out on stool samples which were received from the different schools of karachi during january 2013 to december. the selected area was karachi city which is largest city of pakistan, located in southern part of pakistan within the sindh province. the investigation was carried in 5 schools of karachi including pma school, paf school, sheema public school, maripur government school and bahria foundation school. children’s age between 3-11 years. for the present research work slides were prepared by fresh samples of stool which were collected in the disposable glass bottles using hygienic precaution and were examined in laboratory under the research microscope (40 xs). for the preservation of stool samples a solution was used containing 10% formalin, 20% glycerine, and 70% distilled water. school samples were examined for round and hook worm using the kato katz technique (who,1994). the microscopic examination of samples was made within one hour of collection. results and discussion the present investigation was based on the prevalence of human intestinal parasites among the students of different schools of karachi city. for this purpose total 770 stool samples of male and female students of age 311 years collected from 5 different schools including pma school, paf school, sheema public school, maripur government school and bsahria foundation school during january 2013 to december 2013. in this study only two species of parasites were found and identified namely ascaris lumbricoides and ancylostoma duodenale. single parasitic infection was in high intensity rate i.e. 83.9 % in all samples, from which boys have 39.5 % and girls have 44.4 %. multiple parasitic infection was 42.1 % , with 23.7 % in boys and in girls it was 18.4 % (figure 1). the result shows that parasitic infection occurred more frequently in boys than girls because boys are in touch with the environment and badly affected with the germs. the prevalence of intestinal infection is most common in sheema public school and maripur government school, however low frequency was observed in pakistan bahria foundation school multiple parasites were common in both male and female children and it occurred more frequently in older children as compared to younger because the infection becomes more harmful as the age increases as well as not treated properly and because of the lack of treatment. (figure 2) the significance of round worm & hook worm as major public health problems in school and pre 02 vol 5 (1), january 2014; 01-04 figure 1: prevalence of single and multiple parasitic (ascaris lumbricoides and ancyclostona duodenale) infection by sex in 770 students in 5 schools (dates) ? figure 2 : prevalance of single and multiple parasitic infection by age group school children in the study area is not only as single infections but also as concurrent infections. high prevalence rates and intensity of ascaris and ancylostoma infections were found in the studied population. although variations existed among schools, age groups and sexes with regard to prevalence and intensity as well as associated morbidity, all schools, irrespective of sexes and age groups were affected. multiple parasite infections involved two or more parasites and majority of them involved in ascariasis and acylostomiasis. reference chaudhry zh., afzal m. and malik ma. 2004. epidemiological factors affecting prevelance of intestinal parasites in children of muzaffarabad district. pak.j. zool 36: 267-271 khan, w.; noor-un-nisa and khan, a. 2011. an investigation on incidence intestinal parasites in under above 15 years age in farmers of swat, pak. prok. parasital.,52:43:53. kassem hh.,zaed ha and sadaga ga.2007. intestinal parasitic infection among children and neonatus admitted to ibn-sina hospital, sirt, libya. j.egypt soc. prarasital.37: 371-380. rana ms.and shivanada pg. 2005.prevalance and distribution of intestinal parasitic infestations among school children in kaski district,western nepal.j.med.biomed.res.4: 78-82. sebastiaan j.,van.hal., stark dj.,fotedar r., marriott d., john t., ellis jt.and harkness jl., 2007. amoebiasis:current status in australia m.j.a. 186: 412-416. sehgal r., gogulamudi b. reddy., jaco j. verveij., atluri v.subba rao. 2010. prevelance of intestinal parasitic infection among school children amd pregnant women in a low socio-economic area, chandigarh, north i ndia. rif 1(2):100-103. shaukat n., bilqees f.m., hadi r., ziadi v.a. and nadia a. 2006. a record of intestinal parasitic infections from ehsanullah diagnostic laboratory, nazimabad karachi. j. baqai med. univ. 9: 29-36. siddiqui, m.i., bilqees f.m., ilyas m, and perveen s. 2002. prevalence of parasites infections in a rural area of karachi pakistan j. pak. med. assoc. 52: 315-320. talat r., ibrahim f., mujeeb s., and ghous s., 2012. prevelance of human intestinal parasite in different areas of karachi pakistan b: helminth infection . proceeding ogf . parasitology 53; 67-76 wani s.a., ahmad f, zargar s.a., dar p.a., dar z.a., and jan t.r. 2008. intestinal helminthesis in a population of children from the kashmir valley, india. j. helminthal, 82, 313-317. waqar sn., hussain h., khan r., khawaja a., majid h., malik s., nadee, t. and beg ma 2003.intestinal parasitic infections in children from northern pakistan.pak. medi.net.j., 12:73-77. who (1994). bench for the diagonosis intestinal parasites.who,geneva,italy. 03 vol 5 (1), january 2014; 01-04 biology journal.indd rads journal of biological research & applied sciences volume 10, no. 2, december 2019 table of contents patron-in-chief mr. wajeehuddin ahmad chancellor, jinnah university for women patron prof. dr. naeem farooqui vice chancellor, jinnah university for women chief editor prof. dr. sayyada ghufrana nadeem jinnah university for women managing editor dr. samina khan jinnah university for women section editor dr. iqra munir jinnah university for women editorial board prof. dr. absar alum (usa) dr. samina n. assanie-shivji (usa) prof. dr. omar bagasra (usa) dr. shazia t. hakim (canada) dr. alfred iqbal (usa) prof. dr. reza-ul-karim (usa) dr. khanzadi fatima khattak (pakistan) dr. ghousia lutfullah (pakistan) dr. aashiq mohammad (pakistan) dr. syed muhammad naeem (pakistan) dr. azeem-ul-hasan naqvi (usa) dr. ansaruddin syed (pakistan) published biannually by reyazuddin research & development society, jinnah university for women, 5-c nazimabad, karachi-74600, pakistan e-mail: jbas@juw.edu.pk phone: 3661-9902, 3662-0857-9 fax: 3662-0614 http://jbas.juw.edu.pk original articles 44 study of the modulating interactions of multitrait rhizobacteria using zea mays l. as the host plant sana shakeel, ambreen ahmed, ifrah javaid 54 evaluation of selected pakistani honeys in comparison with manuka honey against vibrio cholerae muhammad barkaat hussain 63 eco-taxonomic study of family poaceae (gramineae) muhammad nauman khan, sajjad ali, tabassum yaseen, sami ullah, akhtar zaman, majid iqbal, sikandar shah 76 serodiagnostic investigation of syphilis in taluka gambat sagheer hussain shah, agha asad noor 82 effect of smoking on the lipid profile of inhabitant smokers of hasilpur, district bahawalpur, pakistan mushtaq hussain lashari, sumbel sumera, umer farooq, zia ur rehman, nuzhat sial, farooq ahmad, fozia afzal, muhammad saleem akhtar, muhammad nawaz, a.b. gulshan 87 improvement in morphology and organic substances of vigna radiata growing under conocarpus erectus and moringa oleifera amended soil shabana askari, ayesha siddiqui, sania razzaque 94 characterization of multidrug resistant bacteria isolated from hospital environment saba irshad, humaira yasmeen 102 studies on the reproduction and growth of hoopoe (upupa epops) in district gujrat, punjab, pakistan muhammad idnan, hafiz muhammad jamil, arshad javid, muddasir hassan abbasi, muhammad altaf, asif shahzad, asma naeem 109 the assessment of depression level among diabetic patients in karachi using bdi (beck’s depression inventory) kiran rafiq, shagufta nesar, zafar saied saify, mohammad azhar mughal, alina rizvi, aleeza raza, alisha hassan instructions to authors microbial iaa: spectral analysis and application vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 53 r a d s j . b i o l . r e s . a p p l . s c i 53 op e n ac c e ss f u l l l e n g t h a r t i c l e microbial iaa: spectral analysis and application to modulate growth of triticum aestivum ammara abid and ambreen ahmed* department of botany, university of the punjab, quaid-e-azam campus, lahore 54590, pakistan a b s t r a c t plant growth promoting rhizobacteria (pgpr) play an essential part in transformation, solubilization, and mobilization of nutrients procured from the soil. plant-microbe interaction can be termed as an eco-friendly approach which not only improves plant growth but helps in sustaining the soil and prevents environmental degradation from agrochemicals. pgpr improve plant growth through various mechanisms. one of the mechanisms involved is phytohormone production by the bacterial strains. in the current study, spectral analysis of thirteen already isolated and identified auxin-producing microbial strains (aal1, ab8, a7b, a5c, a3e, a11e, al2, a9g, a12g, a13g, am10, p4, and s6) was carried out. fourier transform infrared spectroscopy (ftir) of the bacterial iaa exhibited close structural similarity between bacterial iaa and standard iaa. the growth-enhancing capability of strains was verified through the application of these strains on triticum aestivum seedlings and enhancement of growth was statistically analyzed which indicated remarkable improvement in growth and metabolism both under laboratory and field conditions. several bacterial isolates also proved to be very effective in improving biochemical parameters of plants. the current study suggested that the application of iaa-producing pgpr as biofertilizer is effective in enhancing plant growth as well as plant yield. keywords ftir, iaa, pgpr, biofertilizers, triticum aestivum. *address of correspondence ambreenahmed1@hotmail.com article info. received: april 08, 2018 accepted: june 25, 2018 cite this article: abid a, ahmed a. microbial iaa: spectral analysis and application to modulate growth of triticum aestivum. rads j. biol. res. appl. sci. 2018; 9(2): 53-63. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n microorganisms are too small to be seen with the naked eye i.e., app. 1 to 100 μm. in spite of having microscopic size, they have major contributions to the stability of the ecosystem. microbial activities involving growth rate and metabolism take place at microscopic level but exert great impact at macroscopic level 1. the aim behind the implementation of the microbial application is to increase the nutrient availability and improving the growth and development of plants which is an indispensable practice for agriculture. in the recent years, the utilization of plant growth promoting rhizobacteria (pgpr) has been multiplied tremendously in different regions of the world for the sustainable ecosystem and agriculture 2. human population is increasing day by day due to which ample farming has been practiced now-a-days, which excessively depends upon agrochemicals that lead to a great number of hazardous health and environmental issues. hence, environment-friendly methods must be applied to retain the quality of soil and crops 3. plant growth promoting rhizobacteria (pgpr) are capable of enhancing nutrients supply in the rhizosphere and inducing the transport processes in roots 4. the rhizosphere is a hot spot of microbial abundance and activities due to the presence of root exudates and rhizodeposits 5. the plant growth promoting rhizobacteria are generally known as nodule promoting rhizobacteria o r i g i n a l a r t i c l e javascript:opendsc(2694522117,%201840,%20'4719'); javascript:opendsc(2694522117,%201840,%20'4719'); javascript:opendsc(592012883,%201,%20'58'); javascript:opendsc(592012883,%201,%20'58'); javascript:opendsc(592012883,%201,%20'981'); javascript:opendsc(592012883,%201,%20'981'); javascript:opendsc(54947441,%2037,%20'4320'); javascript:opendsc(54947441,%2037,%20'4320'); javascript:opendsc(54947441,%2037,%20'4320'); javascript:opendsc(592012883,%201,%20'2609'); javascript:opendsc(592012883,%201,%20'2609'); javascript:opendsc(592012883,%201,%20'2609'); javascript:opendsc(592012883,%201,%20'2609'); javascript:opendsc(592012883,%201,%20'2609'); javascript:opendsc(592012883,%201,%20'2609'); javascript:opendsc(592012883,%201,%20'2609'); javascript:opendsc(592012883,%201,%20'2609'); javascript:opendsc(592012883,%201,%20'2609'); javascript:opendsc(592012883,%201,%20'2609'); javascript:opendsc(54947441,%2037,%20'4323'); microbial iaa: spectral analysis and application vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 54 r a d s j . b i o l . r e s . a p p l . s c i 54 (npr) or plant health promoting rhizobacteria (phpr) with reference to the soil which is the major environment for plant-microbe interactions 2. pgpr have been proven beneficial for enhancing the plant growth via direct and indirect mechanisms 6. pgpr utilization as biofertilizers is an appropriate practice for producing better crops with minimal use of chemical fertilizers by stimulating different processes in plants such as mobilization of nutrients, biocontrol and phytohormone production 7. m a t e r i a l s a n d m e t h o d s growth of bacterial isolates thirteen already isolated and identified auxin-producing bacterial isolates, bacillus sp. (aal1), bacillus sp. (p4), bacillus sp. (ab8), bacillus sp. (s6), cronobacter sp. (al2), enterobacter sp. (a3e), enterobacter sp. (a5c), enterobacter sp. (a7b), enterobacter sp. (a11e), enterobacter sp. (a9g), enterobacter sp. (am10), enterobacter sp. (a12g) and exiguobacterium sp. (a13g) by ahmed16 were used in the present study. all the bacterial strains were routinely grown at 37 ºc for 24 hours using l-agar and l-broth 8. fourier transform infrared spectroscopy (ftir) the production of auxin by the bacterial isolates was first checked by colorimetric analysis. auxin synthesis by the bacterial isolates was later confirmed by fourier transform infrared spectroscopic (ftir) analysis. spectral analysis was carried out using agilent cary 630 ftir. the bacterial strains were grown using lb-medium for 24 hours at 37 ºc in the presence of tryptophan. after centrifugation of 24 hours incubated bacterial culture, the supernatant was collected and its ph was adjusted to 3 using 6n hcl. bacterial auxin was extracted from the supernatant thrice by using ethyl acetate. after the removal of ethyl acetate through vaporization, the residue was collected in methanol and was analyzed through fourier transform infrared spectroscopy. synthetic iaa (sigma) was used as standard and also analyzed through ftir. effect of bacterial treatment on the growth of triticum aestivum certified seeds of triticum aestivum var. fd-08 were used in the current study. healthy seeds of wheat were obtained from organization in lahore (pakistan) named as punjab seed corporation. sterilization and pregermination inoculation treatments were given to the seeds following ahmed and hasnain 9. under laboratory conditions, pots were filled with 0.2 kg sieved soil, six replicates were taken for each treatment and 11 treated seedlings were transferred to each pot and placed under 10 klux light with a photoperiod of 16 hours at 30 + 2 ºc. harvest was taken after 30 days of inoculation and several growth parameters like root length, shoot length, fresh weight, the number of leaves of plants, as well as biochemical parameters i.e., protein content and auxin content, were estimated. under field conditions, pots were filled with 7.5 kg sieved soil, six replicates for each treatment were taken and 15 seeds were sown per pot after treatment with bacterial cultures. after germination of the seedlings, thinning of plants was done to have 11 seedlings per pot. these wheat plants were grown in pots till maturity. plants were then harvested and various physical parameters were studied like number of leaves, spike length, shoot length, seed weight, spikelet length, number of tillers and grain yield after 140 days of inoculation. biochemical parameters such as protein estimation following lowry et al. 10 and auxin estimation following mahadevan 11 were performed twice, firstly, after 90 days of inoculation and secondly, after 140 days of inoculation i.e., at maturity. both auxin and protein content was estimated using leaves of treated and non-treated plants. statistical analysis the data obtained were analyzed statistically by using the software spss.v.16. duncan’s multiple range test was applied for comparing means through analysis of variance. r e s u l t s fourier transform infrared spectroscopy (ftir) spectral analysis of microbial iaa obtained from the selected auxin-producing isolates i.e., bacillus sp. (aal1), cronobacter sp. (al2), bacillus sp. (p4), bacillus sp. (s6), bacillus sp. (ab8), enterobacter sp. (a5c), enterobacter sp. (a11e), enterobacter sp. (a7b), enterobacter sp. (a12g), enterobacter sp. (a3e), enterobacter sp. (a9g), enterobacter sp. (am10), exiguobacterium sp. (a13g) and synthetic iaa (sigma) as standard was carried out. the interferogram of standard iaa exhibited –oh peak at javascript:opendsc(54947441,%2037,%20'4323'); javascript:opendsc(54947441,%2037,%20'4323'); javascript:opendsc(54947441,%2037,%20'4323'); javascript:opendsc(592012883,%201,%20'2610'); javascript:opendsc(592012883,%201,%20'2610'); javascript:opendsc(592012883,%201,%20'2610'); javascript:opendsc(592012883,%201,%20'2610'); javascript:opendsc(592012883,%201,%20'2610'); javascript:opendsc(592012883,%201,%20'68'); javascript:opendsc(592012883,%201,%20'68'); javascript:opendsc(592012883,%201,%20'68'); javascript:opendsc(592012883,%201,%20'68'); javascript:opendsc(58372459,%2037,%20'4376'); javascript:opendsc(33944742,%2037,%20'4374'); javascript:opendsc(33944742,%2037,%20'4374'); javascript:opendsc(33944742,%2037,%20'4374'); javascript:opendsc(551257142,%201,%20'4659'); javascript:opendsc(551257142,%201,%20'4659'); javascript:opendsc(592012883,%201,%20'1726'); javascript:opendsc(592012883,%201,%20'1726'); javascript:opendsc(592012883,%201,%20'77'); microbial iaa: spectral analysis and application vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 55 r a d s j . b i o l . r e s . a p p l . s c i 55 3384 cm-1. the spectral region ranging between 28003100 cm-1 showed c-h stretching bands indicating the presence of fatty acid chains and amino acid chains, –cn peak is at 737 cm-1 and the aromatic ring was present ranging between 1407-1686cm-1. the interferogram obtained from microbial iaa extracted from bacterial strains bacillus sp. (aal1), bacillus sp. (p4), enterobacter sp. (a3e), cronobacter sp. (al2) exhibited -oh spectral region at 3411 cm-1 and lies in the range of 2400-3400 cm-1. the –ch band recorded in the spectral range of 2800-3100 cm-1, the c-n band was at 2942 cm-1 and the aromatic ring lies within 1400-1600 cm-1. the interferogram obtained from analysis of microbial iaa extracted from the bacterial isolates bacillus sp. (s6), bacillus sp. (ab8), enterobacter sp. (a9g), enterobacter sp. (a5c) and enterobacter sp. (a7b) was also carried out and -oh band showed peaks between 2400-3400 cm1. the –ch band was present between 2800-3100 cm-1 and the aromatic ring was present between 1420-1654 cm-1. the -ch stretching band indicates the presence of fatty acid and amino acid which was in the range of 28003100cm-1 and the –cn band was at 1015 cm-1. bacterial iaa extracted from enterobacter sp. (am10) showed similar results such as the –oh spectral region lies between 2400-3400 cm-1 and the stretching bands of -ch were present between 2800-3100 cm-1 and of -oh band between 2400-3400cm-1. the aromatic group was present ranging between 1420-1670 cm-1 and –cn band was at 1016 cm-1 (fig. 1). interferogram obtained from the bacterial strain enterobacter sp. (a11e), exiguobacterium sp. (a13g) and enterobacter sp. (a12g) also showed that -oh peak lies between 2400-3400 cm-1. the aromatic band ranges between 1420-1458 cm-1. similarly -ch stretching peak observed at 2833 cm-1 and cn peak at 1022 cm-1. interferograms obtained through fourier transform infrared spectroscopy (ftir) revealed that structurally microbial iaa is similar to standard iaa as indicated from the comparable peaks in the interferograms obtained using microbial iaa [extracted from selected bacterial isolates (ab8, a5c, aal1, a7b, a3e, a11e, a9g, a12g, al2, am10, a13g, p4, s6)] and standard iaa. these results were further supported by plant growth enhancement of wheat plants after bacterial treatment both under laboratory and field conditions. a) b) fig. 1: interferogram obtained through ftir analysis. (a) standard iaa (b) microbial iaa extracted from enterobacter sp. (am10). effect of bacterial treatment on growth of triticum aestivum (30 days old plants) in the laboratory trial, enhancement in germination percentage was shown by treatment with bacterial isolates such as bacillus sp. (ab8) exhibited up to 14%, cronobacter sp. exhibited 16% percentage and exiguobacterium sp. (a13g) up to 9.5% increase respectively, in comparison to control (fig. 2). plants inoculated with bacterial strains cronobacter sp. (al2), bacillus sp. (aal1) and enterobacter sp. (a7b) showed significant enhancement in shoot length i.e., 14, 12 and 9% respectively. inoculation with bacterial isolate enterobacter sp. (a7b) exhibited significant improvement in root length up to 27%. plants treated with bacterial isolates such as exiguobacterium sp. (a13g), enterobacter sp. (a5c) and bacillus sp. (ab8) have also exhibited a significant increase in root length up to 19, 20 and 21% over control plants (fig. 3). enterobacter sp. (al2) and cronobacter sp. (al2) showed improvement in the number of leaves i.e., 11 and 12% respectively, over non-inoculated plants (fig. 4). plants treated with the javascript:opendsc(592012883,%201,%20'77'); javascript:opendsc(592012883,%201,%20'77'); javascript:opendsc(592012883,%201,%20'77'); javascript:opendsc(592012883,%201,%20'77'); javascript:opendsc(592012883,%201,%20'77'); javascript:opendsc(592012883,%201,%20'77'); javascript:opendsc(592012883,%201,%20'77'); javascript:opendsc(592012883,%201,%20'77'); javascript:opendsc(592012883,%201,%20'77'); javascript:opendsc(592012883,%201,%20'77'); javascript:opendsc(592012883,%201,%20'77'); javascript:opendsc(592012883,%201,%20'77'); javascript:opendsc(592012883,%201,%20'77'); javascript:opendsc(592012883,%201,%20'494'); javascript:opendsc(592012883,%201,%20'494'); javascript:opendsc(592012883,%201,%20'494'); javascript:opendsc(592012883,%201,%20'494'); javascript:opendsc(592012883,%201,%20'494'); javascript:opendsc(592012883,%201,%20'494'); javascript:opendsc(592012883,%201,%20'494'); javascript:opendsc(592012883,%201,%20'675'); javascript:opendsc(592012883,%201,%20'675'); javascript:opendsc(592012883,%201,%20'675'); javascript:opendsc(592012883,%201,%20'675'); javascript:opendsc(592012883,%201,%20'675'); javascript:opendsc(592012883,%201,%20'675'); javascript:opendsc(592012883,%201,%20'675'); javascript:opendsc(592012883,%201,%20'751'); javascript:opendsc(592012883,%201,%20'751'); javascript:opendsc(592012883,%201,%20'751'); javascript:opendsc(592012883,%201,%20'751'); javascript:opendsc(592012883,%201,%20'751'); javascript:opendsc(592012883,%201,%20'751'); javascript:opendsc(592012883,%201,%20'915'); javascript:opendsc(592012883,%201,%20'915'); javascript:opendsc(592012883,%201,%20'915'); javascript:opendsc(592012883,%201,%20'915'); javascript:opendsc(592012883,%201,%20'65'); javascript:opendsc(592012883,%201,%20'65'); javascript:opendsc(592012883,%201,%20'983'); javascript:opendsc(592012883,%201,%20'1005'); javascript:opendsc(592012883,%201,%20'1005'); javascript:opendsc(592012883,%201,%20'1005'); javascript:opendsc(592012883,%201,%20'1005'); javascript:opendsc(592012883,%201,%20'1128'); javascript:opendsc(592012883,%201,%20'1245'); javascript:opendsc(592012883,%201,%20'1245'); javascript:opendsc(592012883,%201,%20'1245'); javascript:opendsc(592012883,%201,%20'1245'); microbial iaa: spectral analysis and application vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 56 r a d s j . b i o l . r e s . a p p l . s c i 56 bacterial isolates also exhibited a significant enhancement in fresh weight over non-treated plants. treatments with cronobacter sp. (al2) [28.2%], bacillus sp. (aal1) [28.2%], bacillus sp. (s6) [33.3%] showed a significant increase while enterobacter sp. (a9g) showed 13% reduction in fresh weight over control (fig. 5). treatment with the microbial isolates enterobacter sp. (am10) and exiguobacterium sp. (a13g) exhibited a significant increase in protein content i.e., 152% and 175% over control (fig. 7). cronobacter sp. (al2) exhibited 224% significant enhancement in auxin over non-treated plants. moreover, improvement in auxin content was recorded by the treatment with bacterial strains i.e., bacillus sp. (p4) up to 150% and enterobacter sp. (a7b) up to 145% compared to control. few bacterial isolates such as aal1, s6, a11e and a3e exhibited a decrease in auxin content over control (fig. 8). effect of bacterial treatment on the growth of triticum aestivum (90 and 140 days old plants) under field conditions, plants treated with bacterial strain enterobacter sp. (a5c) showed up to 22.5% while cronobacter sp. (al2) showed 22.1% increase in germination percentage over non-treated plants (fig. 2). shoot length enhancement up to 40% was observed after the application of enterobacter sp. (a9g) (fig. 3). plants inoculated with enterobacter sp. (a5c) exhibited significant enhancement in the number of leaves up to 32% over non-inoculated plants (fig. 4). maximum significant improvement in tiller number was observed by enterobacter sp. (a9g) treatment i.e. 35% compared to control (fig. 4). the plants inoculated with the bacterial isolate enterobacter sp. (a3e) exhibited a maximum significant enhancement in spike length up to 15.5% while enterobacter sp. (a5c) showed significant improvement in spikelet length up to 18.1% over control (fig. 4 & 5). significant improvement was observed in the grain yield of treated plants over non-treated plants. plants inoculated with enterobacter sp. (a9g) exhibited significant increase in grain yield up to 37.5% as compared to plants without bacterial treatment. similarly, plants treated with the isolate bacillus sp. (aal1) showed maximum significant increase in seed weight i.e., 29.7% over control (fig. 5 & 6). under field conditions, protein analysis of 90 days old plants, revealed 34% protein enhancement in plants treated with enterobacter sp. (a5c) as compared to noninoculated plants. fig. 2: effect of bacterial inoculations on germination percentage of triticum aestivum (fd-08) under laboratory and field conditions. data represent mean of sixty-six plants. different letters represent a significant difference between treatments using duncan's multiple range test (p=0.05). javascript:opendsc(592012883,%201,%20'1442'); javascript:opendsc(592012883,%201,%20'1442'); javascript:opendsc(592012883,%201,%20'1442'); javascript:opendsc(592012883,%201,%20'1442'); javascript:opendsc(592012883,%201,%20'1442'); javascript:opendsc(592012883,%201,%20'1442'); javascript:opendsc(592012883,%201,%20'1546'); javascript:opendsc(592012883,%201,%20'1558'); javascript:opendsc(592012883,%201,%20'1558'); javascript:opendsc(592012883,%201,%20'1669'); javascript:opendsc(592012883,%201,%20'1669'); javascript:opendsc(592012883,%201,%20'1669'); javascript:opendsc(592012883,%201,%20'1669'); javascript:opendsc(592012883,%201,%20'1669'); javascript:opendsc(592012883,%201,%20'1669'); javascript:opendsc(592012883,%201,%20'1669'); javascript:opendsc(592012883,%201,%20'1669'); javascript:opendsc(592012883,%201,%20'1718'); javascript:opendsc(592012883,%201,%20'1722'); javascript:opendsc(592012883,%201,%20'1722'); javascript:opendsc(592012883,%201,%20'1840'); javascript:opendsc(592012883,%201,%20'1840'); javascript:opendsc(592012883,%201,%20'1840'); javascript:opendsc(592012883,%201,%20'1842'); javascript:opendsc(592012883,%201,%20'1842'); javascript:opendsc(592012883,%201,%20'1842'); javascript:opendsc(592012883,%201,%20'2110'); javascript:opendsc(592012883,%201,%20'2110'); javascript:opendsc(592012883,%201,%20'2110'); javascript:opendsc(592012883,%201,%20'2110'); javascript:opendsc(53129898,%2037,%20'4731'); javascript:opendsc(53129898,%2037,%20'4731'); javascript:opendsc(53129898,%2037,%20'4728'); javascript:opendsc(592012883,%201,%20'2234'); javascript:opendsc(592012883,%201,%20'2234'); microbial iaa: spectral analysis and application vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 57 r a d s j . b i o l . r e s . a p p l . s c i 57 fig. 3: effect of bacterial inoculations on shoot length and root length of triticum aestivum (fd-08) under laboratory and field conditions. data represent mean of sixty-six plants. different letters represent a significant difference between treatments using duncan's multiple range test (p=0.05) [sl: shoot length, rl: root length (lab)]. fig. 4: effect of bacterial inoculations on number of leaves, number of tillers, spike length (cm) of triticum aestivum (fd08) under laboratory and field conditions. data represent mean of sixty-six plants. different letters represent a significant difference between treatments using duncan's multiple range test (p=0.05). microbial iaa: spectral analysis and application vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 58 r a d s j . b i o l . r e s . a p p l . s c i 58 fig. 5: effect of bacterial inoculations on a fresh weight (g), seed weight (g), spikelet length (cm) of triticum aestivum (fd-08) under laboratory and field conditions. data represent mean of sixty-six plants. different letters represent a significant difference between treatments using duncan's multiple range test (p=0.05). fig. 6: effect of bacterial inoculations on grain yield of triticum aestivum (fd-08) under field conditions. data represent mean of sixty-six plants. different letters represent a significant difference between treatments using duncan's multiple range test (p=0.05). microbial iaa: spectral analysis and application vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 59 r a d s j . b i o l . r e s . a p p l . s c i 59 fig. 7: effect of bacterial inoculations on the protein content of 30, 90 and 140 days old plants of triticum aestivum (fd08) under laboratory and field conditions. data represent mean of sixty-six plants. different letters represent a significant difference between treatments using duncan's multiple range test (p=0.05). fig. 8: effect of bacterial inoculations on auxin content of 30, 90 and 140 days old plants of triticum aestivum (fd-08) under laboratory and field conditions. data represent mean of sixty-six plants. different letters represent a significant difference between treatments using duncan's multiple range test (p=0.05). while in 140 days old plants, maximum significant enhancement in protein content was shown in plants treated with bacillus sp. (p4) up to 262% over control (fig. 7). in 90 days old plants, 100 % improvement in auxin content was observed in treatment with exiguobacterium sp. (a13g) over non-treated plants. auxin estimation of 140 days old treated and non-treated plants showed significant improvement in the quantity of auxin when treated with bacillus sp. (ab8) i.e., 40% over control (fig. 8). d i s c u s s i o n in the present world, the environmental issues relevant to increase in crop production are of great concern at the global level. utilization of pgpr in this aspect is one of the most significant approaches in reducing the use of javascript:opendsc(592012883,%201,%20'2612'); javascript:opendsc(592012883,%201,%20'2612'); microbial iaa: spectral analysis and application vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 60 r a d s j . b i o l . r e s . a p p l . s c i 60 agrochemicals, balancing the ecosystem, maintaining soil, mobilizing nutrients and improving the crop quality 7. different mechanisms are involved in plant growth improvement like phosphate solubilization, nitrogen fixation, siderophore production, induced systemic resistance and antibiotic production, etc.12. apart from these mechanisms, one major approach playing an essential part is the synthesis of phytohormones like gibberellins, auxins and cytokinins etc.13. in the class of naturally occurring auxins, the most essential plant growth regulator is iaa which is involved in a number of plant growth mechanisms like cell division, cell differentiation, metabolites production, germination of seed, development of root, pigment formation, adventitious roots initiation and photosynthesis 14. most of the pgprs induce root developing system by maximizing the surface area, number, and size of root hairs 15. in the present work, ftir analysis of microbial iaa obtained from the selected auxin-producing bacterial isolates i.e., s6, p4, a5c, a13g, a9g, a11e, aal1, ab8, al2, a3e, a7b, a12g, and am10 was carried out. the interferograms obtained using bacterial iaa extracted from selected isolates (ab8, aal1, a7b, a5c, a11e, a3e, a9g, al2, p4, am10, a12g, s6 and a13g) were compared with standard iaa verify the similarity between microbial iaa and synthetic iaa (sigma). the interferogram obtained from standard iaa exhibited –oh band at 3384 cm-1. the region of c-h stretching vibrations ranges between 2800-3100cm-1 indicating the presence of amino acid and fatty acid. the –cn band lies near 737 cm-1 and the aromatic ring was in the range of 14051686cm-1. similar values were recorded for the –ch stretching band, -oh band, -cn band and an aromatic group of enterobacter sp. (am10) in comparison with the interferogram of standard iaa (fig. 1). comparable results were recorded with microbial iaa extracted from remaining bacterial isolates. the impact of their auxin production potential to modulate the growth of plants was further studied by application of these isolates on triticum aestivum. in the laboratory experiment, the plants treated with bacterial isolate cronobacter sp. (al2) showed maximum significant enhancement in germination percentage and shoot length i.e., 16 and 14% respectively, as compared to non-treated plants. from the selected bacterial isolates, maximum improvement in root length was shown by enterobacter sp. (a7b) up to 27% over control (fig. 2 & 3). plants inoculated with bacterial isolate enterobacter sp. (a9g) exhibited significant improvement in the number of leaves i.e., 27% over non-treated plants. plants inoculated with bacillus sp. (s6) exhibited an increase in fresh weight i.e., 33.3% in comparison to plants without bacterial inoculation. however, few strains showed a reduction in the fresh weight of plants treated with bacterial isolates in comparison to non-treated plants (fig. 4 & 5). under field conditions, plants treated with enterobacter sp. (a5c) showed maximum increase in germination percentage up to 22.5% while the rest of the strains also showed moderate and slight improvement. enterobacter sp. (a9g) showed an increase in shoot length up to 40% and significant improvement in leaves number was exhibited by enterobacter sp. (a5c) up to 32% in comparison to control (fig. 3 & 4). all the above results indicated that the auxin-producing isolates positively affected triticum aestivum growth with significant improvements recorded in most of the growth parameters when compared with non-bacterial control treatments. pgpr have the ability of aggregating carbon compounds from the root hairs that can be used as an energy reservoir. they are involved in stimulating various growth factors such as inducing germination percentage, increasing root length and shoot length etc. by the synthesis of phytohormones which in turn stimulate the nutrients availability by mobilizing the nutrients absorption through enhancement of water uptake ability and minimizing the pathogenic attack on plants by accelerating the resistance mechanism and improving overall growth of plants 9. growth inducing mechanisms are more significant in roots overshoots. auxins produced by the microbial strains have a remarkable effect on root growth especially in the initiation of roots, apical dominance, ethylene production, cell division and elongation 16. other researchers reported that the germination of tomato seeds by the application of bacterial inoculum showed enhancement in germination rate and root length over control 13. similarly, the seeds of chickpea treated with microbial strains showed improvement in shoot length up to 93% in comparison to control plants 17. javascript:opendsc(592012883,%201,%20'2612'); javascript:opendsc(592012883,%201,%20'2612'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'2615'); javascript:opendsc(592012883,%201,%20'73'); javascript:opendsc(592012883,%201,%20'73'); javascript:opendsc(592012883,%201,%20'73'); javascript:opendsc(592012883,%201,%20'80'); javascript:opendsc(592012883,%201,%20'80'); javascript:opendsc(592012883,%201,%20'2670'); javascript:opendsc(592012883,%201,%20'2670'); javascript:opendsc(592012883,%201,%20'2670'); javascript:opendsc(592012883,%201,%20'1170'); javascript:opendsc(592012883,%201,%20'1170'); javascript:opendsc(592012883,%201,%20'1170'); javascript:opendsc(592012883,%201,%20'1170'); javascript:opendsc(592012883,%201,%20'1170'); javascript:opendsc(592012883,%201,%20'2920'); javascript:opendsc(592012883,%201,%20'2920'); javascript:opendsc(592012883,%201,%20'1462'); javascript:opendsc(592012883,%201,%20'1462'); javascript:opendsc(592012883,%201,%20'2790'); javascript:opendsc(592012883,%201,%20'2790'); javascript:opendsc(592012883,%201,%20'1735'); javascript:opendsc(592012883,%201,%20'1735'); javascript:opendsc(592012883,%201,%20'1735'); javascript:opendsc(592012883,%201,%20'1735'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2739'); javascript:opendsc(592012883,%201,%20'2755'); javascript:opendsc(592012883,%201,%20'2755'); javascript:opendsc(592012883,%201,%20'2755'); javascript:opendsc(592012883,%201,%20'2755'); microbial iaa: spectral analysis and application vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 61 r a d s j . b i o l . r e s . a p p l . s c i 61 under the field grown plants, improvement in various yield parameters was recorded as compared to control. significant enhancement in spike length and spikelet length was recorded in plants treated with enterobacter sp. (a5c) and enterobacter sp. (a3e) upto18.1% and 15.5% over control (fig. 4 & 5). plants treated with enterobacter sp. (a9g) exhibited maximum significant enhancement in tiller number up to 35% and grain yield up to 37.5% in comparison to non-inoculated plants while significant reduction in number of tillers and yield was observed in plants inoculated with exiguobacterium sp. (a13g) and bacillus sp. (s6) (fig. 4 & 6). improvement in seed weight was recorded with bacillus sp. (aal1) and enterobacter sp. (a5c) up to 29.7% and 27% respectively, over control (fig. 5). the increase in spike length, spike number, spikelet length and tiller number might be due to reduced attack and sufficient availability of nutrients by applying plant health promoting rhizobacteria 18, 19. the increased photosynthetic activities and extensive uptake of nutrients and water resulted in better quality crops and increased yield 20. sufficient auxin content poses remarkable effect on plant yield and better development 21. however, the amount of auxin supports plant growth up to a certain concentration. any further increase in iaa above the limit would cause growth retardation especially in case of plant roots 22. enhancement of protein content was recorded in all the treated and non-treated plants at different stages of growth i.e., 30, 90 and 140 days old plants. however, minor difference was recorded in protein content at these three stages but out of all thirteen isolates, enterobacter sp. (am10) exhibited maximum significant increase in protein content in comparison to control up to 152% in 30 days old plants while in 90 days old treated plants, the bacterial isolates enterobacter sp. (a5c) and enterobacter sp. (am10) exhibited maximum increase in protein content up to 34 and 8% respectively, over control. thus, in 30 days old plants, treatment with enterobacter sp. (am10) showed maximum enhancement up to 152% but it was reduced to 8% in 90 days old plants since the proteins might have been utilized in photosynthesis and seed formation so a reduction in proteins was recorded with increasing maturity of plants. protein analysis of 140 days old treated and non-treated fully grown plants exhibited significant improvement in protein content by treatment with bacillus sp. (p4) up to 262% over control. at juvenile and middle stage of growth, proficient amount of soil nutrients were consumed by the plants attributed to protein synthesis in great amount. the increased protein synthesis leads to improved plant growth. however, in 140 days old plants, proteins were still produced but the quantity was decreased in comparison to the early growth phases (30 and 90 days old plants) (fig. 7). pgpr is capable of fixing nutrients from the soil specifically nitrogen which maximizes the protein synthesis which in turn increases the crop yield. during seed development, a large amount of protein is utilized that causes a reduction in protein content at maturity 23, 24. estimation of auxin in 30, 90 and 140 days old control and bacterially inoculated plants was also checked. maximum improvement in auxin level was shown by the application of cronobacter sp. (al2) i.e., up to 224% in 30 days old treated plants over control while in 90days old plants inoculated with exiguobacterium sp. (a13g), a significant increase in auxin content i.e., 100 % over control was observed. treatment with bacillus sp. (ab8) exhibited 40% enhancement in auxin content in 140days old treated plants. cronobacter sp. (al2) showed maximum significant enhancement in auxin content in 30days old inoculated plants. but in 90days old al2 treated plants, auxin content was reduced to 12% and in 140 days old treated plants, the auxin content recorded was up to 1% only (fig. 8). this reveals that auxin is actively synthesized during early growth phases in the meristematic areas such as leaves then translocated to other plant regions which reinforce other mechanisms involved in the development of plants 25. at premature phases of plant development i.e. 30 days old plants, the enhancement in auxin level could be attributed to the presence of meristematic areas in the shoot. likewise, meristems of the root may also contribute to increasing the auxin content by enhancing the tryptophan availability i.e., a precursor for auxin synthesis. in 140 days old plants, wheat was fully grown and active meristematic points are not left in the plants. another justification is that the microbial isolates vigorously synthesize auxin till juvenile stage so when the plants reached the mature stage, the microbes may not be actively responsive for javascript:opendsc(592012883,%201,%20'1816'); javascript:opendsc(592012883,%201,%20'1816'); javascript:opendsc(592012883,%201,%20'2915'); javascript:opendsc(592012883,%201,%20'2915'); javascript:opendsc(592012883,%201,%20'2915'); javascript:opendsc(592012883,%201,%20'2915'); javascript:opendsc(592012883,%201,%20'2915'); javascript:opendsc(592012883,%201,%20'2915'); javascript:opendsc(592012883,%201,%20'2915'); javascript:opendsc(592012883,%201,%20'2919'); javascript:opendsc(592012883,%201,%20'2919'); javascript:opendsc(592012883,%201,%20'2919'); javascript:opendsc(592012883,%201,%20'2240'); javascript:opendsc(592012883,%201,%20'2240'); javascript:opendsc(592012883,%201,%20'3006'); javascript:opendsc(592012883,%201,%20'3006'); javascript:opendsc(592012883,%201,%20'3013'); javascript:opendsc(592012883,%201,%20'3013'); javascript:opendsc(592012883,%201,%20'3013'); javascript:opendsc(592012883,%201,%20'1559'); javascript:opendsc(592012883,%201,%20'1559'); javascript:opendsc(592012883,%201,%20'1559'); javascript:opendsc(592012883,%201,%20'2892'); javascript:opendsc(592012883,%201,%20'2892'); javascript:opendsc(592012883,%201,%20'2892'); javascript:opendsc(592012883,%201,%20'2291'); javascript:opendsc(592012883,%201,%20'2291'); javascript:opendsc(592012883,%201,%20'2291'); javascript:opendsc(592012883,%201,%20'2291'); javascript:opendsc(592012883,%201,%20'3014'); javascript:opendsc(592012883,%201,%20'3014'); javascript:opendsc(592012883,%201,%20'3014'); javascript:opendsc(592012883,%201,%20'3014'); javascript:opendsc(592012883,%201,%20'3014'); javascript:opendsc(592012883,%201,%20'3014'); javascript:opendsc(592012883,%201,%20'3014'); javascript:opendsc(592012883,%201,%20'3021'); javascript:opendsc(592012883,%201,%20'3021'); javascript:opendsc(592012883,%201,%20'3021'); javascript:opendsc(592012883,%201,%20'3021'); javascript:opendsc(592012883,%201,%20'3021'); javascript:opendsc(592012883,%201,%20'3021'); javascript:opendsc(592012883,%201,%20'3023'); javascript:opendsc(592012883,%201,%20'3023'); javascript:opendsc(592012883,%201,%20'3028'); javascript:opendsc(592012883,%201,%20'2433'); javascript:opendsc(592012883,%201,%20'2433'); javascript:opendsc(592012883,%201,%20'2433'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); microbial iaa: spectral analysis and application vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 62 r a d s j . b i o l . r e s . a p p l . s c i 62 auxin synthesis because there is a reduction in auxin level in mature plant regions. furthermore, some auxin-producing bacterial isolates may accelerate the growth of the plants at different phases. some strains like enterobacter sp. (ag9), exiguobacterium sp. (a13g), enterobacter sp. (am10) have improved auxin content at mature stages of plant growth (fig. 8). these bacterial isolates might not be very vigorously involved in auxin production during early phases of growth but become active at later growth stages and then synthesize greater amounts of auxin. other elements involved in biochemical improvement still need to be studied further to comprehend the whole mechanism. abbasi and his coworkers reported results similar to our findings that increase in auxin content of inoculated wheat seeds over non-inoculated seeds 26. vegetables treated by the application of bacterial strains manufacture auxin between 0.78-401.62 μg/ml as compared to control 27. c o n c l u s i o n spectral analysis of the selected auxin-producing bacterial isolates (ab8, a7b, a5c, aal1, a11e, a3e, a9g, al2, p4, a12g, s6, am10, and a13g) revealed that auxin produced by these bacterial isolates is structurally indole-3-acetic acid (iaa). ftir analysis further confirmed that microbial iaa is structurally similar to phytohormonal iaa thus exert beneficial impact similar to plant iaa e.g., increase the meristematic activity in plants which leads to improvement in plant growth and it is verified through in vivo studies using triticum aestivum var. fd-08. pgpr is indigenously present in nature so they are cost-effective and environment-friendly tools so their application as biofertilizer should be encouraged to improve plant growth and development. in addition, these are safer to practice, causing no deleterious effects on the ecosystem and living organisms around and exhibit considerable positive impact on crop quality and yield as demonstrated by the results recorded during the present study. r e f e r e n c e s 1. habig j, hassen ai, swart a. application of microbiology in conservation agriculture. conserv agric. 2015 (doi: 10.1007/978-3-319-11620-4_20). 2. deka h, deka s, baruah ck. plant growth promoting rhizobacteria for value addition: mechanism of action. soil biol. 2015; 42. (doi: 10.1007/978-3-319-134017_15). 3. song x, liu m, wu d, griffiths bs, jiao j, li h, et al. interaction matters: synergy between vermicompost and pgpr agents improves soil quality, crop quality and crop yield in the field. appl soil ecol. 2015; 89: 25–34. 4. vacheron j, desbrosses g, bouffaud m l, touraine b, moënne-loccoz y, muller d, et al. plant growthpromoting rhizobacteria and root system functioning. front plant sci. 2013 (doi: 10.3389/fpls.2013.00356). 5. nandal m, hooda r. plant growth promoting rhizobacteria: a review article. int j curr res. 2013; 5(12): 3863-71. 6. agrawal pk, agrawal s, kundan r, bhatt m. application and perspective of plant growth promoting rhizobacteria in development of sustainable agriculture. int j curr res. 2014; 6 (10): 9044-51. 7. chitraselvi pe, kalidass s, kant r. efficiency of rhizosphere bacteria in the production of indole acetic acid, siderophore and phosphate solubilization. int j chem tech res. 2015; 7(6): 2557-64. 8. gerhardt p, murray rge, wood wa, kreig nr. in: methods for general and molecular bacteriology, 1994. american society for microbiology. washington, d. c. 9. ahmed a, hasnain s. auxin-producing bacillus sp.: auxin quantification and effect on the growth of solanum tuberosum. pure appl chem. 2010; 82(1): 313–9. 10. lowry oh, resebrough nj, farr al. protein measurement with the folin-phenol reagent. j biol chem. 1951; 193: 265-75. 11. mahadevan a. in: growth regulators, microorganisms, and diseased plants. 1984. oxford and ibh publishing company, india, 31. 12. prathap m, ranjitha kbd. a critical review of plant growth promoting rhizobacteria. j plant pathol microbiol. 2015 (doi:10.4172/2157-7471.1000266). 13. mangmang js, deaker r, rogers g. effects of plant growth promoting rhizobacteria on seed germination characteristics of tomato and lettuce. j trop crop sci. 2014; 1(2): 35-40. 14. ahirwar nk, gupta g, singh v, rawlley rk, ramana s. influence on growth and fruit yield of tomato (lycopersicon esculentum mill.) plants by inoculation with pseudomonas fluorescence (ss5): possible role of plant growth promotion. int j curr microbiol appl sci. 2015; 4(2): 720-30. 15. jha ck, saraf m. plant growth promoting rhizobacteria (pgpr): a review. ejard. 2015; 5(2): 0108-19. 16. ahmed a. efficacy of bacterial hormone in plant growth promotion. ph.d. thesis, 2011. the university of punjab. 17. dasgupta d, ghati a, sarkar a, sengupta c. and paul g. application of plant growth promoting rhizobacteria (pgpr) isolated from the rhizosphere of sesbania bispinosa on the growth of chickpea (cicer arietinum l.). int j curr microbiol appl sci. 2015; 4(5): 1033-42. javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3150'); javascript:opendsc(592012883,%201,%20'3159'); javascript:opendsc(592012883,%201,%20'3159'); javascript:opendsc(592012883,%201,%20'3159'); javascript:opendsc(592012883,%201,%20'3159'); javascript:opendsc(592012883,%201,%20'3159'); javascript:opendsc(592012883,%201,%20'3159'); javascript:opendsc(592012883,%201,%20'3165'); javascript:opendsc(592012883,%201,%20'3165'); javascript:opendsc(592012883,%201,%20'3165'); javascript:opendsc(592012883,%201,%20'3165'); javascript:opendsc(592012883,%201,%20'3165'); javascript:opendsc(592012883,%201,%20'3165'); javascript:opendsc(592012883,%201,%20'60'); javascript:opendsc(592012883,%201,%20'60'); javascript:opendsc(592012883,%201,%20'60'); javascript:opendsc(592012883,%201,%20'60'); javascript:opendsc(592012883,%201,%20'3226'); javascript:opendsc(592012883,%201,%20'3226'); javascript:opendsc(592012883,%201,%20'3226'); javascript:opendsc(592012883,%201,%20'3224'); javascript:opendsc(592012883,%201,%20'3224'); microbial iaa: spectral analysis and application vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 63 r a d s j . b i o l . r e s . a p p l . s c i 63 18. shirinzadeh a, soleimanzadeh h, shirinzadeh z. effect of seed priming with plant growth promoting rhizobacteria (pgpr) on agronomic traits and yield of barley cultivars. world appl sci j. 2013; 21 (5): 727-31. 19. haghighi p, habibi d, sani b. wheat response to plant growth promoting rhizobacteria, humic acid and snbrassinolide. int j biosci. 2014; 5(10): 51-60. 20. stefan m, munteanu n, stoleru v, mihasan m, hritcu l. seed inoculation with plant growth promoting rhizobacteria enhances photosynthesis and yield of runner bean (phaseolus coccineus l.). sci hort. 2013; 151: 22-9. 21. aslantaş r, çakmakçi r, şahin f. effect of plant growth promoting rhizobacteria on young apple tree growth and fruit yield under orchard conditions. sci hort. 2007; 111(4): 371-7. 22. ahmed a, hasnain s. auxin as one of the factors of plant growth improvement by plant growth promoting rhizobacteria. pol j microbiol 2014; 63(3): 261-6. 23. fabre f, planchon c. nitrogen nutrition, yield and protein content in soybean. plant sci 2000; 152: 51–8. 24. hassan a, zamir ms, khan i, anjum sa, mahmood a, ahmed auh, et al. sustainable maize production through seed inoculation and different tillage regimes. int j agron agric res. 2014; 4(3): 67-76. 25. tiberia i, doru p, catherine b. auxin control in the formation of adventitious roots. not bot horti agrobio. 2011; 39(1): 307-16 26. abbasi mk, sharif s, kazmi m, sultan t, aslam m. isolation of plant growth promoting rhizobacteria from wheat rhizosphere and their effect on improving growth, yield and nutrient uptake of plants. plant biosys. 2011; 145(1): 159-68. 27. dias a, santos sgd, vasconcelos vgd, radl v, xavier gr, rumjanek ng, et al. screening of plant growth promoting rhizobacteria for the development of vegetable crops inoculants. afr j microbiol res. 2013; 7(19): 2087-92. http://www.sciencedirect.com/science/article/pii/s0304423806005012 http://www.sciencedirect.com/science/article/pii/s0304423806005012 http://www.sciencedirect.com/science/article/pii/s0304423806005012 http://www.sciencedirect.com/science/journal/03044238/111/4 assessing the hepatotoxicity of industrial leachate; histopathology and heavy metal contents in liver of wistar rats vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 82 r a d s j . b i o l . r e s . a p p l . s c i 82 op en ac ces s f u l l l e n g t h a r t i c l e assessing the hepatotoxicity of industrial leachate; histopathology and heavy metal contents in liver of wistar rats muhammad babar khawar1, rabia mehmood1, muddasir hassan abbasi2,* and nadeem sheikh1,2,* 1cell & molecular biology lab, department of zoology, university of the punjab, lahore, pakistan 2centre for applied molecular biology (camb), university of the punjab, lahore, pakistan a b s t r a c t the process of paper production requires a huge quantity of water and energy and in turn contributes a number of effluents in the form of phenolics, toxic organic compounds and heavy metals in wastewater (leachate). the present investigation was aimed to assess the toxic effects of leachate on liver micro-architecture and heavy metal elements of the liver. eighteen (18) healthy male wistar rats (240 ± 10g) were selected and acclimatized prior to experimental treatment. these rats were randomly divided into three groups viz, control group (received 4ml/ kg normal saline), group 1 (4ml/ kg leachate) and group 2 (4ml/ kg 1:10 diluted leachate). all the animals were dissected and liver tissues were collected and processed accordingly after 24 h of leachate treatment. high level of cadmium and chromium were found in group 1 as compared to the control group upon liver metal contents analysis found out by flame atomic absorption spectrometer. a clear disruption of micro-architecture of the liver, congested sinusoids, damaged central vein, and perturbed morphology was observed in group-1 as revealed by h & e staining. moreover, loss of polarity, congestion, and disruption of hepatocytes and pronounced vacuolization in the cytoplasm was observed in group 2 compared to control sections. on the basis of above findings, it can be concluded that paper industry leachate is highly toxic and its intraperitoneal injection results in hepatotoxicity that not only affects the hepatic micro-architecture but also results in perturbed liver metal contents. therefore, proper treatment of such wastewater is required before its disposal. keywords heavy metals, histopathology, leachate, toxicity, wastewater. *address of correspondence s_nadeem77@yahoo.com muddygcs@gmail.com article info. received: september 16, 2018 accepted: october 26, 2018 cite this article: khawar mb, mehmood r, abbasi mh, sheikh n. assessing the hepatotoxicity of industrial leachate; histopathology and heavy metal contents in liver of wistar rats. rads j. biol. res. appl. sci. 2018; 9(2): 82-87. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n pulp and paper industry is involved in the spread of pollution because of the toxicity it poses to the environment. this industry utilizes a large quantity of lignocelluloses materials as well as a large amount of water as a raw material for paper production and in turn results in the discharge of a large quantity of effluents 1;2. these effluents are problematic because of their biochemical oxygen demand (bod), absorbable organic halides (aox), dark coloration, chemical oxygen demand (cod), ph and large amounts of suspended solids (ss). the early stage of the paper production is pulping which is a chief pollution source and results in the processed material. a significant amount of wastewater is produced at various stages of pulping. firstly, mechanical pulping results in 90–95% of the yield of the pulp but the resultant pulp is highly colored, and of poor quality due to presence of short fibers in it. chemical pulping involves some appropriate chemicals with suitable media (alkaline or acidic) are used to process wood chips at a high temperature and pressure within an aqueous solution o r i g i n a l a r t i c l e assessing the hepatotoxicity of industrial leachate; histopathology and heavy metal contents in liver of wistar rats vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 83 r a d s j . b i o l . r e s . a p p l . s c i 83 which converts the wooden chips into a fibrous mass. chemical pulping results in 40–50% pulp yield of the total wood utilized 3. alkaline medium (kraft process) is required to process the woodchips, solutions of sodium hydroxide (naoh) commonly called caustic soda and sodium sulfide (nas2) are used. on the other hand, acidic medium (sulfite process) involves the use of certain acids like a combination of sulfurous acid (h2so3) and some other compounds like bisulfide ions (hso3-) to dissolve the lignin component. these effluents also contain significant amounts of some highly toxic chlorinated compounds i.e. chlorinated hydrocarbon, lignosulphonic acids, chlorinated phenols and some resin acids etc. these chlorinated compounds react with lignin and some of its derivatives resulting in some very strong toxic compounds 4;5. most of the arbitrarily produced organo-chlorinated compounds during the process of pulp bleaching are highly noxious xenobiotics which remain un-degraded for a longer period of time in the environment. if these effluents are discharged to a hypoxic environment or anaerobic water the condition often became worse because of the high toxicity and lipophilic abilities of these xenobiotics upon methylation of chlorinated organic compounds by certain species of anaerobic bacteria. unluckily, this high lipophilicity made them extremely soluble in fats of higher animals. if any of this methylated chlorinated compound is accidentally ingested by an animal species i.e. fowl or fish, it gets deposited in their adipose tissues rather being completely metabolized and excreted through urine, it gradually becomes more and more concentrated (bioaccumulation) as is carried to the higher trophic levels in the food chain 6. so, the present study was aimed to assess the hepatotoxicity of paper industry wastewater by analyzing the heavy metal contents and histopathology of the liver. m a t e r i a l a n d m e t h o d animals healthy wistar rats of eight (8) weeks of age weighing about 240 ± 10g were obtained from the department of zoology, university of punjab (lahore-pakistan), housed in a room of animal house under controlled conditions with temperature (22 ± 1°c), humidity and light (12-h light/ dark cycles) with fresh water and food (rat chow) available ad libitum throughout the course of study period. procedures involving animals and their care were conducted in conformity with international laws and policies. experimental methodology and animal groupings eighteen (18) healthy male wistar rats weighing about 240 ± 10g were taken and randomly divided into three groups i.e. control, group 1 & group 2. four (4) ml/ kg leachate was injected intraperitoneally to group-1 while group-2 received the same quantity of 1:10 diluted leachate. while control group received same quantity of normal saline. animals were euthanized and sacrificed after 24 hours of the leachate induction. liver tissues of all the animals were excised and processed accordingly for further studies. determination of liver metals contents to reduce the chances of contamination, all the materials and chemicals used in the experiment were properly washed with ddh2o, and a sterilized stainless steel scalpel was used to slice the hepatic tissues. before carrying out the analysis, sample tissue (1g) was taken from each liver specimen of control and both the experimental groups. wet digestion method was adopted for the digestion of samples to determine heavy metal contents. briefly, hepatic tissues were placed in a digestion flask with 7ml of conc. nitric acid (hno3) and 1 ml of hydrogen peroxide (h2o2). after that, the flasks were placed on a hot plate for about 4-5 hours for complete digestion. then, they were cooled down to room temperature. the remaining material was then filtered and diluted to make the final volume 15ml. then the analysis of each sample was carried out for two heavy metals viz, cadmium and chromium by using flame emission atomic absorption spectrometry. h & e staining h & e staining was carried out as follows: de-paraffinized slides were dipped in hematoxylin working solution for about 1.5 min followed by rinsing thoroughly with deionized h2o and then dehydration was carried out with assessing the hepatotoxicity of industrial leachate; histopathology and heavy metal contents in liver of wistar rats vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 84 r a d s j . b i o l . r e s . a p p l . s c i 84 70% and 90% ethanol for 5 min each. slides were subsequently stained with working eosin solution for 3-5 min and then rinsed with de-ionized water followed by a rapid dehydration through alcohol and xylene. finally, the sections were subjected to microscopy to capture the images. ethical statement the experimental procedure and methodologies were approved by the ethical committee of the department of zoology, university of the punjab, lahore, pakistan. statistical analysis all the data reported are means ± sem and were statistically calculated by using prism graph pad 5 software (san diego, ca). one-way anova was used to find out the statistical significance with p < 0.05 considered significant. r e s u l t s cadmium and chromium liver contents cadmium, and chromium level in the liver of both the experimental groups, showed statistically significant alterations compared to control. cadmium level showed a significant positive change and was measured as 102.3 mg/ kg in group 1 while a significant negative change with 6.75 mg/ kg was noted in group 2 (p<0.0001) compared to control 42.6 mg/ kg. on the other hand, for chromium, a significant increase of 136.5 mg/ kg was observed in group 1 while group 2 showed a decrement with 2.4 mg/ kg as compared to control 4.05 mg/ kg (p<0.0001) (table 1) (fig. 1). fig. 1: level of metal contents of cadmium and chromium in liver tissues. cadmium level was found to be increased significantly in group 1 while it was decreased in group 2 as compared to the control (p<0.0001). a similar trend was observed in chromium levels in group 1 (p<0.0001), while group 2 showed no significant alterations compared to control. the results are representative of three animal series n=3 (mean ± s.e.m.). table 1: concentration of different heavy metals in liver tissues digested in hno3 and h2o2 (7:1). samples animals concentrations of cd (mg/l) average (mg/l) average cd contents in liver (mg/kg) concentrations of cr (mg/l) average (mg/l) average cr contents in liver (mg/kg) control c1 2.87 2.84 42.6 0.21 0.27 4.05 c2 2.94 0.30 c3 2.72 0.29 group 1 d1 6.49 6.82 102.3*** 8.96 9.10 136.5*** d2 6.95 9.27 d3 7.01 9.06 group 2 e1 0.38 0.45 6.75*** 0.13 0.16 2.4 e2 0.45 0.15 e3 0.53 0.19 assessing the hepatotoxicity of industrial leachate; histopathology and heavy metal contents in liver of wistar rats vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 85 r a d s j . b i o l . r e s . a p p l . s c i 85 histopathological examination microscopic examinations were carried out using the microscope (olympus vanox), which showed various cellular changes in h & e stained liver sections. in the control h&e stained sections, no pathological abnormalities were seen and sections looked normal with regular cellular architecture. the hepatocytes had intact cytoplasm, normal sinusoidal spaces and central vein (fig. 2). the administration of leachate in rats led to disruption in general micro-architecture of the hepatic tissue. sections from group 1 showed thickening of epithelial cells surrounding the central vein, ruptured central vein, congestion of sinusoids, severe necrosis in some hepatocytes and a slight degeneration of a few hepatocytes (fig. 3). while sections of group 2 showed a disturbed architecture characterized with edema of hepatocytes, vacuolization, and degeneration in some hepatocytes but central vein remain intact (fig. 4). fig. 2: h&e stained section of control. hematoxylin & eosin stained liver section of control group showing intact micro-architecture and no sign of pathological anomaly. fig. 3: h&e stained section of group 1. hematoxylin & eosin stained liver section of group 1 showing a disturbed micro-architecture with clear signs of a pathological anomaly. section showing a congestion of sinusoids (c) and degeneration of some hepatocytes (d). moreover in the enlarged view arrowhead is showing the thickening of basement membrane and a ruptured central vein can also be observed (r). fig. 4: h&e stained section of group 2. hematoxylin & eosin stained liver section of group 2 showing an abnormal micro-architecture while central vein (cv) remains intact with some clear signs of pathological problems. section showing an extensive vacuolization (v) and degeneration of some hepatocytes (d). enlarged view showing the edema of hepatocytes represented by an arrowhead. assessing the hepatotoxicity of industrial leachate; histopathology and heavy metal contents in liver of wistar rats vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 86 r a d s j . b i o l . r e s . a p p l . s c i 86 d i s c u s s i o n liver, kidney, and gills are some of the metabolically active organs which are prime sites of heavy metal accumulation because of their functions 7. previous data revealed the role of the liver as a pivotal organ primarily involved in bio-transformations, redistribution of certain materials, storage of various useful as well as harmful substances, detoxification and many metabolic activities 8,9. animals exposed to cadmium can accumulate this metal in various organs of the body primarily in the livers and kidneys because of the presence of free protein-thiol group which is responsible for a strong fixation and subsequent bioaccumulation of heavy metals. despite very efficient excretory mechanism for the elimination of such heavy metals, vertebrates are unable to develop these mechanisms, during the course of evolution, to the extent necessary for today’s anthropogenic sources of pollution 10. liver cadmium and chromium showed a significant increase in group 1 compared to control. this increase in heavy metal contents is alarming because of their toxicity and interference with many important biological phenomena. increased level of cadmium and chromium in the current study are clearly reflective of the effect of leachate induction resulting in bioaccumulation of these heavy metals. liver, one of the largest glands of the body, absorbs the nutrients from the small intestine and stored them as glycogen. in addition to it, the liver is also involved in the biosynthesis of a variety of plasma proteins and in the detoxification and neutralization of various exogenous toxins. so keeping in view the above-said roles, the hepatic cells are one of the highly active cells having eosinophilic cytoplasm and euchromatin nucleus. the capillary plexus of hepatic sinusoids is responsible for the transportation of materials between blood and liver tissue11 and as hepatocytes are continuously exposed to the toxic substances in the blood so majorly these cells could be affected by these noxious substances. in the current study, leachate induction led to prominent alterations in the microarchitecture of the liver tissues in both studied groups which are obvious from the h & e stained histologic sections of the liver. similar changes were observed by ortiz et al., (1994) in the liver of chicks and rats after they were fed with diets high in tannins extracted from faba bean (vicia faba). ortiz et al., (1994) observed extensive vacuolization of hepatocytes, degeneration as well as marked eosinophilia12. similarly, emiola et al., (2007) observed severe degeneration of hepatocytes, congestion of sinusoids, as well as an extensive coagulative necrosis in chickens fed with raw, dehulled, and aqueous and dry-heated kidney bean meals 13. histological findings of the current study are in accordance with the results of the above studies because similar changes have been observed in group 1 and group 2 after the induction of leachate intraperitoneally. c o n c l u s i o n so, it is recommended that before disposal of wastewater in water bodies or onto land, the characteristic dark color and toxic components especially phenols and chlorinated compounds must be eliminated or minimized to the levels below the discharge limits. acknowledgements the authors are thankful to the vice chancellor, university of the punjab, lahore, pakistan for providing financial support to publish the article. competing interests statement for the authors the authors declare that they have no conflicts of interests. r e f e r e n c e s 1. sumathi s, hung yt. treatment of pulp and paper mill wastes. in: wang lk, hung y-t, lo hh, yapijakis c, editors. waste treatment in the process industries illustrated ed. crc press; 2006.p. 453-497. 2. thompson g, swain j, kay m, forster cf. the treatment of pulp and paper mill effluent: a review. bioresour technol. 2001; 77(3):275-286. 3. smook ga. handbook for pulp & paper technologists. vancouver: angus wilde publications; 1992. 4. choudhary sk, jha an, srivastava dk. effect of paper mill effluent on seed germination and seedling growth of maize. environ ecol. 1987; 5: 285-287. 5. pokhrel d, viraraghavan t. treatment of pulp and paper mill wastewater-a review. sci total environ. 2004; 333(1):37-58. 6. kierkegaard a, renberg l. chemical characterization of organochlorine compounds, originating from pulp assessing the hepatotoxicity of industrial leachate; histopathology and heavy metal contents in liver of wistar rats vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 87 r a d s j . b i o l . r e s . a p p l . s c i 87 mill effluents, in fish. water sci technol. 1988; 20(2): 165. 7. dural m, goksu ml, ozak aa, baris d. bioaccumulation of some heavy metals in different tissues of dicentrarchus labrax l, 1758, sparus aurata l, 1758 and mugil cephalus l, 1758 from the camlik lagoon of the eastern cost of mediterranean (turkey). environ monit assess. 2006; 118(1-3):65-74. 8. agah h, leermakers m, elskens m, rez fsm, willy b. accumulation of trace metals in the muscle and liver tissues of five fish species from the persian gulf. environ monit assess. 2009; 157(1-4):499-514. 9. malik n, biswas ak, qureshi ta, borana k, virha r. bioaccumulation of heavy metals in fish tissues of a freshwater lake of bhopal. environ monit assess. 2010; 160(1-4):267-276. 10. pompe-gotal j, crnic ap. cadmium in tissues of roe deer (capreolus capreolus) in croatia. veterinarski arhiv 2002; 72(6):303-310. 11. carneiro j. basic histology: text & atlas. lange medical books, mcgraw-hill, medical pub. division; 2003. 12. ortiz lt, alzueta c, trevino j, castano m. effects of faba bean tannins on the growth and histological structure of the intestinal tract and liver of chicks and rats. br poult sci. 1994; 35(5):743-754. 13. emiola ia, ologhobo ad, gous rm. performance and histological responses of internal organs of broiler chickens fed raw, dehulled, and aqueous and dryheated kidney bean meals. poult sci. 2007; 86(6):1234-1240. choice of maize genotype affects wheat haploid seed and success of embryo rescue vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 1 r a d s j . b i o l . r e s . a p p l . s c i . 1 op en ac ces s f u l l l e n g t h a r t i c l e choice of maize genotype affects wheat haploid seed and success of embryo rescue muhammad ilyas khokhar1,*, abdul razaq1, junaid iqbal2, muhammad jamshaid anwar1, muhammad zaffar iqbal1, sajid ur rehman1 1agricultural biotechnology research institute, ayub agricultural research institute, faisalabad, pakistan 2department plant breeding and genetics, university of agriculture (uaf), faisalabad, pakistan a b s t r a c t in wheat (triticum aestivum) breeding, the use of double haploids plays a vital role by reducing the length of the breeding cycle. the first step in the production of a wheat double haploid is to create a haploid, which in wheat can be achieved via wheat × maize cross, and resulting haploid plants are recovered by embryo rescue. in this study, a wheat segregating population (f2) was emasculated and pollinated with pollen from four maize varieties (sadaf, malka-2016, pearl and mmri yellow). maize genotype affected the outcome of haploid seed production (sadaf = 28.58%; pearl = 28.33%; malka-2016 = 26.42%; mmri yellow = 17.97%) and embryo rescue (malka-2016 = 27.02%; mmri yellow = 25.82%; sadaf = 20.17%; pearl = 16.47%). sadaf produced maximum haploid seed (28.58%) followed by pearl (28.33%) but higher embryo rescued success (27.02%) was observed in malka-2016 followed by mmri (25.82%). we recommend the use of sadaf or malka-2016 to produce haploid seed and to achieve successful embryo rescue in wheat × maize crossing for wheat doubled haploid production. keywords 2, 4-d, haploid embryo induction, embryo rescue. *address of correspondence khokharab@yahoo.com, ilyasabri@yahoo.com article info. received: january 1, 2019 accepted: july 1, 2019 cite this article: khokhar mi, razaq a, iqbal j, anwar mj, iqbal mz, rehman s. choice of maize genotype affects wheat haploid seed and success of embryo rescue. rads j. biol. res. appl. sci. 2019; 10(1): 1-5. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n double haploid (dh) development is a powerful technique to accelerate the conventional breeding program. dhs are extensively used for genetic studies i.e. inheritance of quantitative traits, genomics, gene identification, quantitative trait loci (qtl) mapping, whole genome mapping and production of stable transgenic plants1. several methods including parthenogenesis, apogamy, anther/microspore culture, ovary culture and wide hybridization have been developed to produce haploid plants2. anther-culture and chromosome elimination techniques were used for, haploid wheat plants. but anther-culture technique is not effective as it is greatly influenced by wheat genotype3. chromosome elimination technique was applied by campbell et al. in wheat4. the mechanism behind chromosome elimination in wheat × maize crossing system is that after pollination maize pollen germinates on stigma and reaches to embryo sac to fertilize wheat egg. after the fertilization a hybrid is produced with 21 wheat chromosomes and 10 maize chromosomes. centromeres on chromosomes lose their strength to attach with the spindle fiber, maize chromosomes eliminate after 2 to 3 cell division ultimately haploid embryo with 21 wheat chromosomes are formed5. kazi et al.,6 stated that maize pollen is an effective factor for haploid seed development. khan et al.,5 studied the effect of five maize genotypes (neelum, sadaf, sultan, agaiti-2002 and agaiti-85) as male parent in wheat × maize crossing and observed significant effects on haploid seeds formation and embryos production. other o r i g i n a l a r t i c l e choice of maize genotype affects wheat haploid seed and success of embryo rescue vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 2 r a d s j . b i o l . r e s . a p p l . s c i . 2 factors that affect haploid seeds production in wheat × maize technique are growth conditions of the wheat and maize plants, mode of growth regulator application, genotype, time of embryo rescue, and embryo rescue medium5. the present study was conducted to examine the effect of different maize genotypes on haploid seed formation and to assess the success of survival of rescued embryos. m e t h o d o l o g y the research was conducted in cytogenetic laboratory and experimental area of agricultural biotechnology research institute (abri), ayub agricultural research institute (aari) faisalabad, pakistan during 2017-18. the experiments were laid out as completely randomized design (crd), replicated thrice and 20 spikes emasculated for each replication and treatment. plant material wheat segregated material (f2) of the desirable cross (ufaq-2002×aari-11) was sown in an open field during november, 2017. four maize commercial varieties i.e. malka-2016, pearl, mmri yellow and sadaf obtained from maize and millet research institute, yousafwala, pakistan and were sown in a high tunnel (artificial structure made with iron and covered with polythene sheet, length 50 feet and width 10 feet) (figure 1a) with 8 days interval i.e. 8, 16, 24, 32, 40 and 48 days after 1st date of sowing (31 october, 2017) to ensure maize pollen availability throughout the reproductive stage of wheat crop for pollination from february to march 2018. emasculation the first set of wheat tillers were selected for emasculation from 19th february 2018 when booting occurred and cut from the base with appropriate length (92-95cm). all leaves were removed from the tiller except flag leaf to avoid evapotranspiration and also detached central floret of each spikelet with the help of forceps to easily emasculate the lateral florets. one-third portion of lateral florets also cut from the top to accelerate the emasculation and pollination. then anthers removed with forceps and emasculated spikelets were covered with butter paper bag to avoid from outcrossing, tagged and mentioned date of emasculation. twenty spikes per replication were emasculated and crossed with four maize genotypes. emasculated tillers were kept in the tap water jar and placed in a growth room under controlled temperature, light7. pollination after 72 hours of emasculation, fresh maize pollens were collected in a petri dishes (figure 1b) and wheat spikelets were pollinated with the help of camel hair brush within 10-12 minutes (figure 1c). pollinated spikes were kept in medium (a) (table 1) for two days and these tillers were taken out from medium (a) and kept into medium (b) detailed in table 1 (figure 1d). growth media changed every 72 hours for twelve days then haploid seeds were separated from spikes. table 1. three different media composition used for haploid seed production. medium composition medium (a) 10 mg/l 2,4-d + 6% h2so3 + 40 g/l sucrose medium (b) 6% h2so3 + 40 g/l sucrose ½ ms medium (c) 2.22 g/l ms + 2.0 g/l phyta gel + 30 g/l sucrose embryo rescue developed haploid seeds had whitish in colour, small in size and absent of endosperm as described by khan et al.,5, placed in a petri dish (figure 1f) then seeds were surface sterilized with 1% clorex (clorox chemical co., oakland, usa), added two drops of tween-20 and placed for 15 minutes on water bath shaker. haploid seeds were washed three times with autoclaved distilled water in laminar air flow cabinet (gelaire, italy, model hf96). haploid seed (figure 1g) was dissected under stereomicroscope (model: tl2, meiji techno, japan), haploid embryo rescued and put into autoclaved half strength ms8 as earlier described by khan et al.,5. the whole procedure was performed under a laminar flow cabinet (streamline laboratory product, singapore). the ph of the ½ ms medium was adjusted at 5.8 by adding 1n hcl or 1n naoh before autoclave. the media were then autoclaved at 15 psi for 20 min at 120°c according to khokhar et al.,9. haploid embryo rescued by examining under stereomicroscope in laminar flow cabinet and put choice of maize genotype affects wheat haploid seed and success of embryo rescue vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 3 r a d s j . b i o l . r e s . a p p l . s c i . 3 into ½ ms medium test tubes (length 15cm and diameter 2.5cm), covered with polypropylene paper and placed in an incubation room at 22c0 under dark period for two weeks according to khan et al.,5. figure 1. (a) maize tassel; (b) maize pollen collection; (c) pollination; (d) pollinated spikes; (e) haploid seeds in spikelets; (f) haploid seeds; (g) haploid embryo. data collection number of haploid seeds: haploid seeds developed were counted and found sum of total haploid seeds from three repeats of each cross. number of embryos rescued: number of embryos rescued were recorded as sum of total embryos rescued of each cross per repeat. haploid seeds produced (%): haploid seeds production percentage were counted by using this formula. haploid embryo rescued (%): haploid embryos rescued percentage were found by using this equation. embryo formed in florets (%): embryo formed in florets was counted by using this formula. statistical analysis data were analyzed with statistics 8.1 software program (table 2). mean variance of wheat × maize crosses with a success percentage of haploid embryo rescue was calculated by the least significant difference (lsd) at α = 0.05 (table 3). table 2. analysis of variance four maize genotypes on haploid embryos rescue success percentage. sov df ss ms f value p value treatment 3 206.267 68.7556 5.15* 0.0285 error 8 106.900 13.3625 total 11 313.167 cv 16.08 table 3. lsd test for haploid seed production and embryo rescue success (%) in wheat × maize crosses. crosses haploid seed production embryo rescue success (%) wheat (f2) x sadaf 74.33 b 21.20 b wheat (f2) x malka-2016 74.00 b 27.13 a wheat (f2) x pearl 85.00 a 16.67 c wheat (f2) x mmri yellow 50.33 c 25.93 a lsd (α = 0.05) 2.306 2.985 r e s u l t s a n d d i s c u s s i o n 240 spikes of wheat in the filial generation (f2) were emasculated and pollinated with four maize varieties. 3360 florets (about 14 florets/ spike) were pollinated which produced 861 haploid seeds (25.34%) table 2 and out of these 186 embryos were rescued. it was observed maximum haploid seeds produced 223 from 780 florets (28.58%) in “sadaf” followed by 222 from 840 (28.33%) in pearl, 255 from 900 (26.42%) in “malka-2016”, while minimum 151 out of 840 (17.97%) in “mmri yellow” were noted as explained in table 4. niroula et al.,10 analyzed different maize genotypes and the results showed that “arun-2” formed 24 embryos out of 111 pollinated floret followed by “khumal yellow” which resulted in 22 embryos from 116 whereas “rampur composite” resulted in a minimum number of 5 embryos from 91 pollinated floret. our study showed better results were obtained 223 choice of maize genotype affects wheat haploid seed and success of embryo rescue vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 4 r a d s j . b i o l . r e s . a p p l . s c i . 4 haploid seed out of 780 florets when “sadaf” used as maize source followed by 255 out of 900 florets from “pearl” with 28.58% and 28.33%, respectively. these studies revealed that different maize genotypes helped in better haploid seed development. khan et al.,5 studied haploid seed production using different maize genotypes and observed that average performance of haploid seed production in “neelum” (56.41%) as compared to other varieties i.e. agaiti-2002 (48.69), sultan (46.98), agaiti-85 (46.41%) and sadaf (45.17%). xynias et al.,2 studied maximum haploid seed 11.3% in cross penios × acheloos followed by 10.6% in penios × kvz/cgn while minimum in vergina × acheloos (5.6%) after 12-14 days of pollination. in the current study, 28.58% haploid seeds were developed after 12 days so this study confirmed that haploid seed production is the best in 12 days after pollination. maximum embryos rescued 27.02% in “malka-2016” followed by 25.82% in “mmri yellow”, 20.17% in “sadaf” and lowest 16.47% in “pearl” (table 4). niroula et al.,10 analyzed different maize genotypes and the results showed that “arun-2” formed 24 embryos out of 111 pollinated floret followed by “khumal yellow” which resulted in 22 embryos from 116 whereas “rampur composite” resulted in a minimum number of 5 embryos from 91 pollinated florets. in our studies the highest number of embryos were rescued when maize genotype malka-2016 was used as pollinator. the rescued embryos percentage ranges 4.64% to 7.69% in “mmri yellow” and “malka-2016”, respectively. khan et al.,5 observed the highest average percentage of haploid embryos setting in “sadaf” (19.13%) followed by “neelum” (13.20%) while lowest in “agaiti-85” (9.11%) followed by “sultan” (12.23%). our study revealed that maize genotypes also had significant role on embryo rescue performance. average embryo rescue best percentage was found in “malka-2016” (27.02%) followed by “mmri yellow” (25.82%) and lowest result observed in “pearl” (16.47) table 4. embryo rescue percentage in our study is (27.02%) found better comparatively (19.13%) in sadaf used by khan et al.,5. male pollen source of malka-2016 enabled us to produce higher number of haploid embryos rescued. xynias et al.,2 reported maximum haploid embryos percentage after 12 days of pollination (10.9%) while 9.8% and 7.6% after 14 and 16 days of pollination, respectively. this study confirmed our finding regarding maximum embryos formation after 12 days of pollination. khan and ahmad11 emasculated wheat spikes and pollinated after 24, 48 and 72 hours. embryo rescue ranged 0.0-9.52% after 72 hours, 0.0 to 7.48% after 48 hours and 2.22 to 9.09% after 24 hours at various temperature. in our study, embryos rescued range 4.64 to 7.69% after 72 hours of pollination at 22±2˚c in the growth room. it was concluded the time of emasculation had vital role in embryos rescue percentage when pollinated after 72 hours. these finding conceded the previous work. c o n c l u s i o n this study concluded specific maize genotypes are suitable for haploid seed production. maize genotypes sadaf and malka-2016 are specific to produce haploid table 4. response of wheat × maize crosses on haploid seed production, embryo rescued, haploid embryo rescued from haploid seeds and embryo formed in florets. crosses no. spikes no. florets no. haploid seeds haploid seeds produced (%) no. embryos rescued haploid embryo rescued (%) embryo formed in florets (%) wheat (f2) x sadaf 60 780 223 28.58 45 20.17 5.35 wheat (f2) x malka2016 60 840 222 26.42 60 27.02 7.69 wheat (f2) x pearl 60 900 255 28.33 42 16.47 4.66 wheat (f2) x mmri yellow 60 840 151 17.97 39 25.82 4.64 total 240 3360 861 25.34 186 22.24 5.58 choice of maize genotype affects wheat haploid seed and success of embryo rescue vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 5 r a d s j . b i o l . r e s . a p p l . s c i . 5 seed and embryo rescue, respectively so these genotypes are recommended as the best pollinators in wheat × maize crossing for obtaining maximum haploid seed formation and embryo rescue. r e f e r e n c e s 1. hussain b, khan ma, qurban a, shadab s. double haploid production is the best method for genetic improvement and genetic studies of wheat. ijavms. 2012;6(4):216-28. 2. xynias l, koufalis a, vavdinoudi eg, roupakias d. factors affecting doubled haploid plant production via maize technique in bread wheat. acta biologica cracoviensia, botanica. 2014;56(2):67-73. 3. daniel g, baumann a, schmucker s. production of wheat doubled haploids (triticum aestivum l.) by wheat x maize crosses using colchicine enriched medium for embryo regeneration. cereal research communications. 2005; 33:461-68. 4. campbell aw, griffin wb, burritt dj, conner aj. production of wheat doubled haploids via wide crosses in new zealand wheat. new zeal j crop hort. 2000;28(3):185-94. 5. khan ma, kashif m, ahmad j, khan as, khaliq i, bilquees f, shaukat s. sadafa potential donor for enhancing frequency of doubled haploids in wheat × maize crossing system. pak j agri sci. 2014;51(2):363-7. 6. kazi am, gul a, ahmed j, mirza ji. a simplified and effective protocol for production of bread wheat haploids (n = 3x = 21, abd) with some application areas in wheat improvement. pak j bot. 2006;38(2):393-406. 7. campbell aw, griffin wb, conner aj, rowarth js, burritt dj. the effects of temperature and light intensity on embryo numbers in wheat doubled haploid production through wheat× maize crosses. ann bot. 1998;82(1):29-33. 8. murashige t, skoog f. a revised medium for rapid growth and bioassy with tobacco tissue culture. physiol plant. 1962; 15:473-97. 9. khokhar mi, iqbal mz, rehman s, akhtar s, javed k. steps and factors in double haploid wheat plant production through wheat × maize cross a review. j agric res. 2017;55(2):267-78. 10. niroula rk, bimb hp, thapa db, sah bp, nayak s. production of haploid wheat plants from wheat (triticum aestivum l.) x maize (zea mays l.) cross system. himalayan journal of sciences. 2007;4(6):65-9. 11. khan ma, ahmad j. in vitro wheat haploid embryo production by wheat x maize cross system under different environmental conditions. pak j agri sci. 2011;48(1):49-53. effect of cadmium levels on the growth curve of candida albicans aziz fatima1*,qudsia hussain1, shazia tabassum hakim1, sayyada ghufrana nadeem1 1department of microbiology, jinnah university for women, karchi-74600, pakistan. abstract environmental pollution by toxic heavy metals is one of the most pressing problems. metals are released in the environment in industrial effluent. cadmium is one of the heavy metal toxic to microorganisms; however, there are yeast strains resistant to this metal. one of yeast species candida albicans is a diploid fungus (a form of yeast) that is a causal agent of opportunistic oral and genital infections in humans. in the present study, effect of cadmium on the growth curve of candida albicans was demonstrated. candida albicans was grown both in the presence and absence of cadmium in yepd media. growth curves of the candida albicans were plotted to study the growth pattern of the yeast isolate. it was found that candida albicans grow well at 50 µg/ml concentration of cadmium. the organism was found to be resistant to the used heavy metal. growing metal resistant cells is very important as it can ensure better removal through the process of biosorption. such approaches may help in the removal of toxic metals from the environmental thus reducing environmental pollution. keywords: biosorption, candida albicans, cadmium, detoxification, heavy metals. introduction rapid industrialization and urbanization have enhanced the levels of organic and inorganic contaminants in the environment (chaudhari et al., 2009). the inorganic minerals like sodium, potassium, calcium, magnesium and heavy metals like iron, manganese, lead, mercury, chromium, cadmium, nickel, cobalt, beryllium copper etc., when present above the permissible limit are harmful. agricultural water pollution is caused by fertilizers, insecticides, pesticides, farm animal wastes and sediments (begum et al., 2009). industrial activities have led to large-scale contamination of the environment with toxic heavy metals and radio nuclides (lloyd and lovely, 2001). heavy metals are chemical elements with a specific gravity that is at least 5 times the specific gravity of water. some well-known toxic metallic elements with a specific gravity that is 5 or more times that of water are arsenic, 5.7; cadmium, 8.65; iron, 7.9; lead, 11.34; and mercury, 13.546. toxic heavy metals in air, soil, and water are global problems that are a growing threat to humanity. there are hundreds of sources of heavy metal pollution, including the coal, natural gas, paper, and chore-alkali industries. in response to the growing problems, federal and state governments have instituted environmental regulations to protect the quality of surface and ground water from heavy metal pollutants, such as cd, cu, pb, hg, cr, and fe. interactions between microorganisms and metals can be conveniently divided into three distinct processes, a) intracellular interactions, b) cell-surface interactions, and extracellular interactions (gaylarde and videla, 1995). microbes have evolved to deal with toxic metals using several mechanisms. heavy metals such as mercury, lead, copper, and arsenic are metabolic poisons in that they inhibit the activity of certain enzymes. heavy metal ions often damage viable cell severly if they play essential role in many metabolic processes at low concentrations. candida albicans is yeast that showed resistance to certain*corresponding author: azizfatima1988@gmail.com 38 vol 4 (2), july 2013; 38-41 heavy metals. it lives in the mouth, throat, intestines and genitourinary tract of most humans and is usually considered to be a normal part of the bowel flora (the organisms that coexist with us in our lower digestive tract). it is actually a member of a broader classification of organisms known as fungi. candida albicans is a diploid organism which has eight sets of chromosome pairs. interestingly, candida is one of the few microorganisms that have a diploid gene controlling the same protein – this means that is capable of pleomorphic activity being able to mutate forms from the budding form to the mycelial, pathogenic form (bruins et al., 2009). cadmium is an important toxic metal whose in vivo metabolism and cellular mechanisms of toxicity appear to be highly complex (fowler, 1978). cadmium is a lustrous, silver-white, ductile, very malleable metal. its surface has a bluish tinge and the metal is soft enough to be cut with a knife, but it tarnishes in air. it is soluble in acids but not in alkalis. it is similar in many respects to zinc but it forms more complex compounds. cadmium metal exhibits excellent resistance to corrosion, particularly in alkaline and seawater environments, possesses a low melting temperature and rapid electrical exchange activity, and has both high electrical and thermal conductivity (lenntech, 2010). in mammals, cadmium exerts multiple toxic effects and has been classified as a human carcinogen by the international agency for research on cancer. cadmium affects cell proliferation, differentiation, apoptosis and other cellular activities. the inhibition of dna repair processes by cadmium represents a mechanism by which cadmium enhances the genotoxicity of other agents and may contribute to the tumor initiation by this metal. cadmium modulates also gene expression and signal transduction, reduces activities of proteins involved in antioxidant defenses (waisberg et al., 2003). cd depletes many essential metal antioxidants including selenium in the body. oxidative stress occurs as a result of an increase in cd-induced peroxidation of membrane lipids in the organs when it accumulates. (chen et al., 1995). fungal sporulation was more sensitive to cd than was mycelia growth, as spore formation was inhibited at cd concentrations that were no inhibitory to mycelia proliferation. in c. utilis there is a sharp decline in cell growth even at very low concentrations of cadmium (up to 0.0625mm) followed by a slow decrease in cell growth from 0.125 to 1mm cadmium (bertin and averbeck, 2006). cadmium-binding proteins have an important role in moderating cadmium toxicity in some fungi. in a previous study, candida was exposed to cadmium via soil or food. they demonstrated that the effect of cadmium on juvenile production of candida decreased with time (smit et al., 2004). c. tropicalis was found to be resistant to cd up to a concentration of 2,800 mg l-1. c. albicans and c. tropicalis are known for high levels of resistance to the water-soluble ions hg2 ,pb2+, cd2+, arsenate ,and selenite (rehman and anjum, 2010). in this study effect of cadmium levels on the activity of candida albicans was observed by plotting the growth curve. materials and methods culture: candida albicans. chemical: cadmium chloride solution (cdcl2) media: yeast extract peptone dextrose media (yepd) glass wares and instruments: petri plates, justers, tips, conical flasks, hot air oven, spectrophotometer, incubator, autoclave. procedure: inoculate 100ml of yepd media with 16 hour old culture of candida albicans and mark as control. incubate at 37oc for 24 hours. prepare stock solution of cadmium chloride in 100ml of distilled water. 50ug of cadmium chloride is dissolved in 100ml of distilled water and autoclave. inoculate another 100ml yepd media with 16 hour old culture of candida albicans and solution of cadmium. incubate at 30oc for 48 hours. after 48 hours, take the optical density of both control and test at 600 nm 39 vol 4 (2), july 2013; 38-41 after interval of 2 hours. results and discussion the use of yeast as biosorbent is particularly important because of the ease of genetic manipulation. cadmium is one of the important heavy metal present in industrial effluent, so there is need to grow metal resistant cells for the removal of cadmium. normally at higher concentration the metal can form certain complex compounds inside the microbial cell and may lead to toxic effects. but certain micro organisms are the potent agents for bioremediations such as yeast isolates accumulate toxic metals in significant values. the yeast can transform the absorbed metal into complex polymeric compounds non toxic for the cells. in yeast, several resistance mechanism can activated on exposure to toxic metal and identify certain genes involved in the metal detoxification. the resistance of yeast cell by cadmium is increase by the metallotheinoine and gluthione. yeast cells containing cad 2 exhibit a resistance to cadmium intracellular level through enhanced cadmium efflux system. in the present study, candida albicans was used to observe the effect of cadmium on its growth curve. microscopic examination showed that the growth of yeast isolates and the size of cell become smaller in the presence of heavy metal i.e. cd but the growth of the organism was not inhibited by the cadmium and the organism continues to grow in the presence of 50 ug/ml of cadmium (table i). the organism removed about 80% of cadmium from the yepd after 96 hours of incubation. in previous study it was observed that prolonged exposure of certain candida albicans strains to inhibitory concentrations of cadmium resulted in the appearance of resistant colonies (malavasic and cihlar, 1992). previous studies revealed that certain strains of yeast can show high resistance to cadmium in the yepd medium. at 50ug/ml concentration of cadmium in the yepd medium, there was a decline and a number of colonies start decreasing after 48 hours of incubation (muneer et al., 2007). while in the present study the growth of candida albicans in the 50ug/ml concentration of cadmium appears to be slower as compared to the control in which no cadmium was present but the growth was not inhibited by the heavy metal and the organism continues to grow at this concentration of cadmium. clinical isolates of c. albicans and candida glabrata exhibit high levels of resistance to both copper and cadmium salts, although the molecular basis of this resistance is not known (oh et al., 1999). whether the yeast isolates studied in this investigation, possess reduction abilities or they simply uptake the metal from the medium and accumulate with resultant lowering of the metal concentration in the medium has to be determined. these yeast isolates can be exploited for bioremediation of cadmium containing wastes, since they seem to have the potential to accumulate the toxic metal from the environment. figure 1. growth curve to show the effect of cadmium on candida albicans. 40 vol 4 (2), july 2013; 38-41 table i. optical density of candida albicans in control and in 50µg/ml cadmium concentration at 600 nm. 1 2 3 4 5 6 1 2 3 4 5 6 0 . 3 7 0.587 0.615 0.623 0.861 0.904 0.131 0.188 0.203 0.228 0.375 0.499 s. no. time (hrs) control cd (50) ug/ml references begum a, ramaiah m, khan hi, veena k. 2009. heavy metal pollution and chemical profile of cauvery river water. e-j. chem., 6 (1): 47-52. bertin g, averbeck d. 2006. cadmium: cellular effects, modifications of biomolecules, modulation of dna repair and genotoxic consequences (a review) biochimie., 88(11):1549–1559. bruins mr, kapil s & oehme fw. 2000. microbial resistance to metals in the environment. ecotox environ safety, 45: 198-207. chaudhari td, eapen s, fulekar mh. 2009. characterization of industrial waste and identification of potential micro -organism degrading tributyl phosphate. j toxicol environmental health sci, 1:1 7. chen h, yao j, zhou y, wang f, gai n, zhuang r et al. 2008. the toxic effect of cadmium on pure microbes using a microcalorimetric method and a biosensor technique. j. environ. sci. health a environ. sci. eng., 43:1639-1649. chen t, li w, schulz pj, furst a, chien pk. 1995. induction of peroxisome proliferation and increase of catalse activity in candida albicans by cadmium. biol trace element res, 50: 125-133. fowler ba. 1978. general subcellular effects of lead, mercury, cadmium, and arsenic. environ health perspect., 22:37–41. gaylarde cc and videla ha. 1995. bioextraction and biodeterioration of metals. cambridge univ. press, pp. 51-55. lenntech. 2010. cadmium. available from: http://www.lenntech.com/periodic/ elements/cd. htm#ixzz15mignbpt. lloyd jr, lovley dr. 2001. microbial detoxification of metals and radionuclides. curr. opin. biotechnol., 12: 248–253. malavasic cw and cihlar rl. 1992. growth response of several candida albicans strains to inhibitory concentrations of heavy metals. j med vet mycol, 30(6): 421-432. muneer b, shakoori fr, rehman a and shakoori ar. 2007. chromium resistant yeast with multimetal resistance isolated from industrial effluents and their possible use in microbial consortium for bioremediation of wastewater. pak j zool, 39(5): 289-297. oh kb, watanabe t, matsuoka h. 1999. a novel copper-binding protein with characteristics of a metallothionein from a clinical isolate of candida a l b i c a n s . m i c r o b i o l o g y 1 4 5 : 2 4 2 3 – 2 4 2 9 . rehman a and anjum ms. 2010. cadmium uptake by yeast, candida tropicalis, isolated from industrial effluents and its potential use in wastewater cleanup operations. water, air, and soil pollut., 205: 149-159. smit ce, stam em, baas n, hollander r, van gestel ca. 2004. effects of dietary zinc exposure on the life history of the parthenogenesis’ spring tail folsima candida (collebola: isotomida). environ toxicol chem, 23(7):1719-1724. waisberg m, joseph p, hale b, beyersmann d. 2003. molecular and cellular mechanisms of cadmium carcinogenesis. toxicology, 192 : 95-117. 41 vol 4 (2), july 2013; 38-41 prevalence and risk factors for diabetes mellitus in urban population of karachi (a short study) sobia jawaid1*, sayyada ghufrana nadeem1 1department of microbiology, jinnah university for women, karachi-74600, pakistan. abstract a descriptive observational study has been conducted on diabetes among male and female patients in karachi. data has been collected from april to june 2011. total number of patients were (n=1690). 64.7 % (n=1095) were male patients while 35.2% (n=595) were female patients. statistical analysis has been carried out separately for both male & female patients. 23.9% patients have target range for hba1c, 27.0% patients have target range for fasting plasma glycemia and 8.0% patients have target range for random blood sugar. it indicates they are pre diabetic patients. 12.5% patients have high values of hba1c, 12.5% patients have high values of fasting plasma glucose, and 4.0% patients have high values of random blood sugar. it indicates that they have diabetes. 13.5% patients have very high value for hba1c, 10.3%patients have very high value for fasting glucose, and 2.3% patients have high value for random blood sugar. they have diabetes mellitus condition. in this observational hospital based study prevalence of diabetes among male is greater than female and those patients at target ranges may at risk of developing diabetes. keywords: diebetes mellitus, prevalence, rbs, fbs, hba1c. introduction pakistan currently ranking at 7th position in the list of countries with major burden of dm and it is expected to move to 4th position (khuwaja et al., 2003). glycated hemoglobin (hba1c) expressed as a percentage of total blood hemoglobin concentration gives a good retrospective assessment of the mean plasma glucose concentration during the preceding 6 –8 weeks while the recent glycaemic level has the highest influence and the preceding 30 days contribute only up to 50% ( akinloye et al.,2007; alam et al., 2006). the higher the percentage of circulating hba1c in the diabetes, poorer is the mean diabetic control (akinloye et al., 2007) fasting plasma glucose level estimation has its limitations like the person has to fast for a specified period of time (gillet, 2009). similarly for random plasma glucose level the health care provider is not sure about the actual number of hours passed after the meal or any history of recent intake of any hypoglycemic or hyperglycemic drug. type 1 diabetes (t1dm, iddm, or, formerly, juvenile diabetes) is a form of diabetes mellitus that results from autoimmune destruction of insulin-producing beta cells of the pancreas. diabetes mellitus type 2– formerly non-insulindependent diabetes mellitus (niddm) or adult-onset diabetes– is a metabolic disorder that is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency (kumar et al., 2005). materials and methods descriptive observational hospital based study has *corresponding author: sobi_butter@yahoo.com 64 vol 4 (2), july 2013; 64-66 been carried out, separately for both male and female. hba1c, random blood sugar level, fasting blood glucose level values has been calculated. data has been collected from ankle saria hospital karachi, from april 2011 to june 2011.total number of patients were 1690. 595 were females and 1095 were male patients. 96 test request for hba1c, (66 male patients, 30 female patients), 1346 test request for random blood sugar, (851 male patients, 495 female patients), 248 fasting plasma glucose, (178 male patients, 70 female patients). data collected and was subjected to analysis in spss v. 19 software. results and discussion the diagnosis of diabetes mellitus is established with fasting plasma glucose = 7.0 mmol/l (126 mg/dl) or random plasma glucose = 11.1 mmol/l (200 mg/dl). in asymptomatic patients two samples are required to confirm diagnosis. this showed that 23.9% patients have target range for hba1c, 27.0% patients have target range for fasting plasma glycemia and 8.0% patients have target range for random blood sugar. target ranges of blood glucose level are pre diabetic ranges. it indicates that patients were at risk of developing diabetes. blood glucose targets should be in a healthy range to prevent diabetes complications. if the range of blood glucose level exceeds from target value than hyperglycemia occurs and if it decreases than hypoglycemia condition occur. 12.5% patients have high values of hba1c, 12.5% patients have high values of fasting plasma glucose, and 4.0% patients have high values of random blood sugar. it indicates that these patients have diabetes. 13.5% patients have very high value for hba1c, 10.3%patients have very high value for fasting glucose, and 2.3% patients have high value for random blood sugar. it indicates that these patients have diabetes mellitus. our study showed that males have high ratio of having dm as compared to females. although females are also at high risk. we also concluded from the present study that the proportion of individuals which are found at target risk needs further screening for diabetes and health education regarding diabetes mellitus. diabetes mellitus can be controlled and prevented by maintaining healthy eating plan, maintaining weight, because obesity can be a cause of 65 vol 4 (2), july 2013; 64-66 table i. total count for male & female patients 31 (46.9%) 18 (27.2%) 9 (13.6%) 8 (12.1%) n=66 17 (56.6%) 5 (16.6%) 3 (10%) 5 (16.6%) n=30 48 (50%) 23 (23.9%) 12 (12.5%) 13 (13.5%) n=96 85 (47.%7) 52 (29.2%) 27 (15.1%) 14 (7.8%) n=178 38 (54.2%) 15 (21.4%) 4 (5.7%) 13 (18.5%) n=70 123 (49.5%) 67 (27.0%) 31 (12.5%) 27 (10.8%) n=248 719 (88.2%) 75 (8.8%) 35 (4.1%) 22 (2.5%) n=851 433 (87.4%) 33 (6.6%) 19 (3.8%) 10 (2.0%) n=495 1152 (85.5%) 108 (8.0%) 54 (4.0%) 32 (2.3%) n=1346 normal ranges target ranges high ranges very high ranges total male female total male female total male female total ranges glycated hemoglobin (hba1c) fasting plasma glucose (fpg) random blood sugar (rbs) 66 vol 4 (2), july 2013; 64-66 diabetes, avoiding alcohol and smoking. if a person have pre diabetic range than he should take proper treatment, and control blood sugar level in order to prevent diabetes mellitus. proper medication and awareness about the seriousness of disease can also prevent the epidemic of diabetes among people in pakistan. refrences akinloye oa, adaramoye oa, akinlade ks, odetola aa, raji aa. (2007). relationship between fasting plasma glucose and glycated hemoglobin in adult diabetic nigerians. african journal of biomedical research,10:127-132. alam t, weintraub n, weinreb j. (2006).what is the proper use of hemoglobin a1c monitoring in the elderly? j am med dir assoc, 7: s60–s64. gillet mj. 2009. guidelines review-international expert committee report on the role of the a1c assay in the diagnosis of diabetes. diabetes care, 32(7): 1327-1334. khuwaja ak, fatmi z, soomro wb, khuwaja nk. (2003).risk factors for cardiovascular disease in school children: a pilot study. j pak med assoc, 53: 396-400. kumar v, nelson f, abul k, ramzi s, stanley l. (2005). robbins and cotran pathologic basis of disease (7th ed.). philadelphia, pa.: saunders. pp. 1194–1195. isbn 0-7216-0187-1. figure 1. graphical representation of target range, high range, very high ranges of hba1c, bsr & bsf of female patients figure 2. graphical representation of target range, high range, very high ranges of hba1c, bsr & bsf of male patients effect of time-lapse administration of panadol (paracetamol) on spleen and kidney functions of adult albino mice vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 88 r a d s j . b i o l . r e s . a p p l . s c i 88 op en ac ces s f u l l l e n g t h a r t i c l e effect of time-lapse administration of panadol (paracetamol) on spleen and kidney functions of adult albino mice muddasir hassan abbasi1,*, komal david2, muhammad idnan2, zaira ahmad3 and asif mahmood qureshi2 1centre for applied molecular biology (camb), university of the punjab, lahore, pakistan 2school of zoology, minjah university, lahore, pakistan 3environmental science department, lahore college for women university, lahore, pakistan a b s t r a c t panadol is a remarkable pain and fever reducing non opioid drug. it is known to be completely safe and tolerant medicine throughout the globe among people of all age groups. the goal of this study was to elaborate the histopathological effects of reduced interrupted regime of panadol on spleen and kidney functions of mice (mus musculus). the animals were categorized into four groups, the control group (c), and rest of the three were labeled as 1, 2 and 3, made on the basis of time interval of pandol administration via gavage (n=7). matched volume of panadol (15000 µg/0.1ml) and normal saline was given to mice. tissue samples were collected after sacrifice of the mice and processed for assessment. the findings of the current study reflected the histopathological damage of kidney and spleen caused by panadol in reduced interval of time. the kidney section illustrated clear distortion in glomeruli integrity, marked increase in interstitial spaces, damaged epithelia, and degeneration in tubules in all the groups. the spleen histology exhibited the degradation of white pulp, depopulation, activation of follicles, cellular disruption thereby overall disorganized stature. the raised values of serum creatinine and blood urea examinations also revealed the deleterious effects of panadol overconsumption. it is inferred from the above mentioned outcomes that though panadol is considered to be a safe drug even then its intake prior to four hours can account for adverse effects on kidney and spleen. keywords creatinine, kidney, mice, overdose, panadol. *address of correspondence muddygcs@gmail.com article info. received: october 03, 2018 accepted: november 14, 2018 cite this article: abbasi mh, david k, idnan m, ahmed z, qureshi am. effect of time-lapse administration of panadol (paracetamol) on spleen and kidney functions of adult albino mice. rads j. biol. res. appl. sci. 2018; 9(2): 88-93. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n the analgesic effect of panadol among painkilling remedies is quite well established but it won't be inappropriate to call it a mystery drug because even after a decade of research its exact mechanism of action is still to be elucidated1. mazaleuskaya acquainted that paramount of panadol efficacy in terms of its analgesic and antipyretic dimensions depend chiefly on the administration of its appropriate dose. recent evidence expounds that panadol, when used at a recommended dose of 4g/day in adults and 50-75mg/kg/day in young ones, is far more beneficial and tolerable as compared to other over the counter medication2. overdosing of panadol accounts for a major cause of drug-related toxicities including, acute liver damage and other complications throughout the world (kanabar)3. the half-life of panadol at standard dose is 1.5-2.5 hours, but in case of overdosing the metabolism retards thus extending the half-life to 4-8 hours. the fever and pain o r i g i n a l a r t i c l e effect of time-lapse administration of panadol (paracetamol) on spleen and kidney functions of adult albino mice vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 89 r a d s j . b i o l . r e s . a p p l . s c i 89 ceasing properties of panadol depend on its concentration reaching the brain cells requiring an exit of panadol from the bloodstream to the nervous system tissues before it implies its effect4. toxicity of panadol gets explicit generally in three phases. in the first twenty-four hours’ mild symptoms like nausea, vomiting, profuse sweating, malaise and diffused abdominal discomfort appears according to thapa et al5. in the next seventy-two hours’ liver malfunctioning initiates as it is the main site of metabolism of drugs, hepatic transaminase and aspartate aminotransferase ast level is enhanced and acute renal impairment occurs. the next phase about ninety-six hours after overdose is characterized by severe vomiting, jaundice, gastrointestinal disturbance, coagulation failure, low blood glucose, encephalopathy, acidosis of metabolic system and acute kidney failure. later on, the high-level resolution of hepatic and renal complications and multiorgan damage and death occurs. steps involved in liver damage evoked by panadol overdose are associated with increased formation of free radicals leading to heavy oxidative stress. the putative metabolite of panadol, napqi gets accumulated leading to consumption of glutathione (gsh) thereby gsh stores in liver reduce. excess napqi interact with cellular protein mercapto groups. a result is an overgrowing number of reactive oxygen species (ros) thereby increase peroxidation of lipids. nitration of tyrosine, a chief biomarker in peroxynitrite formation finally causes liver necrosis as stated by el morsey et al6. kidneys are the major homeostatic organs as they regulate not only blood pressure but also the blood composition and fluid volume of the blood. therefore, drug overdose not only affects the liver but also put kidneys under irretrievable stress. panadol causes acute and chronic kidney failure. the mechanism, which leads to liver injury has been well studied however the molecular level of panadol-induced nephrotoxicity is poorly encompassed as mentioned by lorz et al7. abraham et al8 found out that there is no special treatment for panadolinduced kidney damage. vitamin c is a good chain-breaking antioxidant and a free radical absorber. super-dose of vitamin c may prove beneficial in the treatment of panadol induced nephrotoxicity. the basic mechanism for protection by vitamin c apparently seems to be the rejuvenation of nonprotein thiol. panadol is an extensively used remarkable drug, however, the main point is to use it according to the recommended dosage to avoid any kind of damage or toxicity to the body. in this regard time delay between repeated doses is the hallmark to achieve steady state plasma levels in due time. the major emphasis of the current study is to elucidate the histopathological effects of a reduced interrupted regime of panadol on spleen and kidney function of mice and correlate the changes with that of a human. this study will be important in the prognostic evaluation of dose interval of panadol in humans. m a t e r i a l a n d m e t h o d materials all the materials/chemicals were obtained from commercial sources as indicated: panadol 500mg tablets, (glaxosmithkline consumer healthcare) were taken from a local market. chemicals including formalin, ethanol, and chloroform were from sigma-aldrich chemie (munich, germany) or merck (darmstadt, germany). colonies of female mice (mus musculus) female albino mice were nurtured for two weeks in the well-maintained animal house of school of zoology in minhaj university lahore (mul). the mice were scrutinized for their weight thrice a week and experimental work was initiated when the animals attained the weight of 30g. the availability of fresh water and food (the chow i.e., 20% unrefined protein in its composition) was made possible all the time until the onset of the experimental procedure. methodology dose calculation and preparation the standard human dose of panadol (500mg/kg) was converted and prepared for mice (30g). three standard tablets of 500 mg/kg were powdered and mixed with 10 ml of distilled water to prepare the required dose. effect of time-lapse administration of panadol (paracetamol) on spleen and kidney functions of adult albino mice vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 90 r a d s j . b i o l . r e s . a p p l . s c i 90 experimental design the animals were categorized into four groups (n=7), the control group (c), and rest of the three were experimental groups labelled as 1, 2 and 3, made on the basis of the time interval of 1 hour, 2 hours and 3 hours respective of the initial dose administration. the control group was provided with normal saline of the matched volume of panadol (15000 µg/0.1ml) which was given to the experimental group at a time lapse of 1, 2 and 3 hours respective of the initial dose. all of the doses were given to each of the adult female mice via gavage. tissue sampling and processing the blood samples were extracted from the heart right after dissection, and all the viscera were placed in a petri dish with 0.9 % saline. kidneys & spleen from each animal were collected in separate glass vials. all the organs were rinsed with physiological sodium saline and portion was fixed in 10% formalin. paraffin-embedded sections were prepared, processed followed by microscopic imaging as described by abbasi et al 9. r e s u l t s renal profile in the current study blood urea, nitrogen (bun) and serum creatinine level were measured. the rationale behind the measurement of urea and creatinine to check the kidney functioning is that glomerular filtration rate (gfr) is reflected both by plasma as well as serum levels, the key parameters that characterize renal function. bun shows a comparative rise in group 1 and 3 while group 2 exhibits a mild decrease. an opposite trend can be noted in the case of serum creatinine level for all the comparable groups (table 1). table 1: comparison of blood urea nitrogen (bun) and creatinine levels between control(c) and experimental groups of panadol administrated mice. groups bun (mg/dl) creatinine level (mg/dl) c 10.14 0.43 1 13.29 0.36 2 12.29 0.39 3 15.14 0.37 values represent a mean of 7 replicates histopathology kidney kidney histology of the control group depicted normal sized renal capsule with fuzzy proximal as well as clear distal tubules (fig. 1a). repeating the dose after one hour of the administration of initial dose the renal tissues elucidated widening of interstitial spaces, and some darkly stained areas demonstrating damage to the tissues, moreover the glomerulus stays no more intact (fig. 1b). dose repetition after two hours of initial dose the histological findings demonstrated much widened interstitial spaces, the glomerular space markedly enhanced and the renal capsules not only got reshaped but a great variation arose among the size of renal capsules (fig. 1c). likewise, the third experimental group exhorts much deterioration in renal structure when it was given the repeat dose of panadol after three hours of the first dose (fig. 1d). fig. 1: haematoxylin-eosin staining of kidney sections of panadol administered mice after 1 (b), 2 (c), and 3h time interval compared with control (a). renal corpuscles indicated by red arrow, intact glomeruli (black arrow), normal glomerular space (blue arrow), proximal tubules (green arrow) and distal tubules (orange arrow). enlarged bowman’s space (red arrows), architectural deterioration (yellow and blue arrows) can be evident in treated mice after 1, 2 and 3hrs (magnification: 160×). effect of time-lapse administration of panadol (paracetamol) on spleen and kidney functions of adult albino mice vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 91 r a d s j . b i o l . r e s . a p p l . s c i 91 spleen morphologically prominent red pulp (rp) and the white pulp (wp) were obvious in the control section of the spleen (fig. 2a). the spleen section of the first experimental group elaborated deterioration of splenic follicles (fig. 2b). while second and third group manifest not only necrosis but also depopulation and distortion of follicles (fig. 2c-d). fig. 2: haematoxylin-eosin staining of spleen sections of panadol administrated female mice after 1 (b), 2 (c) and 3h time interval compared with control (a). white pulp (wp) & red pulp (rp) can be marked in (a). in panadol induced mice after 1, 2, 3 hours’ distortion of follicles (yellow arrows), depopulation (blue arrow), darkly stained areas (red arrows) elucidate structural damages (magnification: 160×). d i s c u s s i o n the current study is focused on renal and spleen impairment due to panadol overdose in terms of the reduced interrupted regime. the kidney is the second targeted entity of any metabolite formed. overdosing of drugs like panadol can malfunction kidney even in the normal appearing subjects. however, nephrotoxicity pertaining to panadol after a supra-therapeutic dose may occur even in the absence of hepatotoxicity. in the present work, histological deterioration in the renal tubules along with cellular distortion was evident at each time point during the course of study. the histopathological assessment of experimental groups revealed not only glomerular shrinkage and enhanced space in bowman's capsule but also desquamation of epithelial layer, vacuolar distortion of tubules and increase in interstitial spaces these results correlate with those explained by zhou 10. our results are in accordance with those reported by gulnaz11 that overdosing of panadol leads to the small size of the glomerulus, vacuoles and cellular desquamation in distal and proximal convoluted tubules. severe necrosis in tubules, degeneration of epithelium and distortion of the glomerular structure was elucidated in another study reported by fouad 12 that is in relevance with the current study. excessive glomerular damage evident by bleeding along with epithelial rupture, deterioration of cell boundary and damage in brush border in proximal tubules as a result of panadol overdose was explained by khorsandi13. these changes might have occurred due to the interaction of toxic metabolites with the architectural integrity of renal tubules and glomeruli leading to release of lysosomal enzymes thereby causing damage. the results of the current study also stand in agreement with those of ucheya14 that elaborated hypoplasia, wider capsule space some even without glomeruli, vascular congestion and tubular necrosis due to panadol overdose. in another study by odigie15 the histopathological results revealed tubular expansion and enlargement of glomerular space due to prolonged overconsumption of panadol. panadol induced renal toxicity has been attributed chiefly to cytochrome p-450 oxidase isoenzymes found in kidney however other mechanisms like the role of prostaglandin synthetase and n-deacetylase enzymes must not be ignored. paradoxically, the major element in detoxification of panadol and its metabolites is glutathione but its conjugates are involved in the synthesis of nephrotoxic molecules as mentioned by mazer16. regarding splenic sections, the experimental groups elucidate not only shrinkage but also a cellular distortion of white pulp at each time point as compared to the control sections. moreover, depopulation of lymphocytes was notable in white pulp these changes might be due to the interference of toxic metabolites of panadol overdose. effect of time-lapse administration of panadol (paracetamol) on spleen and kidney functions of adult albino mice vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 92 r a d s j . b i o l . r e s . a p p l . s c i 92 the current results are in line with another investigation conducted by gomaa17 that explains nonsteroidal antiinflammatory drugs (nsaid) and panadol induced disastrous effects on immunity related organs i.e. bone marrow, spleen, and lymph nodes. moreover, the toxic metabolites of panadol overdose damage hepatocytes thereby triggering an innate response of the immune system, the over activity of cells and activation of leucocytes may be in relation with tissue damage induced by an overdose of the drug. in addition to histopathological assessment, the extent of renal damage is also characterized by the rise in urea and creatinine level, whose concentration in serum helps in an overview of glomerular filtration rate (gfr) and ultimately renal function. the normal wear and tear of muscles of the body synthesize a chemical waste product creatinine that goes into the bloodstream. in the current study increase in blood, urea was observed in group 1 and 3, while group 2 showed a slight decrease in value this might be the reason of the self-corrective mechanism of the body. the serum creatinine values, however, showed a decrease in values within a narrow range, these results were quite similar with those elaborated by sener18 thus ratify the idea that some minimum time is required to take next dose otherwise an irreparable loss of integrity of kidney and spleen can be observed. c o n c l u s i o n in this study, detrimental histopathological effects of a reduced interrupted regime of panadol conjure with the notion of a completely safe drug and it is highly recommended that the correct dose within time frame must be taken. it is further recommended that the second dose must not be administered before four hours to avoid any substantial damaging effect of this drug and still more detailed studies are required to crack the mystery of this drug in biological medium. funding disclosure statement there is no research grant for this particular research. acknowledgement the authors are thankful to ms. sana fatima, for her valuable comments on histological sections. disclosure statement the authors declare no potential conflict of interest. contributors’ statement muddasir hassan abbasi & komal david contributed equally to this study. muhammad idnan is involved in the troubleshooting. zaira ahmad & asif mahmood qureshi are involved in manuscript drafting. muddasir hassan abbasi supervised the research project and approved the final draft. r e f e r e n c e s 1. borst p, de wolf c, van de wetering k. multidrug resistance-associated proteins 3, 4, and 5. pflug arch eur j phy 2007;453(5):661-73. 2. mazaleuskaya ll, sangkuhl k, thorn cf, fitzgerald ga, altman rb, klein te. pharmgkb summary: pathways of acetaminophen metabolism at the therapeutic versus toxic doses. pharmacogenet genomics 2015;25(8):416. 3. kanabar d. a practical approach to the treatment of low-risk childhood fever. drugs r d 2014;14(2):45-55. 4. hodgman mj, garrard ar. a review of acetaminophen poisoning. crit care clin 2012;28(4):499-516. 5. thapa b, walia a. liver function tests and their interpretation. indian j pediatr 2007;74(7):663-71. 6. el morsy em, kamel r. protective effect of artichoke leaf extract against paracetamol-induced hepatotoxicity in rats. pharm. biol. 2015;53(2):167-73. 7. lorz c, justo p, sanz a, subirá d, egido j, ortiz a. paracetamol-induced renal tubular injury: a role for er stress. j am soc nephrol 2004;15(2):380-89. 8. abraham p. vitamin c may be beneficial in the prevention of paracetamol-induced renal damage. clin exp nephrol 2005;9(1):24-30. 9. abbasi mh, fatima s, khawar mb, jahan s, sheikh n. an in vivo study on intoxicating effects of nerium oleander water based extract on multiorgan of wistar rat. can. j. gastroenterol. hepatol 2018;2018. 10. zhou y-d, hou j-g, liu w, ren s, wang y-p, zhang r et al. 20 (r)-ginsenoside rg3, a rare saponin from red ginseng, ameliorates acetaminophen-induced hepatotoxicity by suppressing pi3k/akt pathwaymediated inflammation and apoptosis. int immunopharmacol 2018;59:21-30. 11. gulnaz h, tahir m, munir b, sami w. protective effects of garlic oil on acetaminophen-induced effect of time-lapse administration of panadol (paracetamol) on spleen and kidney functions of adult albino mice vol 9 (2), december 2018 issn (print): 2305 – 8722 issn (online): 2521 – 8573 93 r a d s j . b i o l . r e s . a p p l . s c i 93 nephrotoxicity in male albino rats. biomedica 2010;26(7):9-15. 12. fouad aa, yacoubi mt, el-bidawy mh. therapeutic potential of hemin in acetaminophen nephrotoxicity in rats. environ toxicol pharmacol 2009;27(2):277-82. 13. khorsandi l, orazizadeh m. protective effect of curcuma longa extract on acetaminophen induced nephrotoxicity in mice. daru 2008;16(3):155-59. 14. ucheya r, igweh j. histological changes in kidney structure following a long–term administration of paracetamol (acetaminophen) in pregnant sprague dawley rats. niger. j. physiol. sci. 2006;21(1-2). 15. odigie eb, achukwu pu. histopathological effects of acetaminophen abuse in male wistar rats, and prevalence in human subjects: an experimental and cross-sectional study. j exp clin anat 2015;14(2):81. 16. mazer m, perrone j. acetaminophen-induced nephrotoxicity: pathophysiology, clinical manifestations, and management. j. med. toxicol. 2008; 4(1):2-6. 17. gomaa s. adverse effects induced by diclofenac, ibuprofen, and paracetamol toxicity on immunological and biochemical parameters in swiss albino mice. j basic appl zool 2018;79(1):5. 18. şener g, şehirli aö, ayanoğlu‐dülger g. protective effects of melatonin, vitamin e and n-acetylcysteine against acetaminophen toxicity in mice: a comparative study. j. pineal res.2003; 35(1):61-68. effect of burn injury on the dissemination of candida albicans from the skin of mouse qudsia hussain1*, amna shafiq1, shazia tabassum hakim1, sayyada ghufrana nadeem1 1department of microbiology, jinnah university for women, karchi-74600, pakistan. abstract burn injury is associated with the greatly increased susceptibility of thermally injured patients to infection from a variety of pathogens. candida albicans is a commensal of skin and has been reported as a severe pathogen in thermally injured patients. systemic candidiasis has emerged as a major cause of death in burn patients. in this study, the ability of candida albicans to infect thermally injured host and the host response to systemic infections with candida albicans was studied by using burned mice. the c. albicans suspension was spreaded over the burn surface. mice were sacrificed ten days after burn to check the presence of yeasts in the kidneys, lung, liver and spleen or burn wounds. after 48 hr. of incubation of culture plates yeasts were recovered from the kidneys, lung, liver, spleen of burned mice. this study describes the enhanced susceptibility of burned mice to systemic candidiasis and shows that a systemic infection with candida can lead organisms to contaminate the wound. keywords: burn patients, candida albicans, candidiasis, swiss albino mice, thermal injury. introduction the genus candida includes about 150 different species, however, only a few are known to cause human infections. c. albicans, is a normal constituent of the human flora, a commensal of the skin and the gastrointestinal and genitourinary tracts. c. albicans is the most significant pathogenic specie which can cause infections [called candidiasis or thrush] in humans and other animals (kaminski et al., 1995). other candida species pathogenic in humans include c. tropicalis, c. glabrata, c. krusei, c. parapsilosis, c. dubliniensis, and c. lusitaniae (fader et al., 1985). c. albicans remains the most common infective species, other candida spp. are becoming increasingly significant. in a range of large-scale studies of candidaemia between 1999 and 2006, about 52% of 9717 cases involved c. albicans, about 30% involved either c. glabrata or c. parapsilosis and less than 15% involved c. tropicalis, c. krusei or c. guilliermondii (marissa et al., 2012). candidiasis is a common infection of the skin, oral cavity and esophagus, gastrointestinal tract, vagina and vascular system of humans. most infections occur in immunocompromised patients. c. albicans, expresses several virulence factors that contribute to pathogenesis. these factors include host recognition biomolecules (adhesions), morphogenesis (the reversible transition between unicellular yeast cells and filamentous, growth forms), phenotypic switching, secreted aspartyl proteases and phospholipases (murciano et al., 2006). c. albicans is a dimorphic fungus. normal room temperatures favor the yeast form of the organism, but under physiological conditions (body temperature, ph and the presence of serum) it may develop into a hyphal form. pseudohyphae, composed of chains of cells, are also common. however, the ability to assume various forms may be related to the pathogenicity of the organism. the yeast form is 10-12 µm in diameter. pseudohyphae [chains of cells] may be formed from budding yeast cells which remain attached to each other. chlamydospores may be formed on the pseudomycelium (calum et al., 2009). to infect host tissue, the usual unicellular yeast-like form of c. albicans reacts to environmental cues and switches*corresponding author: qudsiahussain@hotmail.com 33 vol 4 (2), july 2013; 33-37 into an invasive, multicellular filamentous form. the ability of c. albicans to form hyphae has been proposed as a virulence factor as these structures are often observed invading tissue and c. albicans strains which are unable to form hyphae are defective in causing infection. although candida most frequently infects the skin and mucosal surfaces, it can cause systemic infections manifesting as pneumonia, septicaemia or endocarditis in severely immunocompromised patients. systemic fungal infections [fungemias] have emerged as important cause of morbidity and mortality in immunocompromised patients. burn injury is associated with the greatly increased susceptibility of thermally injured patients to infection from a variety of pathogens (kobayashi et al., 1998) compared with other trauma patients, because of loss of the skin barrier (ruth, 1980). skin integrity is of importance for the protection and separation of body tissues from the surrounding environment. the loss of skin due to burns or trauma exposes the body to severe stress; impairing or even eliminating the many vital functions this organ performs (alice et al., 1985). c. albicans has been reported as a severe pathogen in thermally injured patients (kobayashi et al., 1998).the pathogenesis of fatal candida infections in burn patients is complicated (alice et al., 1985). severe burn injury induces an immunosuppressive state which is responsible for the increased susceptibility to development of opportunistic infections that can lead to sepsis and increase mortality (patenaude et al., 2010). the susceptibility of thermally injured mice to c. albicans infection was 50 times greater when compared to the sensitivity of normal mice (kobayashi et al., 1998). burn injury leads to suppression of nearly all aspects of immune response. chemotaxis, phagocytosis, and killing function of neutrophils, monocytes, and macrophages are impaired. systemic candidiasis and chronic mucocutaneous candidiasis are common infections in burn patients (calderone and fonzi, 2001). systemic candidiasis has emerged as a major cause of death in burn patients. host defense against systemic candidiasis relies mainly on the ingestion and elimination of c. albicans by cells of the innate immune system, in particular macrophages, monocytes and neutrophils. increased incidence of infection is due to defects in non-specific defense functions, such as neutrophil and macrophage phagocytosis and bacterial activity, as well as to an inhibition of specific immune functions, such as cellmediated immunity. the decrease in host resistance to infection is obviously related to a depression of both humoral and cellular components of the host defense system (okawa et al., 2004). the present study was performed by burning the skin of mouse and investigating the dissemination of c. albicans from skin to different organs of the mouse. materials and methods organisms: the c. albicans was maintained on sabouraud dextrose agar slants at 4°c. to prepare cultures for animal inoculation, the stock culture was inoculated into 25 ml of sabouraud dextrose broth and incubated for 48 hr at 37°c in an environmental shaker incubator at 125 rpm. the organisms were harvested by centrifugation and suspended to a concentration of 108 organisms per ml in sterile saline. mouse burn model: swiss albino mice of either sex weighing 20 to 25 g. the animals were acclimated for 1 week before burning and given water and standard laboratory chow ad libitum throughout the study. the mouse was anesthetized with chloroform. the back of the mice was shaved, and the animals were anesthetized with chloroform. ethanol (0.5 ml) was evenly spread over the area of the back, ignited, and allowed to burn for 10 sec. the animals were given 1 ml of normal saline intraperitoneally as fluid replacement therapy. this method, which produced a partial-thickness burn of approximately 30% of the body surface, was not fatal for any of the animals. animal inoculation consisted of 0.1 ml of the candida suspension (108 organisms per ml) spread onto the surface of the wound. the animal was sacrificed after 10 days post burn. biopsies were obtained for quantitation of organisms in the tissue. quantitation of organisms in tissue: biopsies (skin, 34 vol 4 (2), july 2013; 33-37 wound, or kidneys) were weighed and homogenized in tissue grinders containing 5 ml of sterile saline. the samples were diluted in saline and 0.1-ml portions were plated in duplicate by the agar pour plate technique. sabouraud dextrose agar containing chloramphenicol to suppress bacterial growth was used as the growth medium. the plates were incubated for 48 hr at 37°c, and colonies were counted. cfu/g of tissue was determined by the following formula: [(mean no. of cfu/ml)/ (weight of biopsy)] x [(5 ml)/ (dilution)]. results and discussion candidiasis is the most common fungal infectious disease in burn patients. colonization of c. albicans in burn patients is found to be very high i.e., about 63% (macmillan et al., 1972). the present investigation was undertaken to study the dissemination ability of c. albicans by using thermally injured mouse as possible animal model of infection. the study of alice et al (alice et al.,1985). initially described the enhanced susceptibility of thermally injured mice to an intravenous injection of candida. concerning the depth of burn, our previous study demonstrated that a deeper burn increased the susceptibility of mice to wound invasion after topical application of candida (avniel et al., 2006). this finding agreed with a study on candida infections in burn patients, which report that candida invaded fullthickness wounds more often than partial-thickness burns (kobayashi et al., 1998). the deeper burn in our model may account for the apparent increased mortality we observed at lower concentrations of candida, although the strain of mouse used may also have affected the outcome of infection. the result of the studies emphasize the importance of a careful strain selection when doing animal studies, and each study describes an animal model that can be used to study the effect of thermal injury on the pathogenesis of candidiasis (fader et al., 1986). the morbidity and mortality of burned mice in response to systemic infection with c. albicans were examined in mice given a burn and suspension of c. albicans by spreading over the surface of wound. the scald burn, of 7 sec duration, resulted in a uniform full-thickness injury, at ten days after infection, the kidneys, wound, liver ,lung, spleen or skin were assayed for the presence of the organisms. in burned mice, organisms were uniformly isolated from the organs at the time of sacrifice (table i and ii). c. albicans that was spread over the surface of the wound disseminate quickly and large number of colonies was observed on culture media by culture different organs of the burned mice (figure 1-7). 35 vol 4 (2), july 2013; 33-37 figure 1. culture from lung sample after 24hr. figure 2. culture from kidney sample after 24hr. 36 vol 4 (2), july 2013; 33-37 figure 3. culture from liver sample after 24hr. figure 7. culture from kidney sample after 48 hr. figure 4. culture from spleen sample after 24hr. figure 5. culture from spleen sample after 48 hr. figure 6. culture from liver sample after 48 hr. table i. isolation of c.albicans from tissue after 24 hrs of incubation. kidney sample after 48 hr. organ culture revealed that the organisms arrived in the organ via the blood stream. in a previous study the mice were injected via an intraperitoneal injection, contiguous spread from the peritoneum was not observed as no yeasts were observed in the lower skeletal muscle of the dorsum (okawa et al., 2004). this study demonstrated that thermal injury enhances the ability of c. albicans to infect mice and that the depth of burn appears to be an important factor in determining whether the organisms can invade the burn wound to cause systemic infection. this animal model should be valuable in elucidating the virulence factors of c. albicans that play a role in the pathogenesis of candidiasis after thermal injury. references alice nn, law ej and holder ia.1985.increased susceptibility to lethal candida infections in burned mice preinfected with pseudomonas.aeruginosa or pretreated with proteolytic enzymes. infect immun, 49:780-784. avniel s, arik z, maly a, sagie a, basst hb, yahana hd,et al. 2006. involvement of the cxcl12/cxcr4 pathway in the recovery of skin following burns. j invest dermatol., 126:468-476. calderone ra and fonzi wa. 2001. virulence factors of candida.albicans. trends, 9:327-35. calum h, moser c, jensen po, christophersen j, maling ds,van gennip m.2009. thermal injury induces impaired function in polymorphonuclear neutrophil granulocytes and reduced control of burn wound infection. clin exp immunol., 156: 102–110. fader rc, nunez d, unbehagen j, and linares ha.1985. experimental candidiasis after thermal injury. infect. immun., 49: 780-784. faderr c, carlile jc, unbehagen j and linares ha.1986.systemic candidiasis after thermal injury. curr microbiol,14: 231-234. kaminski h, miyaguchi h, tamaki t, suenaga n, hisamatsu m, mihashi i, et al. 1995. degradation of humoral host defense by candida albicans pro teinase. infect. immun., 63:984-988. kobayashi m, kobayashi h, herndon dn, pollard rb and suzuki f.1998. burn-associated candida.albicans infection caused by cd30+type 2 t cells. j. leukoc. biol., 63: 723731. macmillan bg, law ej and holder ia. 1972. experience with candida infection in the burn patient. arch. surg., 104:509-514. marissa ps, tristram s, and brown s. 2012. the contribution of growth rate to the pathogenicity of candida spp. inter j biol life scie, 8:80-86. murciano c, villamón e, yáñez a, o’connor je, gozalbo d and gil ml .2006. impaired immune response to candida.albicans in aged mice. j med microbiol, 55: 1649-1656. okawa y, kobayashi m, sakai k and suzuki m.2004. role of polymorphonuclear leukocytes in the resistance of tumor-bearing mice against candida.albicans infection. biol. pharm. bull., 27: 674—678. patenaude.j, elia md, hamelin c, bernie j.2010. selective effect of burn injury on splenic cd11c+ dendritic cells and cd8a+cd42cd11c+ dendritic cell subsets. cell. mol. life sci., 67:1315–1329. ruth e.1980. in vitro phagocytosis of candida.albicans by peritoneal mouse macrophages.infect.immun., 28:963971. 37 vol 4 (2), july 2013; 33-37 table ii. isolation of c.albicans from tissue after 48 hrs of incubation. rads journal of biological research & applied sciences volume 11, no. 1, june 2020 table of contents patron-in-chief mr. wajeehuddin ahmad chancellor, jinnah university for women patron prof. dr. naeem farooqui vice chancellor, jinnah university for women chief editor prof. dr. sayyada ghufrana nadeem jinnah university for women managing editor dr. taqdees malik jinnah university for women section editor dr. iqra munir jinnah university for women ms. fasiha saeed jinnah university for women ms. farkhanda afaque jinnah university for women editorial board prof. dr. salvatore rubino (italy) prof. dr. omar bagasra (usa) prof. dr. tashmeen razzaki (pak) prof. dr. mmm najim (srilanka) prof. dr. shazia tabassum hakim (usa) prof. dr. mahmood akhter (canada) prof. dr. samina n. assanie shivji (usa) prof. dr. m. tariq malik (usa) prof. dr. ghosia lutfullah (pak) prof. dr. hamid mukhtar (pak) prof. dr. saeed khan (pak) prof. dr. agha asad noor (pak) prof. dr. shamsul arfin qasmi (pak) dr. faisal akhlaq (pak) dr. akinrotimi ojo andrew (nigeria) dr. daniela giannetto (italy) dr. alfred iqbal (usa) dr. muhammad ayaz mustafa (pak) dr. hayder fouad (iraq) dr cigdem ozen (turkey) published biannually by reyazuddin research & development society, jinnah university for women, 5-c nazimabad, karachi-74600, pakistan e-mail: jbas@juw.edu.pk phone: 3661-9902, 3662-0857-9 fax: 3662-0614 http://jbas.juw.edu.pk original articles 1 the action of cytomegalovirus (cmv) promoter on expression of genetically engineered insulin in rat hepatocytes gehad elsayed mahmoud 9 prevalence of newcastle disease virus and avian influenza virus (h7n3) in poultry at karachi amjad ali channa, nazeer hussain kalhoro, zaheer ahmed nizamani, ayaz hussain mangi, jamila soomro 15 small dense low-density lipoprotein as risk factor for atherosclerosis in type 2 diabetes mellitus haji muhammad rashid, hiza hassan, mudassar khan, jamal khan, hasanat khan 19 preliminary behavior of chinkara (gazella bennettii) under captive conditions with future conservation strategies muhammad idnan, arshad javid, muhammad nadeem, ali hussain, sajid mansoor, waqas ali, syed mohsin bukhari 27 meta-analysis of prevalence of depression, anxiety and stress among university students shifa shaffique, sehar shahzad farooq, haseeb anwer, hafiz muhammad asif, muhammad akram, soon ki jung 33 consequences of microwave electromagnetic radiation exposure on germination and free proline content of green gram and red bean saima ibrahim, yasra bashir, salma zaki, samia mehmood, umul mustafa, afreen khanum, kainat ishtiaq, fabiha maheen, amber rehmat 39 machine learning based statistical analysis of emotion recognition using facial expression aqib ali, jamal abdul nasir, muhammad munawar ahmed, samreen naeem, sania anam, farrukh jamal, christophe chesneau, muhammad zubair, muhammad saqib anees 47 phytochemical evaluation and anti-inflammatory activity of ethanolic extract of calotropis procera leaves naveed aslam dogar, m. hamza shahid, hafiz usama shaukat, m. abubakar khan, farooq saleem 53 investigating age dependent diversification of bone biomarkers in females farah jabeen, s. ayesha sehrish, afshan zeeshan wasti, farha aziz review articles 61 a preliminary up-to-date review on pakistani medicinal plants with potential antioxidant activity adil hussain instructions for authors the assessment of depression level among diabetic patients vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 109 r a d s j . b i o l . r e s . a p p l . s c i . 109 op e n ac c e s s f u l l l e n g t h a r t i c l e the assessment of depression level among diabetic patients in karachi using bdi (beck’s depression inventory) kiran rafiq1,*, shagufta nesar2, zafar saied saify3, mohammad azhar mughal4, alina rizvi1, aleeza raza1, alisha hassan1 1institute of pharmaceutical sciences, jinnah sindh medical university, karachi, pakistan 2faculty of pharmacy, hamdard university, karachi, pakistan 3international center of chemical & biological sciences, hej, university of karachi, karachi, pakistan 4department of pharmacology, jinnah sindh medical university, karachi, pakistan a b s t r a c t background: diabetes is co-morbid with various metabolic and psychological disorders. diabetes and depression are both chronic conditions with allied adverse and complicated outcomes. objectives: the current study was designed and planned accordingly to elucidate the associated frequency of depression with dm (diabetes mellitus) type 2 and prescribing practice of doctors depending on the distressing reality and threatening figures. methodology: a prototype prospective study was monitored in different public and private sector hospitals and clinics of outpatient settings in karachi after taking approval from the ethical review board of hamdard university. in this context, seven hundred patients having diabetes were covered in the study and their data were analyzed through the statistical software version 20. beck depression inventory (bdi) was the preferred approach to evaluate the depression level. results: the outcome of the study established the fact of high prevalence for the co-morbidity of stress, anxiety and depression among patients with diabetes in karachi, specifically among age bracket from forty to sixty years. unfortunately, 80% (544) of diabetic patients in the study were suffering from depression and anxiety, while only 22.05% (120) patients were taking antidepressants and they were not counseled for the root cause of the disease. severe depression (bdi score=38) was present in 100 subjects, moderate depression (bdi score=27) in 200 subjects, and mild depression was present in 175 of subjects. being a patient of dm is itself a strong reason for contracting a depressive disorder. the result highlight improper treatment of depression in diabetic patients. conclusion: however, the current situation demands to follow proper treatment through effective and appropriate drugs to treat the disease from progressing. moreover, efforts should be undertaken to strengthen the healthcare system where practitioners, either doctors, pharmacists or nurses are liable for patient care in both the physical and psychological domains. keywords diabetes mellitus, depression, bdi (beck depression inventory), outpatients. *address of correspondence kiranrafiq@hotmail.com article info. received: may 2, 2018 accepted: october 25, 2019 cite this article: rafiq k, nesar s, saify zs, mughal ma, rizvi a, raza a, hassan a. the assessment of depression level among diabetic patients in karachi using bdi (beck’s depression inventory). rads j biol res appl sci. 2019;10(2):109-113. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n depression is a ubiquitous condition that frequently and mostly co-exists with chronic conditions. the diabetes association of america and diabetes canada recommends the systematic study of depression in hyperglycemic patients to come up with roots cause of stress1,2. according to a report, approximately sixth part of o r i g i n a l a r t i c l e the assessment of depression level among diabetic patients vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 110 r a d s j . b i o l . r e s . a p p l . s c i . 110 the worlds’ population is facing the continual consequences of diabetes that disrupt the metabolism of carbohydrates, lipids and proteins. the disease is considered as highly convoluted state that ultimately demands intensive self-care, (involving adaptability to the prescribed medications by the physician, along with timely monitoring of blood glucose), healthy and low carbohydrate diet, physical workout, preventive measures for foot care, regular screening for associated complications and follow-up visits to the consultant3-6. preferably, a comprehensive therapy with a strong emphasis on addressing depression is required for optimal outcome7,8. for a decade, it has been a debatable question whether the monitoring of depression can be supportive to improve diabetes and its associated severity. data obtained from different research studies sustain the reality, and importance of treating depression can be an indirect method to overcome the disease as depression itself is significantly responsible for reduced quality of life9,10. in primary care units, depression and diabetes are usually assessed as two commonly confronted clinical conditions. approximately, 13% to 18% of people with diabetes mellitus are reported to have depression who visit clinical settings, and this combination could be bidirectional. consequently, diabetes is more probably accountable for psychological conditions like depression or anxiety11-13. although, the early stage of diabetes is mostly asymptomatic probably leading to longterm complications, various treatment regimens are required to maintain glucose and lipid levels along with blood pressure status of subjects. feelings of anger, offense, destruction and social isolation are the factors highly accountable for intensifying the disease14-17. consequently, co-morbidity of diabetes and stress threaten the lifespan rather than depression or diabetes alone and the condition is reported to have a prolonged duration of sickness and is sometimes associated with frequent hospitalization as compared to individuals having diabetes alone. unfortunately, around 50% of patients remain undiagnosed and an even greater proportion cannot obtain management options of the disorder with reference to stress. another study revealed frequent occurrence of depression in the diabetic group as compared to non-diabetics18-20. a bidirectional adverse interaction has been established through strong data between diabetes and depression21. m a t e r i a l s a n d m e t h o d s sample collection in the current study, a prototype prospective study was conducted in outpatient settings of the public and private sector hospitals and diabetic clinics in karachi, pakistan after seeking due permission. the duration of the study was from february to july 2018. seven hundred (n=700) diabetic patients who had been receiving various regimens of drugs were recruited in the study. only 680 subjects agreed to join the survey. in the current study, the “beck’s depression inventory” was applied to examine the anxiety level of type 2 diabetic patients. the method adopted was a psychiatric measuring mechanism with expressive words and phrases with the relevancy of depression severity for a time period. the scale includes a total of 21 questions. each question is given 0-3 points, and the total score is obtained with their addition. the entire gain ranges from 0 to 63. the tool was found satisfactory for evaluating the depression level and for the purpose of the beck depression inventory form was answered by the patients at the clinics22,23. exclusion criteria for the study population patients having no will to participate, either male or female, were not recruited in the survey study. all females who were pregnant were also excluded from the present study. hospitalized patients were also not included in the study. data assessment after collection of all information, the data were cleaned, coded and entered in spss version 20, through descriptive statistics consisting of percentages and frequency calculated. depression levels of patients were evaluated through the bdi scale. r e s u l t s out of 680 diabetic patients, 54.1% male and 45.9% were female. the result showed 41 to 60 years age group was more prone to diabetes (table 1 and 2). the assessment of depression level among diabetic patients vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 111 r a d s j . b i o l . r e s . a p p l . s c i . 111 table 1. frequency of diabetes occurrence according to gender. s. no. gender frequency % 1 male 368 54.1 2 female 312 45.9 table 2. frequencies of diabetes occurrence according to age. s. no. age (years) frequency (%) 1 1-20 64 9.4 2 21-40 20 2.9 3 41-60 372 54.7 4 61-80 200 29.4 5 above 80 24 3.5 6 total 680 100.0 among 680 diabetic patients, 544 suffered from depression and interestingly, physicians had not prescribed an antidepressant to all the patients having high level of depression with prominent symptoms, whereas only 120 patients received an antidepressant (table 3). the current study was designed depending on the background and common social causes of diabetes and helped to establish depression as an important root cause of the disease. gender distribution for the disease was observed slightly higher for males. during the study, a prescribing pattern for antidepressants was found to be followed by physicians which included bromazepam, alprazolam, clomipramine and escitalopram to some extent (table 4). however, no practice of patient psychoanalysis was observed as the practice could help in declining anxiety but the present data show that the majority of population under the study was experientially untreated for depression (table 3). table 3. prescribing frequencies of antidepressants in diabetic patients. s. no. prescribing status of antidepressant frequency (%) 1 no 424 (77.94) 2 yes 120 (22.05) table 4. commonly prescribed antidepressants to diabetic patients. s. no. medicine prescribing percentage 1 bromazepam 60 2 alprazolam 20 3 clomipramine 15 4 escitalopram 05 table 5. level of depression according to bdi in diabetic patients. s. no. number of patients total score level of depression 1 175 14 mild mood disturbance 2 200 27 moderate depression 3 100 38 severe depression 4 69 49 extreme depression the beck depression inventory form22 was given to patients to fill out at the clinic. the activity result revealed that 544 (80%) of diabetic patients suffered from depression that exhibited a very large number compared to the standard24,25. interestingly, according to the results of a present study, the majority of the diabetic patients suffered from moderate depression (bdi score=27) and minimum number of patients fell in severe depression range (bdi score=49) according to bdi scale calculation (table 5). d i s c u s s i o n the present study covers the prescribing pattern of drugs to treat diabetic complications and also its co-morbidity especially depression. the chances of diabetes alone were found in below frequency as compared to an association with depression. the research reveals that anxiety and depression are coupled with age and tend to peak in the forties and fifties. then it drops after the age of sixty as anxiety issues are seen more common in the middle aged patients. furthermore, other reasons for the the assessment of depression level among diabetic patients vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 112 r a d s j . b i o l . r e s . a p p l . s c i . 112 development of anxiety are different socio-economic problems such as high expectations from employers, children and relatives in various scenarios along with social pressure mounting to a severity of the condition. the study reveals the correlation of life satisfaction with age and accordingly was found high before and after middle age. during the current and various other studies on the correlation of hyperglycemic conditions and depression, self-care along with diabetes works significantly to prevent the upcoming multiple health consequences. diet, exercise and glucose monitoring are the basic tools to fight the disease. however, monitoring anxiety and stress interestingly plays a vital role and influences the spectrum of diabetes. physicians and pharmacists are highly accountable for analyzing the depressive symptoms in diabetics that should be addressed at the earliest either via counseling or through antidepressive medications. in preventive care; regular exercise, socialization, reading and other healthy activities keep the patient away from stress and its co morbidities26. the collected data reveals that co-morbidity of depression with diabetes unfortunately remain unrecognized and untreated in two-thirds of patients approximately. in certain cases, both condition reach up to the chronic stage and severity, and such patients have no other choice than adding antidepressant medication to the regimen27-29. moreover, prescribing practice must be focused on to individualize the antidepressant regimens for patients having diabetes. however, the practice should be carefully handled depending on the patience and restraint to support adaptability for diabetic patients as they are already sufferers of other complications and also burdened with extreme care regarding diet and lifestyle. diabetes care associations suggest regular counseling to carefully address anxiety and stress among diabetics due to the prevalent association of depression with diabetes30. c o n c l u s i o n the present data and outcome explicitly exhibit that the mental health co-morbidities are major consequences of risk for the deprived health status in diabetes, however, screening and monitoring of stress factors can be vital for reducing unavoidable health issues and psychological screening, especially for anxiety, that is significant for good health among patients with diabetes. l i s t o f a b b r e v i a t i o n s bdi beck depression inventory dm diabetes mellitus r e f e r e n c e s 1. american diabetes association. lifestyle management: standards of medical care in diabetes. diabetes care. 2018; 41(1):s38-50. 2. robinson dj, coons m, haensel h, vallis m, yale jf. diabetes and mental health. can j diabetes. 2018; 42(1):s130-41. 3. russel lb, suh dc, safford ma. time requirements for diabetes self-management: too much for many? j fam pract. 2005; 54(1):52-6. 4. lustman pj, clouse re. treatment of depression in diabetes: impact on mood and medical outcome. j psychosom res. 2002; 53(4):917-24. 5. egede le, nietert pj, zheng d. depression and allcause and coronary heart disease mortality among adults with and without diabetes. diabetes care. 2005; 28(6):1339-45. 6. black sa. increased health burden associated with comorbid depression in older diabetic mexican americans. results from the hispanic established population for the epidemiologic study of the elderly survey. diabetes care. 1999; 22(1):56-64. 7. bryan c, songer t, brooks mm, rush aj, thase me, gaynes b, et al. the impact of diabetes on depression treatment outcomes. general hospital psychiatry. 2010; 32(1):33-41. 8. ali s, stone ma, peters jl, davies mj, khunti k. the prevalence of co‐morbid depression in adults with type 2 diabetes: a systematic review and meta‐analysis. diabet med. 2006; 23(11):1165-73. 9. barnard kd, skinner tc, peveler r. the prevalence of co‐morbid depression in adults with type 1 diabetes: systematic literature review. diabet med. 2006; 23(4):445-8. 10. pan a, lucas m, sun q, van dam rm, franco oh, manson je, et al. bidirectional association between depression and type 2 diabetes mellitus in women. arch intern med. 2010; 170(21):1884-91. 11. ciechanowski ps, katon wj, russo je. depression and diabetes: impact of depressive symptoms on adherence, function, and costs. arch intern med. 2000; 160(21):3278-85. 12. gazmararian ja, ziemer dc, barnes c. perception of barriers to self-care management among diabetic patients. diabetes educ. 2009; 35(5):778-88. the assessment of depression level among diabetic patients vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 113 r a d s j . b i o l . r e s . a p p l . s c i . 113 13. ali s, stone m, skinner tc, robertson n, davies m, khunti k. the association between depression and health‐related quality of life in people with type 2 diabetes: a systematic literature review. diabetes metab res rev. 2010; 26(2):75-89. 14. goldney rd, phillips pj, fisher lj, wilson dh. diabetes, depression and quality of life: a population study. diabetes care. 2004; 27(5):1066-70. 15. jacobson am, groot m, samson ja. the effects of psychiatric disorders and symptoms on quality of life in patients with type i and type ii diabetes mellitus. qual life res. 1997; 6(1):11-20. 16. vamos ep, mucsi i, keszei a, kopp ms, novak m. comorbid depression is associated with increased healthcare utilization and lost productivity in persons with diabetes: a large nationally representative hungarian population survey. psychosom med. 2009; 71(5):501-7. 17. katon wj, simon g, russo j, von korff m, lin eh, ludman e, et al. quality of depression care in a population-based sample of patients with diabetes and major depression. medical care. 2004; 24(12):1222-9. 18. li c, ford es, zhao g, ahluwalia ib, pearson ws, mokdad ah. prevalence and correlates of undiagnosed depression among us adults with diabetes: the behavioral risk factor surveillance system, 2006. diabetes res clin pract. 2009; 83(2):268-79. 19. anderson rj, freedland ke, clouse re, lustman pj. the prevalence of comorbid depression in adults with diabetes: a meta-analysis. diabetes care. 2001; 24(6):1069-78. 20. sun n, lou p, shang y, zhang p, wang j, chang g, et al. prevalence and determinants of depressive and anxiety symptoms in adults with type 2 diabetes in china: a cross-sectional study. bmj open. 2016; 6(8):e012540. 21. golden sh, lazo m, carnethon m, bertoni ag, schreiner pj, roux av, et al. examining a bidirectional association between depressive symptoms and diabetes. jama. 2008; 299(23):27519. 22. beck at, ward ch, mendelson m, mock j, erbaugh j. an inventory for measuring depression. arch gen psychiatry. 1961; 4(6):561-71. 23. pouwer f, geelhoed‐duijvestijn ph, tack cj, bazelmans e, beekman aj, heine rj, et al. prevalence of comorbid depression is high in out‐patients with type 1 or type 2 diabetes mellitus. results from three out‐patient clinics in the netherlands. diabet med. 2010; 27(2):217-24. 24. sweileh wm, abu-hadeed hm, al-jabi sw, sa’ed hz. prevalence of depression among people with type 2 diabetes mellitus: a cross sectional study in palestine. bmc public health. 2014; 14(1):2-11. 25. lin eh, katon w, von korff m, rutter c, simon ge, oliver m, et al. relationship of depression and diabetes self-care, medication adherence, and preventive care. diabetes care. 2004; 27(9):2154-60. 26. wayne j. katon. the comorbidity of diabetes mellitus and depression. the american journal of medicine. 2008; 121:s8-15. 27. katon w, cantrell cr, sokol mc, chiao e, gdovin jm. impact of antidepressant drug adherence on comorbid medication use and resource utilization. arch intern med. 2005; 165(21):2497-503. 28. american diabetes association. standards of medical care in diabetes. diabetes care. 2007; 30(1):s4-41. 29. lustman pj, clouse re. depression in diabetic patients: the relationship between mood and glycemic control. j diabetes complicat. 2005; 19(2):113-22. 30. park h, hong y, lee h, ha e, sung y. individuals with type 2 diabetes and depressive symptoms exhibited lower adherence with self-care. j clin epidemiol. 2004; 57(9):978-84. prevalence and resistance profile of clinical isolates of acinetobacter species from karachi, pakistan vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 6 r a d s j . b i o l . r e s . a p p l . s c i . 6 op en ac ces s f u l l l e n g t h a r t i c l e prevalence and resistance profile of clinical isolates of acinetobacter species from karachi, pakistan qurat ul ain, asma naim*, asma saeed department of microbiology, university of karachi, karachi, pakistan a b s t r a c t acinetobacter baumannii causes a variety of infections including pneumonia, urinary tract infection, bacteremia, peritonitis etc. this organism is developing resistance to a number of antibiotics due to various intrinsic and acquired antibiotic resistance genes. the aim of the present study was to determine the prevalence of antibiotic-resistant acinetobacter species from karachi, pakistan. a total of 111 strains of acinetobacter baumannii and 8 strains of non-baumannii acinetobacter were isolated from various hospitals of karachi from september 2013 to december 2014. identification of the isolates was based on the standard biochemical tests and detection of oxa-51 and oxa-23. antibiotic resistance profile of the isolates was determined by kirby-bauer disc diffusion method and minimum inhibitory concentration (mic) was also determined by broth macro-dilution method. among 111 acinetobacter baumannii isolates, 8 were pan-drug resistant (pdr) and 103 isolates were multidrug resistant (mdr) while all non-baumannii acinetobacter were mdr. the effective antibiotics against a. baumannii were colistin, gentamicin, trimethoprim/sulfamethoxazole and ciprofloxacin with mic50 value 1, 256, 256, 256µg/ml, respectively. these findings strongly suggest the proper detection and reporting of pdr/mdr acinetobacter from clinical samples and also the judicious use of broad-spectrum antibiotics is necessary to prevent the further spread of resistant strains of acinetobacter. keywords acinetobacter baumannii, oxa-51, minimum inhibitory concentration, pandrug resistant, multidrug resistant, broadspectrum antibiotics. *address of correspondence anaeem@uok.edu.pk article info. received: october 10, 2018 accepted: february 26, 2019 cite this article: ain q, naim a, saeed a. prevalence and resistance profile of clinical isolates of acinetobacter species from karachi, pakistan. rads j. biol. res. appl. sci. 2019; 10(1): 6-13. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n acinetobacter spp. are gram-negative, aerobic, emerging opportunistic pathogens with an exceptional ability to develop resistance to different groups of antibiotics and associated with a wide range of iatrogenic infections including pneumonia, meningitis, bacteremia, and urinary tract infections1. the taxonomy of genus acinetobacter has been revised extensively and the species level identification by phenotypic characterization is difficult2. among the acinetobacter spp., a. baumannii has become one of the top seven pathogens threatening the health care settings, particularly the intensive care setting (icus). because of its remarkable capability to colonize patients in the hospital environment, it causes hospital outbreaks due to cross-transmission between patients3. it is also increasingly exhibiting multiple antibiotic resistance and several prevalent strains are resistant to nearly all antibiotics currently in use. excessive use of antimicrobials in the clinical environment has contributed to the emergence and spread of nosocomial infections. acinetobacter baumannii presents an array of antibiotic resistance mechanisms which result in limited treatment options for clinicians. acinetobacter baumannii exhibits resistance by both natural and acquired drug resistance mechanisms. multidrug-resistant acinetobacter baumannii (mdrab) are often associated with co-infection by other pathogenic organisms which make it difficult to determine o r i g i n a l a r t i c l e prevalence and resistance profile of clinical isolates of acinetobacter species from karachi, pakistan vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 7 r a d s j . b i o l . r e s . a p p l . s c i . 7 its attributable mortality4. in pakistan, acinetobacter baumannii has emerged as one of the most common nosocomial pathogen5 and there is very limited data regarding the persistence of this notorious organism in developing countries like pakistan. due to the higher incidence of nosocomial infections caused by mdrab, there is a need to pay attention to the detection of this organism within the hospital environment and also in the general population. the present study was designed to determine the prevalence of drug-resistant acinetobacter baumannii isolates in the clinical settings in karachi, pakistan. m a t e r i a l s a n d m e t h o d s bacterial isolation and identification along with 111 strains of acinetobacter baumannii and 8 strains of non-baumannii acinetobacter species were obtained from various hospitals and diagnostic laboratories of karachi from september 2013 to december 2014. for this study, strains were collected and inoculated on mcconkey's agar, and gram staining was performed. the pure cultures were maintained on trypticase soy agar (tsa), stored at 4˚c and can be available for routine testing. the isolates were further identified on the basis of the standard biochemical tests including oxidase test, catalase test, temperature growth test (44˚c), glucose fermentation, hemolysis on blood agar, citrate utilization test and gelatin liquefaction6. for additional confirmation, oxa-23 and oxa-51 genes were detected by pcr7,8 using specific primers (table 1). dna preparation and pcr conditions for the detection of oxa-23 and oxa-51 genes the boiling method was used for the dna preparation, by adding 200 µl of endonuclease free water in 1.5 ml eppendorf tube (cornell). take 2-3 colonies of bacteria from nutrient agar plate to make a suspension in endonuclease free water. heat this suspension in a water bath at 90˚c for 10 minutes. then cool to ambient temperature24. the reaction mixture contains twelve and half microliter master mix (2x) (merck), 0.5 µl of reverse primer, 0.5 µl of forward primer (idt, usa) and 9 µl of endonuclease free water were mixed in pcr tubes (cornell) two and half microliter of dna template was added in this 22.5 µl of reaction mixture (total volume of reaction mixture in each pcr tube was 25 µl) and subjected to thermocycler and set to perform 30 to 35 cycles. the isolates possessing these genes were referred to as acinetobacter baumannii, while only oxa-23 positive strains were categorized as non-baumannii acinetobacter8. antibiotic susceptibility testing the antimicrobial susceptibility profile was determined using the kirby-bauer disk diffusion technique according to the protocol of clinical and laboratory standards institute (clsi)9. sensitivity to colistin was interpreted according to the criteria defined by galani and co workers10. a total of 12 antibiotics belonging to seven classes of antibiotics were used in this study including cefepime (30μg), ceftriaxone (30μg), ceftazidime (30μg), table 1. primers and pcr conditions for oxa-51 and oxa-23 genes. target gene sequence pcr conditions amplicon size (bp) reference denaturation annealing extension number of cycles blaoxa51 taatgctttgatcggccttg 94˚c for 1 minute 50˚c for 1 minute 72˚c for 90 seconds 30 353 8 tggattgcacttcatcttgg blaoxa23 cttgctatgtggttgcttctc atccattgcccaaccagtc 94˚c for 1 minute 50˚c for 1 minute 72˚c for 90 seconds 30 650 7 prevalence and resistance profile of clinical isolates of acinetobacter species from karachi, pakistan vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 8 r a d s j . b i o l . r e s . a p p l . s c i . 8 cefotaxime (30μg), trimethoprim/ sulfamethoxazole (25μg), gentamicin (10μg), amikacin (10μg), imipenem (10μg), meropenem (10μg), ciprofloxacin(5μg), piperacillin/tazobactam (100μg) and colistin (10μg) [oxoid]. minimum inhibitory concentration minimum inhibitory concentration (mic) of 100 a. baumannii and eight non-baumannii acinetobacter isolates was determined by broth macro-dilution method using at least one antibiotic from each class including cefotaxime, trimethoprim/ sulfamethoxazole, gentamicin, meropenem, ciprofloxacin, piperacillin/tazobactam, and colistin following the guidelines provided by clsi9. test concentrations of antibiotics used are mentioned in table 6. the lowest antibiotic concentration which inhibited growth was considered as the minimum inhibitory concentration (mic)2. r e s u l t s in total, 119 acinetobacter strains were isolated from 160 clinical specimens. of these, 111 strains were identified as acinetobacter baumannii and eight were nonbaumannii acinetobacter with the prevalence rate of 69% and 5% respectively. species identification was confirmed after the successful pcr amplification of oxa-23 and oxa-51 genes. the highest number of isolates were obtained from tracheal aspirates (62%) followed by sputum (14%), pus (9%), wounds (5%) and single isolate were recovered from urine, blood, central venous catheter (cvc) tip, endotracheal tubes (ett) tip, bronchoalveolar lavage (bal) and peritoneal fluid (table 2). a higher frequency of acinetobacter species was isolated from males (68/111). high-risk age groups ranged from neonates to teenagers whereas patients with the age above fifty also had a higher frequency (figure 1 and 2). after successful pcr amplification of oxa-23 and oxa51 genes, isolate were easily identified and categorized up to the specie level. the presence of other organisms in the study samples was also detected and a. baumannii was found coexisting with other pathogenic organisms in 30 samples whereas non-baumannii acinetobacter spp. we’re not associated with other pathogens (table 3). table 2. frequency of acinetobacter baumannii and non-baumannii acinetobacter species from various clinical samples. specimens no. of isolates (%) acinetobacter baumannii (n= 111) non-baumannii acinetobacter spp. (n =8) tracheal aspirate 69 (62) 5 (62) sputum 16 (14) 1 (12) pus 9 (8) 0 (0) wounds 6 (5) 1 (12) urine 2 (1) 0 (0) blood 2 (1) 1 (12) cvc tip 2 (1) 0 (0) ett tip 2 (1) 0 (0) bal 2 (1) 0 (0) peritoneal fluid 1 (0.9) 0 (0) figure 1. distribution of age and gender of study population w.r.t. isolation of acinetobacter baumannii. figure 2. distribution of age and gender of study population w.r.t isolation of non-baumannii acinetobacter prevalence and resistance profile of clinical isolates of acinetobacter species from karachi, pakistan vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 9 r a d s j . b i o l . r e s . a p p l . s c i . 9 table 3. co-infecting organisms in the hospitalized patients. organisms no. % methicillin sensitive s. aureus 2 6 methicillin resistant s. aureus 5 16 pseudomonas aeruginosa 7 23 escherichia coli 3 10 klebsiella pneumoniae 6 20 streptococcus fecalis 1 3 burkholderia cepacia 1 3 enterobacter sp. 3 10 enterococcus sp. 1 3 citrobacter freundii 1 3 table 4. antibiotic resistance profile of acinetobacter baumannii by kirbybauer disc diffusion method (n=111). antibiotic groups name of antibiotic symbols (potency) s r β-latams/β-lactamase inhibitor combinations tazobactam/pipercillin tzp (110μg) 0 111 cephems ceftazidine caz (30 μg) 0 111 cefepime fep (30 μg) 0 111 cefotaxime ctx (30 μg) 0 111 ceftriaxone cro (30 μg) 0 111 carbapenems imipenem imp (10 μg) 0 111 meropenem mem (10 μg) 0 111 lipopeptides colistin ct (10 μg) 109 2 aminoglycosides gentamicin cn (10 μg) 1 110 amikacin ak (10 μg) 0 111 fluoroquinolones ciprofloxacin cip (5 μg) 0 111 folate pathway inhibitor trimethoprim/ sulfamethoxazole sxt (25 μg) 10 101 prevalence and resistance profile of clinical isolates of acinetobacter species from karachi, pakistan vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 10 r a d s j . b i o l . r e s . a p p l . s c i . 10 table 5. antibiotic resistance profile of non-baumannii acinetobacter (n=8). antibiotic groups name of antibiotic symbols (potency) s i r β-latams/β-lactamase inhibitor combinations tazobactam/ pipercillin tzp (110μg) 1 0 7 cephems ceftazidine caz (30 μg) 1 0 7 cefepime fep (30 μg) 1 0 7 cefotaxime ctx (30 μg) 0 1 7 ceftriaxone cro (30 μg) 0 1 7 carbapenems imipenem imp (10 μg) 2 0 6 meropenem mem (10 μg) 3 0 5 lipopeptides colistin ct (10 μg) 8 0 0 aminoglycosides gentamicin cn (10 μg) 1 0 7 amikacin ak (10 μg) 1 0 7 fluoroquinolones ciprofloxacin cip (5 μg) 2 1 5 folate pathway inhibitor trimethoprim/ sulfamethoxazole sxt (25 μg) 1 1 6 table 6. minimum inhibitory concentration of acinetobacter baumannii by broth macro-dilution method (n=100). antibiotics range (µg/ml) mic mic50 mic90 mbc (µg/ml) ctx 0.5-1024 >1024 512 >1024 tzp 0.5-1024 >1024 512 >1024 ct 0.625-16 2 0.5 1 4 cn 0.5-512 >512 256 >512 sxt 0.5-512 >512 256 >512 cip 0.25-512 >512 256 >512 mem 0.25-512 >512 512 >512 table 7. minimum inhibitory concentration of nonbaumannii acinetobacter spp. by broth macro-dilution method (n=8). antibiotics range (µg/ml) mic mic50 mic90 mbc (µg/ml) ctx 0.5-512 512 128 512 tzp 0.5-512512 128 512 ct 0.625-16 1 0.25 0.5 2 cn 0.5-512 >128 32 >128 sxt 0.5-512 64 32 64 cip 0.25-512 64 32 64 mem 0.25-512 64 32 64 prevalence and resistance profile of clinical isolates of acinetobacter species from karachi, pakistan vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 11 r a d s j . b i o l . r e s . a p p l . s c i . 11 all a. baumannii strains were resistant to 9 of the 12 antibiotics tested i.e., piperacillin/ tazobactam, ceftazidime, cefepime, cefotaxime, ceftriaxone, imipenem, meropenem, amikacin, and ciprofloxacin while 90% strains were found resistant to trimethoprim/ sulfamethoxazole and 99% to gentamicin. importantly in this study, we observed that two strains were resistant to colistin (table 4). in case of non-baumannii acinetobacter all strains were found to be sensitive against colistin, while 75% were found resistant to trimethoprim/ sulfamethoxazole, 72% to imipenem, 62% to meropenem and ciprofloxacin, while 87.5% to piperacillin/ tazobactam, cefepime, ceftriaxone, ceftazidime, cefotaxime, gentamicin and amikacin (table 5). mic results showed very high level of resistance among a. baumannii against the tested antibiotics except colistin which showed promising results (table 6). the mic 50 values for ctx, tzp, ct, cn, sxt, cip, and mem were 512 μg/ml, 512 μg/ml, 0.5 μg/ml, 256 μg/ml, 256 μg/ml, 256 μg/ml, and 512 μg/ml respectively, and mic 90 values were >1024 μg/ml, >1024 μg/ml, 1 μg/ml, >512 μg/ml, >512 μg/ml, >512 μg/ml, >512 μg/ml respectively. whereas, in case of non-baumannii acinetobacter mic 50 values for ctx, tzp, ct, cn, sxt, cip, and mem were 128 μg/ml, 128 μg/ml, 0.25 μg/ml, 32 μg/ml, 32 μg/ml, 32 μg/ml, and 32 μg/ml respectively, while mic 90 values were 512 μg/ml, 512 μg/ml, 0.5 μg/ml, >128 μg/ml, 64 μg/ml, 64 μg/ml, 64 μg/ml respectively (table 7). d i s c u s s i o n due to the increasing reports of the involvement of a. baumannii in human infections, it is the most extensively studied among the acinetobacter species. in recent years, it has become a significant pathogen causing infections with higher morbidity and mortality rate4. other than acinetobacter baumannii, stenotrophomonas maltophilia, pseudomonas aeruginosa, and burkholderia cepacia complex are the most important clinical aerobic, nonfermenting and gram-negative rods11. the emergence of resistance against major classes of antibiotics has been reported globally. the definition of mdrab and pdrab for acinetobacter varies in the literature. generally, an isolate is considered mdrab if it shows resistance to ≥ 3 classes of antibiotics while pdrab describes acinetobacter strains that show resistance to all standard antimicrobial agents (except colistin)12. review of the literature reveals that a. baumannii is mostly involved in nosocomial infections, especially the immunocompromised, chronically ill or debilitated individuals or the patients with underlying medical problems such as diabetes and cancer are at higher risk11. the local surveillance of drug-resistant organisms in clinical samples enables us to monitor the emergence of opportunistic pathogens and their antimicrobial susceptibility patterns provide the most suitable treatment options. molecular methods were useful to identify the genus acinetobacter up to the species level. as oxa-51 gene is intrinsic to a. baumannii8, plays the key role in the identification. in this study, oxa-51 was also detected for this purpose. in this study, 7.2% acinetobacter baumannii strains were multidrug resistant and 92.7% pan-drug resistant, moreover two strains were found to be colistin resistant indicating the emergence of colistin resistance, while all strains of non-baumannii acinetobacter were mdr. the antibiogram of the isolates shows the high resistance profile against almost all antibiotics. in our report, a. bauammii were more resistant to antibiotics than nonbaumannii acinetobacter spp. we observed high mic values against all tested antibiotics particularly against carbapenems which are considered to be a good choice for acinetobacter infections. however, in our study high resistance against meropenem (>512μg/ml) was observed. based on other similar studies from pakistan, it is evident that antibiotic resistance has been increasing among a. baumannii strains in our region. saleem and co-workers reported 21 mdr and 87 pdr strains of acinetobacter species3. a study by kaleem et al. reported 27 (84.3%) metallo-β lactamase (mbl) producing a. baumannii13. in another study by begum et al., 100% resistance was observed against cephalosporins, fluoroquinolones, carbapenems, and β lactam drugs, but minocycline and tigecycline were found to be active against mdr a. baumannii14. in pakistan (2010) hasan and colleagues reported 87 mdr isolates, 26 xdr isolates, and 19 pdr a. baumannii isolates from hospitals of islamabad and lahore5. pan-drug resistant a. baumannii outbreaks have also been reported from other regions of the world. fallah et prevalence and resistance profile of clinical isolates of acinetobacter species from karachi, pakistan vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 12 r a d s j . b i o l . r e s . a p p l . s c i . 12 al. found a high degree of resistance in a. baumannii isolates against various groups of antibiotics including colistin with 1.8% resistance15. kou et al. reported 100% resistance against carbapenems, cephalosporins, fluoroquinolones, and β lactam drugs while no resistance against colistin16. in a study from china, wang and coworkers (2018) found that 34 isolates were nonsusceptible to both imipenem and meropenem but a single isolate was resistant to meropenem only. resistance against other antibiotics was detected as 58.2% to ceftazidime, 52.2% to sulbactam, ciprofloxacin 64.2%, and 70.1% resistance were observed against cotrimoxazole. whereas, no resistance was found against polymixin and rifampicin, but one isolate was nonsusceptible to minocycline23. büyük et al (2017) reported 84 mdr acinetobacter strains and these isolates showed resistance against amikacin (50%), imipenem (58.33%), moxifloxacin (22.62%), ciprofloxacin (90.47%) and rifampicin (47.62%). while no resistance was found against colistin and tigecycline22. in 2013, an iranian study reported resistance against 21 antibiotics including colistin resistance in 11% isolates, which is higher than our isolates17. indian research by badave and dhananjay (2015) reported, >84% a. baumannii strains resistant against six antibiotics (ampicillin-sulbactam, piperacillin, amikacin, ciprofloxacin, ceftazidime, and imipenem)18. in 2016, chinese scientists reported multidrug or extensive drug resistance in 72.4% of the isolates19. a study by solomon reported a high degree of resistance among a. baumannii where >80% of the isolates were resistant to cefepime, sulfamethoxazole, ciprofloxacin, and ceftriaxone20. our current findings are in accordance with these international reports. colistin and tigecycline are considered as the last resort drugs against mdrab. there are increasing reports of colistinresistant a. baumannii world-wide which is a growing concern among the medical community as this could lead to treatment failure21. c o n c l u s i o n pan-drug resistant acinetobacter baumannii infections are life-threatening for neonates and elderly patients. this study shows that clinical isolates of acinetobacter baumannii are highly resistant to most of the currently used antibiotics. colistin is the last resort drug for treating mdrab infections. gentamicin and trimethoprim/sulphamethoxazole can be used in combination with colistin. early detection of acinetobacter spp. in hospital care settings requires adequate monitoring of the outbreaks using the modern molecular biology, strict infection control that is the most costeffective preventive measure and also to control the indiscriminate use of broad-spectrum antibiotics without any identification of organism and susceptibility testing. however, the lack of standardized laboratory resources makes this an underreported pathogen in developing countries. r e f e r e n c e s 1. camp c, tatum ol. a review of acinetobacter baumannii as a highly successful pathogen in times of war. lab medicine. 2010; 41(11):650-7. 2. looveren vm, goossens h, the arpac steering group. antimicrobial resistance of acinetobacter spp. in europe. clin microb infect. 2004; 10:684-704. 3. saleem af, ahmed i, mir f, ali sr, zaidi ak. panresistant acinetobacter infection in neonates in karachi, pakistan. j infect dev ctries. 2010; 4(1):307. 4. dent ll, marshall dr, partap s, hulette rb. multidrug resistance acinetobacter baumannii: a descriptive study in a city hospital. bmc infect dis. 2010; 10:196. 5. hasan b, perveen k, olsen b, zahra r. emergence of carbapenem-resistant acinetobacter baumannii in hospitals in pakistan. j med microbiol. 2010; 63(11):50–5. 6. holt jg, krieg nr, sneath ph. a, staley jt, williams st. bergey's manual of determinative bacteriology. baltimore: williams and wilkins. 1994:787. 7. mak jk, kim mj, pham j, tapsall j, white, pa. antibiotic resistance determinants in nosocomial strains of multidrug-resistant acinetobacter baumannii. j antimicrob chemother. 2009; 63:47-54. 8. turton jf, woodford n, glover j, yarde s, kaufmann m e, and pitt t l: identification of acinetobacter baumannii by detection of the blaoxa51-like carbapenemase gene intrinsic to this species. j of clin microb. 2006; 44(8):2974-6. 9. clsi. performance standards for antimicrobial susceptibility testing; twenty-second informational supplement. clsi document m100-s22. wayne pa: clinical and laboratory standards institute; 2012. 10. galani i, kontopidou f, souli m, rekatsina p-d, koratzanis e, deliolanis j, et al. colistin susceptibility testing by etest and disk diffusion methods. int j antimicrob agents. 2008; 31(5):434-9. prevalence and resistance profile of clinical isolates of acinetobacter species from karachi, pakistan vol. 10 (1), july 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 13 r a d s j . b i o l . r e s . a p p l . s c i . 13 11. senkyrikova m, husickova v, chroma m, sauer p, bardon j and kolar m: acinetobacter baumannii producing oxa-23 detected in the czech republic. springerplus. 2013; 2:296. 12. munoz-price ls, weinstein ra. acinetobacter infection. n engl j med. 2008; 358(12):1217-81. 13. kaleem f, usman j, hassan a, khan a. frequency and susceptibility pattern of metallo-beta-lactamase producers in a hospital in pakistan. infect dev ctries. 2010; 4(12):810-3. 14. begum s, hasan f, hussain s, shah aa. prevalence of multidrug-resistant acinetobacter baumannii in the clinical samples from tertiary care hospital in islamabad, pakistan. pak j med sci. 2013; 29(5):1253-8. 15. fallah f, noori m, hashemi a, goudarzi h, karimi a, erfanimanesh s, et al. prevalence of blandm, blaper, blaveb, blaimp, and blavim genes among acinetobacter baumannii isolated from two hospitals of tehran, iran. scientific. 2014; doi.org/10.1155/2014/245162. 16. kuo s, yang sp, lee yt, chuang hc, chen cp, chang cl, chen tl, et al. dissemination of imipenem-resistant acinetobacter baumannii with new plasmid-borne blaoxa-72 in taiwan. bmc infect dis. 2013; 13(319):1471-2334. 17. mohajeri p, farahani a, feizabadi mm, ketabi h, abiri r, najafi f. antimicrobial susceptibility profiling and genomic diversity of acinetobacter baumannii isolates: a study in western iran. iran j microbiol. 2013; 5(3): 195-202. 18. badave gk, dhananjay k. biofilm producing multidrug-resistant acinetobacter baumannii: an emerging challenge. j clin diagn res. 2015; 9(1):810. 19. qi l, li h, zhang c, liang b, li j, wang l, du x, et al. relationship between antibiotic resistance, biofilm formation, and biofilm-specific resistance in acinetobacter baumannii. front microbiol. 2016; 7:483. 20. solomon fb, wadilo f, tufa eg, mitiku m. extended spectrum and metalo beta-lactamase producing airborne pseudomonas aeruginosa and acinetobacter baumanii in restricted settings of a referral hospital: a neglected condition. antimicrobial resistance & infection control. 2017;6(1):106. 21. cai y, chai d, wang r, liang b, bai n. colistin resistance of acinetobacter baumannii: clinical reports, mechanisms and antimicrobial strategies. j antimicrob chemother. 2012; 67(7):1607–15. 22. büyük a, yilmaz ff, yurtsever sg, limoncu mh. antibiotic resistance profiles and genotypes of acinetobacter baumannii isolates and in vitro interactions of various antibiotics in combination with tigecycline and colistin. turk j pharm sci. 2017; 14(1):13-8. 23. wang r , dorp lv , shaw lp , bradley p, wang q , wang x , jin l , zhang q, liu y, rieux a, doraischneiders t, weinert la, iqbal z, didelot x, wang h and balloux f. the global distribution and spread of the mobilized colistin resistance gene mcr-1. nature communications. 2018; doi: 10.1038/s41467-01803205-z. 24. hujer am, hujer km, hulten ea, bajaksouzian s, adams jm, donskey cj, ecker dj, massire c, eshoo mw, sampath r, thomson jm, rather pn, craft dw, fishbain jt, ewell aj, jacobs mr, paterson dl and bonomo ra. analysis of antibiotic resistance genes in multidrug-resistant acinetobacter sp. isolates from military and civilian patients treated at the walter reed army medical center. j antimicrob chemother. 2006; 50(12): 4114-23. preliminary behavior of chinkara under captive conditions vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 19 r a d s j . b i o l . r e s . a p p l . s c i . 19 op e n ac c e s s f u l l l e n g t h a r t i c l e preliminary behavior of chinkara (gazella bennettii) under captive conditions with future conservation strategies muhammad idnan1,3,*, arshad javid1, muhammad nadeem4, ali hussain1, sajid mansoor2, waqas ali1, syed mohsin bukhari1 1department of wildlife and ecology, university of veterinary & animal sciences, lahore, pakistan 2department of microbiology, faculty of life science, university of central punjab, lahore, pakistan 3department of zoology, faculty of sciences, university of central punjab, lahore, pakistan 4department of dairy technology, university of veterinary & animal sciences, lahore, pakistan a b s t r a c t background: study of behavioral conformations play a significant role in ex-situ conservation of ungulates particularly of deer to propagate deer farming. due to advancement in animal hunting techniques, captive breeding is the best solution to propagate animals for sports or other traditional & medicinal uses. due to the lack of management methods, captive breeding programs have been used on a trial and error basis for the rearing of wild animals like chinkara. objectives: the present study was planned to investigate behavioral patterns in captivity to explore factors over the reproductive success of the chinkara and the potential of this wild animal (chinkara) for deer farming practices in pakistan. methodology: present study was conducted on the behavior of chinkara (gazella bennettii) [wild-caught (wc)=20, captive-bred (cb)=10] for a period of one year from april 2013 to march 2014 at captive breeding facilities for ungulates, ravi campus pattoki, the university of veterinary and animal sciences (uvas), pakistan. results: the behavior of captive-born and wild-caught animals was observed by focal sampling pattern to frame conservation strategies for successful management practices for the promotion of deer farming in pakistan. similar behavioral patterns were observed in both wc and cb animals but wc male chinkara displayed a higher degree of agonistic interaction than cb males. from these results, it is predicted that there is no obvious immediate effect of captivity on behavioral configurations up to 10 generations in chinkara. conclusion: it is suggested that chinkara is not suitable for domestication like goat until further studies on the ethology of chinkara. keywords chinkara (gazella bennettii), ex situ conservation, deer farming, wildlife management, captivity, domestication. *address of correspondence adnan264@gmail.com article info. received: october 29, 2018 accepted: july 26, 2020 cite this article: idnan m, javid a, nadeem m, hussain a, mansoor s, ali w, bukhari sm. preliminary behavior of chinkara (gazella bennettii) under captive conditions with future conservation strategies. rads j biol res appl sci. 2020; 11(1):19-26. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n the chinkara (gazella bennettii) is one of the ungulates belonging to the genus gazelle which is found in different habitats like flat plains, grasslands, sand deserts, hilly areas, dry scrubs and light forest of south asia, india, o r i g i n a l a r t i c l e preliminary behavior of chinkara under captive conditions vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 20 r a d s j . b i o l . r e s . a p p l . s c i . 20 parts of iran and pakistan1. this animal is distributed much around central and western india, spreading through pakistan, south-western afghanistan into northcentral iran. in the thar desert of india, about 1000,000 chinkara has been estimated2. while a much less number is estimated in iran 2,818. the current population status of chinkara in afghanistan is unknown but they are considered to be scarce3. due to overhunting in pakistan, the numbers of chinkara are declining with no exact estimate4. massive hunting has severely embellished gazelle’s population in afghanistan, iran and pakistan, where they are hunted for meat and trophy. chinkara is a facultative drinker and can survive in very arid areas. in deserts, they occasionally graze cultivated fields with rape seeds and sorghum3. the animal is susceptible to feral dogs, habitat destruction and poaching for meat consumption5. management of endangered species in captivity is of vital importance for a sustainable wildlife management practice; nonetheless, an increase in captive production can be a challenging task for wildlife managers due to insufficient information regarding the breeding behavior, biology of the species concerned and the difficulty of estimating different management patterns. even with vulnerability and complexity, decision-analytic approaches can be used to recognize optimum management decisions to increase the captive production6. in asia, the majority of the herbivores is under threat due to hunting, poaching, grazing competition with livestock and habitat fragmentation etc. conservation of asiatic ungulates is frequently apprehensive by poor information regarding the population dynamics and delicate evaluation of specific threats. wildlife conservation programs involve species-specific information and understanding of all concerned complications that may reduce population growth7. the present study is therefore planned to investigate behavioral patterns in captivity to explore those factors over the reproductive success of the chinkara and the potential of this wild animal (chinkara) for deer farming practices in pakistan. m a t e r i a l s a n d m e t h o d s study area the study was conducted at captive breeding facilities for ungulates at the university of veterinary and animal sciences (uvas), lahore, ravi campus, district kasur, pakistan. the development of the irrigation system like the canal’s system has resulted in the development of vast agricultural areas for farming and gardens or flower’s nurseries of the city at the expense of the sub tropical thorn forest eco-zone. agricultural farmlands, flower farms and canals in adjoining areas constitute a subtropical thorn forest biome in the study area. animals studied a total of 30 animals were observed in this study. animals were housed in two separate enclosures a and b with an equal ratio of males and females. the animals were captured from a semi-wild habitat and housed in enclosures (100 ft. × 200 ft) with a well-ventilated shelter (20 ft. × 20 ft). the shelter was constructed for animals to seek a save place in harsh weather condition. enclosures were also having brick’s wall of four feet in height with wire netting separated the enclosures. it assisted the animals to smell, hear and sight each other. one year before the start of the study chinkara were housed in these enclosures. during the study period, eight chinkara gave birth to young ones in a healthy condition. the animals were observed by similar people on each day and animals were identified with visual cues or identification marks by the observer. randomly a focal animal was selected from the group to observe various behavioral frequencies and different behavioral parameters as mentioned in table 1, were recorded continuously for 5min. a researcher started to observe the animals after 20min of his entrance into the enclosure as the animals at their first notice of an invader started to alert others by a tail wagging, producing a sound of cheenk-cheenkcheenk, which is the reason that the animals are named as chinkara and erecting their ears about the arrival of an observer. after a period of 20-30min, animals started to take fodder and performed other activities normally without showing any obvious notice of the intruders. the behavioral sampling did not affect the normal activities of the animals. different behavioral parameters observed by preliminary behavior of chinkara under captive conditions vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 21 r a d s j . b i o l . r e s . a p p l . s c i . 21 a focal person 05 times in a day, 6 days in a week and for 12 weeks in a period of one year. opportunistic focal animal sampling all opportunistic recordings were considered independent observations. chinkara lives in a group of 3 or 5 or sometimes solitary9 and the same behavior of grouping were observed in captivity. random encounters of chinkara were sought, followed by efforts to get the animal habituated to the observer’s presence. once sited, individuals were seen as long as the observer was tolerated by the animal. those animals that ran away immediately after being noticed were excluded from the analysis as it was difficult to judge their activity in the moments before they escaped. the animals have not separated aside from required therapeutic care or planned accomplishments for management purpose. feeding and enclosure management the animals were provided with fresh fodder, leaves of trees and grasses during the study period. as the leaves of melia azedarach (dhreik) and zizyphus jujube (beri) were abundant and were easily collected from the university area so that they were provided in addition to other forage. on daily basis, fresh and clean water was provided to the animals' ad libitum. statistical software spss 22 versions were used to analyze the data. r e s u l t s a comparison of the wild-caught (wc) and captive-bred (cb) chinkara was observed. the wc males show significantly more agonistic behavior (0.280 ± 0.019) as compared to the individuals, who were born in captivity (cb) males (0.213 ± 0.071) (p < 0.05). usually, the wc males displayed more agonistic interaction, anogenital sniffing, environmental sniffing, self-directed behavior and affinitive interaction than the cb chinkara. on the other hand, the cb animals tended to have a higher frequency of standing-alert and feeding/drinking activities than to wc chinkara as mentioned in (fig. 1) however, there is no statistically significant difference (p > 0.05) observed in behavioral parameters of chinkara. table 1. the average values of behavioral parameters for wild-caught (wc-no.=20) and the captive-born (cbno.= 10) chinkara. s. no. behavioral parameters wild born (wb) captive born (cb) p-value 01 resting behavior (re) 0.391 ± 0.031 0.375 ± 0.168 0.873 02 standing alert condition (sa) 2.727 ± 1.835 2.919 ± 1.267 0.156 03 locomotive behavior (lo) 4.436 ± 1.401 4.405 ± 2.906 0.913 04 feeding/drinking behavior (fd) 3.608 ± 1.690 4.250 ± 1.731 0.021 05 rumination activities (ru) 0.463 ± 0.136 0.114 ± 0.473 0.022 06 tail pasting behavior (tp) ---------------------------------- 07 urinating/defecation process (ud) 0.136 ± 0.034 0.166 ± 0.087 0.283 08 environmental sniffing activities (es) 1.219 ± 0.253 1.057 ± 0.495 0.111 09 self-directed behavior (sd) 1.138 ± 0.430 1.036 ± 0.522 0.351 10 anogenital sniffing activities (as) 0.363 ± 0.132 0.294 ± 0.105 0.280 11 affinitive interaction (ai) 0.202 ± 0.135 0.147 ± 0.092 0.153 12 agonistic interaction (ci) 0.280 ± 0.019 0.213 ± 0.143 0.071 13 miscellaneous behavior (mb) 5.657 ± 2.155 5.527 ± 1.206 0.683 preliminary behavior of chinkara under captive conditions vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 22 r a d s j . b i o l . r e s . a p p l . s c i . 22 the current study also prevails a difference in rumination behavior of wc chinkara as compared to the captive breed (fig. 2). the ruminants seek a safe place from predators and easily chew their cud in wild. that is why we have observed a high degree of rumination in wildcaught animals as compared to the captive breed individuals. contrary to this behavior, captive-born chinkara spent more time in foraging activities as compared to the wild-caught animals as depicted from fig. 3, this might be due to non-exposure to their predators. such a significant change in behavior plasticity provides the patterns for behavioral modifications to tame such wild animals and formulate strategies for deer farming, and subsequently ex-situ conservation for multipurpose aims, which may be discussed in other industrybased research strategies. figure 1. behavioral parameters frequencies between captive-bred (cb=10) and wild-caught (wc=20) chinkara. behavioral parameters include standing-alert (sa), feeding & drinking (fd), locomotion (lo), urinating/defecating (ud), self -directed behavior (sd), resting (re), environmental sniffing (es), ruminating (ru), affinitive interaction (ai), agonistic interaction (ci), ano-genital sniffing (as) and tail-pasting (tp). as and tp were not included in the figure due to their increased frequency. cb= captive born wb =wild born wc=wild caught (meng, et al. 2010) preliminary behavior of chinkara under captive conditions vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 23 r a d s j . b i o l . r e s . a p p l . s c i . 23 figure 2. a comparison of rumination in wild-caught (wc) & captive-breed (cb) chinkara. figure 3. a comparison of feeding in wild-caught (wc) & captive-breed (cb) chinkara. d i s c u s s i o n the behavioral modifications and genetic adaptations may be promoted by captive management through adaptations based on captive phenotypes10. transfer of wild animals to captive conditions may result in differential selection pressures, change in environmental parameters such as availability of resources (water, food and mating partners) and proximity to perceived dangers is maximized by human exposure. in domestic animals, few behavioral traits are lost as compared to the observation of new behavioral traits. the behavioral differences are quantitative in wild and domestic stocks and could be described by threshold response or behavioral frequency11. in the present study, no significant differences were observed for various behavioral categories in wild and captive-born chinkara. rumination in wild-born animals preliminary behavior of chinkara under captive conditions vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 24 r a d s j . b i o l . r e s . a p p l . s c i . 24 was more as compared to the captive breed individuals (figure 2). this may be due to the reason from an evolutionary perspective that in a wild environment the ruminants find some safe place to hide from predators and chew their cud too. in captive conditions wc and cb born chinkara were housed in identical enclosures, where they have no control over the number of co-inhabitants, but it was observed that the dominant male used to defend the female for mating with other males. this agonistic behavior has caused serious fights that resulted in deaths. it has been observed that the dominant male hits other males with horns at the abdominal and thoracic regions. it is noted that the social environment has a key role in the interaction of animals12. results of preliminary agonistic behavioral interactions indicated that an unrestricted environment is not necessary by the factor for captive management. under captive conditions both the wild-born and captiveborn chinkara, which are confined to a similar condition in the study area, lacking ecological enrichment, are expected to involve more in agonistic interactions. this hypothesis is supported by current findings which showed the wb deer are considerably more aggressive than the cb chinkara. fear related behavior in wild animals has a genetic background and it plays an important role in the survival of animals in wild conditions and evolution as well12. besides, close interaction with people can be relied upon to cause an alteration of behavior, particularly distressing, where “selection pressure” may change i.e. aversion from people, who encounter the animals on daily basis for feeding and management related issues13. wallace recommended that, after some generations, the absence of regular “selective pressure” may modify the genetic make-up of vital ethological characteristics14. in the present investigation, the focal person and guardians had to interact with chinkara on daily basis for feeding and managing the enclosures of the animals. such observations are encouraged by zhang, who gave preparatory reports with regards to the domestication of musk deer15. in our findings of various behavioral patterns in chinkara, there were no apparent variations in general behavioral parameters like feeding, drinking, chasing, sniffing etc. but the differences in the social behavior i.e., affinitive (0.202 ± 0.135) and agonistic (0.280 ± 0.019) behavior in wb chinkara were more than those observed in the cb chinkara. on the contrary, it is shown in figure 3 that captive breed individual spent more time on feeding which maybe because they are facing no predator exposure to predators under captive conditions while in wild conditions the organisms have to face such threats. in this study, no age effect was observed due to the small sample size. taming and domestication of animals have been developed over a period of thousands of generations by human interaction and habituation with these animals for their commercial use. however, it is a difficult task to determine the degree of domestication of an animal because the phenotype of the animal not only depends on its genetics but also on the environment in which it is reared. like in the study reported on foxes, it was observed that the foxes which were selected for domestication started to take food from caretakers and used to eat in human presence16. nonetheless, it can be suggested from the results of our preliminary findings on behavioral modes for chinkara in captivity that it has not been adaptive for behavioral modifications even for 10 generations born in captive conditions without proper taming practices. so, these results indicated that chinkara is not suitable for domestication. but it has been observed that if the calves of chinkara are kept from day one with other livestock and human beings they are tamed well and show no fear of human presence. moreover, the animals reared under captive conditions do not learn how to survive in wild conditions, and by the presence of a significant predator, they may also face extinction. to protect such a vulnerable species other conservation measures should be taken into consideration so that the economics of deer farming would not be a question17. to ensure the long-term conservation of endangered animals a healthy stock of captive breed population should be maintained for release of captive animals in wild environment12. in most social mammals, some females disperse from their natal group while others remain and breed there throughout their lives. in a few cases, females typically disperse after adolescence but the rest of them remain and breed in their natal group. this behavior is also observed in chinkara deer under captive conditions which lessens the time to find a new mate for breeding. these preliminary behavior of chinkara under captive conditions vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 25 r a d s j . b i o l . r e s . a p p l . s c i . 25 contrasts in philopatry and dispersal have an important consequence on the kinship structure of groups which in turn, affects forms of social relationships between females18. conservation behavior is a relatively new interdisciplinary field that aims to investigate how proximate and ultimate aspects of animal behavior are formulated to prevent biodiversity loss. the usefulness of this new discipline is to promote practical conservation-matters, which is a topic to debate with some scientists arguing that the importance of behavior in conservation practice is exaggerated 19. commonly it is observed that the captive-born animals are having deficiencies in foraging activities and locomotion as compared to the wild-born animals. few of these discrepancies are continued in reintroduced animals for up to two years in the wild. results of this research, however, provided little signs of farming or domestication and minor changes in behavior of captive and wild-born animals. these results showed the possibilities of reintroduction programs for conservation approaches, especially for the accomplishment of deer farming. c o n c l u s i o n domestication of chinkara is a difficult task as various factors are involved in their behavioral modifications; the elements affecting the domestication process may be biological or ecological, enclosure size or management system and the development of a complicated relationship with human beings as well. from the results of this preliminary study, it is difficult to propose that chinkara is a suitable species for domestication. further studies are recommended to find out stress level in wild chinkara to manipulate suitable managing practices. a c k n o w l e d g m e n t the authors are thankful to all contributors and others from where the literature was cited. l i s t o f a b b r e v i a t i o n s ai affinitive interaction as ano-genital sniffing cb captive-born es environmental sniffing fd feeding & drinking lo locomotion sa standing alert sd self-directed behavior tp tail-pasting ud urinating-defecating wb wild born wc wild-caught r e f e r e n c e s 1. khampariya pk, singh sp. status and feeding ecology of the chinkara in panna forest, panna (mp). life sci. bul. 2011; 8:251-4. 2. mallon dp, kingswood sc. antelopes: north africa, the middle east, and asia: iucn; 2001. 3. habibi k. mammals of afghanistan: zoo outreach organisation with assistance from us fish and wildlife service; 2003. 4. akbari h, moradi hv, sarhangzadeh j, esfandabad bs. population status, distribution, and conservation of the chinkara, gazella bennettii, in iran (mammalia: bovidae). zool middle east. 2014; 60(3):189-94. 5. dookia s, rawat m, jakher g, dookia b. status of the indian gazelle (gazella bennettii sykes, 1831) in the thar desert of rajasthan, india. faunal ecology and conservation of the great indian desert: springer. 2009; 193-207. 6. smith dh, converse sj, gibson kw, moehrenschlager a, link wa, olsen gh et al. decision analysis for conservation breeding: maximizing production for reintroduction of whooping cranes. j wildlife manage. 2011; 75(3):501-8. 7. bhowmik m. disease spectrum and fawn mortality of hog deer (axis porcinus) in eastern himalayan region. tigerpaper. 2000; 27(4):17-20. 8. meng x, yang h, yang q, feng z, peng x, perkins gc. preliminary findings of behavioral patterns in captive alpine musk deer (moschus sifanicus) and prospects for future conservation. turk j vet anim sci. 2010; 34(2):111-7. preliminary behavior of chinkara under captive conditions vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 26 r a d s j . b i o l . r e s . a p p l . s c i . 26 9. jakher g, dookia s, dookia b. herd composition and population dynamics of indian gazelle (gazella bennetti) (sykes, 1831) in gogelao enclosure (nagaur), rajasthan. zoos print j. 2002; 17(11):9368. 10. curio e. conservation needs ethology. trends ecol evol. 1996; 11(6):260-3. 11. andersen il, nævdal e, bøe ke, bakken m. the significance of theories in behavioral ecology for solving problems in applied ethology-possibilities and limitations. appl anim behav sci. 2006; 97(1):85-104. 12. håkansson j, bratt c, jensen p. behavioural differences between two captive populations of red jungle fowl (gallus gallus) with different genetic background, raised under identical conditions. appl anim behav sci. 2007; 102(1):24-38. 13. price eo. behavioral genetics and the process of animal domestication. 1999; 65(3):245-71. 14. wallace m. retaining natural behaviour in captivity for reintroduction programmes. conservation biology series-cambridge-2000; 300-14. 15. zhang b. the taming and raising of musk deer. agriculture, press, beijing. 1979. 16. harri m, mononen j, ahola l, plyusnina i, rekilä t. behavioural and physiological differences between silver foxes selected and not selected for domestic behaviour. anim welfare. 2003; 12(3):305-14 17. parry-jones r, wu jy. musk deer farming as a conservation tool in china: traffic east asia; 2001. 18. clutton-brock th, lukas d. the evolution of social philopatry and dispersal in female mammals. mol ecol. 2012; 21: 472-92. 19. berger-tal o, polak t, oron a, lubin y, kotler b p, saltz d. integrating animal behavior and conservation biology: a conceptual framework. behav ecol. 2011; 22(2), 236-9. prevalence of nematode worms and associated risk factors rukhsana talat1*, farzana ibrahim1, quratul ain1, zaira1 1department of zoology, jinnah university for women, karachi-74600 pakistan. abstract the prevalence of nematode worms with special references to ascaris lumbricoide, enterobius vermicularis and trichuris tricura were studied in different areas of karachi city. a total 223 cases were recorded from seven different hospitals of karachi city during january to december 2011. the total patients of ascaris lumbricoides were 109 including 49 male with 44.95% and 60 female with 55.04% while the total number of patients having enterobius vermicularis were 95 from which 47 were male with 49.47% and 48 female with 50.52%. the total patients suffering from trichuris trichiura were 19 including 9 male with 47.36% and 10 female with 52.63%. keywords: nematode worms, intestine, patients, hospitals, karachi. introduction parasitic intestinal infections caused by nematode worms are most prevalent infections in human in developing countries and cause a significant morbidity and mortality in endemic countries. these nematode worms also known’s as geohelminths and soiltransmitted helminthes which are most prevalent in tropical and subtropical regions due to improper facilities of sanitation (savioli and albonico, 2004 and cappello, 2004) a lot of research work has been done on intestinal infection albonico et al., (1999) worked on control strategies of nematode infection. drake et al., (2000) studied on the cognitive and development impacts of ascariasis, trichiuriasis and hook worm. stephenson et al., (2000) observed malnutrition due to helminth infection. bundy et al., (2001) observed the global epidemiology of nematode infection. crompton and nesheim (2002) observed the impact of nutrition on helminthiasis. siddiqui et al., (2002) studied the distribution of intestinal parasites in a rural area of karachi. cappello (2004) observed the global health impact of nematodes. bonsal et al., (2004) recorded the incidence of intestinal parasites among the people of low socio-economic area of chandigarh (north india) while okyay et al., (2004) observed the prevalence and related factors in school children of turkey. khurana et al., (2005) made comparison among rural and urban population in chandigarh. bethony et al., 2006 worked on ascariasis, trichuriasis and hook worm. shaukat et al., (2006) observed the intestinal parasitic record from ehsanullah diagnostic laboratory in karachi. jacobsen et al., (2007) estimated the prevalence of intestinal infection among young children of highlands of rural ecuator. kabatereine et al., (2007) observed the impact of helminth control programme in uganda children. mehray et al., (2008) studied the prevalence and associated factors of intestinal parasites among children in an urban slum of karachi. hotoz et al., (2008) observed the helminth infection as a neglected tropical disease. wani et al., (2008) recorded the helminthic infection in children of kashmir valley. guadalupe et al., (2009) reported the ascaris lumbricoides in newborns of infected mothers. who, 2010 present the updated global target of soil transmitted helminthiasis. steinmann et al., (2010) observed rapid appraisal of helminth infection among school children in oshoblist, kyrgyzstan. figueiredo et al., (2010) studied that chronic helminth infection are associated with immune hypo responsiveness.*corresponding author: rukhsanatalat4@gmail.com 11 vol 4 (1), january 2013; 11-16 hsieh et al., (2010) detected the parasitic intestinal infection among laboures kaohsiung. talat et al., (2012) observed the prevalence of helminth infection in different areas of karachi. materials and methods the present research work was carried out for one year i.e. january to december 2011. the information was collected from pathological laboratories of different hospitals of karachi city as saifee hospital situated in nazimabad, sindh govt. hospital in khokrapar malir, usman memorial hospital in federal b area, ibn-e-sina hospital in gulshan-eiqbal, haleem hospital in sakhi hasan north nazimabad while khizar hospital and the hospital in shah faisal town. results and discussion the results represent information collected from 223 patients during a period of one year (from january to december 2011) from 7 different hospitals of karachi i.e. ibn-e-sina hospital, saifee hospital, haleem hospital, usman memorial hospital, sindh govt. hospital, khizra hospital and the hospital. nematodes collectively reffered to as soil transmitted helminths parasites causing greatest worldwide cause of illness and are linked to lack of proper sanitation, lack of use of safe water and lack of hygiene therefore intestinal parasites prevails wherever there is poverty and effect the people of all ages. the summarized results are presented in table iv which showed the prevalence of nematode parasites 12 vol 4 (1), january 2013; 11-16 table i: over all patients infected with ascaris lumbricoides diagnosed from seven different hospital of karachi city from jaunary to december 2011. months iben-eseena hospital saifee hospital haleem hospital usman memorial hospital sindh govt. hospital khizar hospital the hospital jan feb mar april may june july aug sept oct nov dec total male female male female male female male female male female male female . 13 vol 4 (1), january 2013; 11-16 months iben-eseena hospital saifee hospital haleem hospital usman memorial hospital sindh govt. hospital khizar hospital the hospital jan feb mar april may june july aug sept oct nov dec total male female male female male female male female male female male female male female table iii: over all patients infected with trichuris trichiura diagnosed from seven different hospital of karachi city from jaunary to december 2011. table ii: over all patients infected with enterobius vermicularis diagnosed from seven different hospital of karachi city from jaunary to december 2011. months iben-eseena hospital saifee hospital haleem hospital usman memorial hospital sindh govt. hospital khizar hospital the hospital jan feb mar april may june july aug sept oct nov dec total male female male female male female male female male female male female male female in all parts of karachi city. the rate of infection of ascaris lumbricoides (table-i) is most common parasite, and was found in all parts of karachi. the rate of intensity in females was found higher (table iv) i.e. 44.95% in males and 55.04% in females. enterobius vermicularis also cause a worldwide disease called enterobiasis which is a second widest distribution and found also common in karachi city (table ii ). the intensity of infection due to e. vermicularis was found 49.47% in males and 50.50% in females (table iv). the distribution of trichuris trichiura in different hospitals is represented in table iii and the overall summarized infection due to trichuris trichiura was found 47.36% in males and 52.63% in females. (table-iv). the geohelminth infections are highly prevalent due 14 vol 4 (1), january 2013; 11-16 table iv: over all summarized record of patients of ascaris lumbricoides, enterboius vermicularis and trichuris trichiura collected from seven different hospital of karachi during jaunary to december 2011. hospitals name iben-eseena hospital saifee hospital haleem hospital usman memorial hospital sindh govt. hospital khizar hospital the hospital grand total observed cases total positive infection cases ascaris lumbricoides enterobius vermicularis trichuris trichiura male female male female male female total male total female total patients to the conditions which are most frequently associated with water source, defecation site, and especially personal hygiene and for the control of infection it is very important to improve the sanitary system references albonico, m., crompton, d. w., and savioli. l. 1999. control strategies for human intestinal nematode infection. adv parasitol, 42, 277-341. bansal d., sehgal r., bhatti h.s., shrivastava., s.k., khurana s., mahajan r.c. and malla n. 2004. intestinal parasites and intra familial incidence in a low socio-economic area of chandigarh (north india). nepal med coll j, 6, 28-31. bethony j, brooker s, albonico m, geiger sm, loukas a, diemert d, hotez pj. 2006. soiltransmitted helminth infections: ascariasis, trichuriasis and heskworm. lancet, 2006, 367:1521-1532. bundy, d.a.p., chan m.s., medley g.f., jamison d and savioli l. 2001. “intestinal nematode infections.” the global epidemiology of infectious diseases (c.j.l. murray and a .d. lopez.eds). cambridge harvard university press. cappello m. 2004. global health impact of soil transmitted nematodes. pediatr infect dis j. 23: 663664 [pub med] crompton, d.w.t., and m.c. nesheim. 2002. nutritional impact of intestinal helminthiasis during the human life cycle. annu. rev. nutr. 22:35-59. de silva nr., brooker s., hotez pz., montresor a, engles d, savioli l 2003. soil transmitted helminth infection updating the global picture. trends parasitol. 19: 547-551 [pub med] drake lj., jukes mch., sternberg rj., bunday dap. 2000 geohelminth infections (ascariasis, trichiuriasis, and hookworm): cognitive and development impacts. sem paediatr infect dis. 11: 245-51 figueiredo ca, barreto ml, rodrigues lc, cooper pj, silva nb, amorim ld, alcantara-neves nm. 2010. chronic intestinal helminth infection are associated with immune hyporesponsiveness and induction of a regulatory network. infect immune, 78: 3160-3167. guadalupe i, mitre e, benitez s, chico me, cordova x, rodriguez j, nutman tb, cooper pj. 2009. evidence of intrauterine sensitization to ascaris lumbricoides infection in newborns of infected mothers. j infect dis. 199:1846-1850 hotoz pj, brindley pj, bethony jm, king ch, pearce ej, jacobson. 2008. j: helminth infections: the great neglected tropical diseases. j clin. invest, 118:13111321. hsieh m-h., lin w-y., dai c-y., huang j-f., huang c-k., chien h-h., wang c-l., chung w-l., wujr., chem e-r., ho c-k., yu m-l. 2010. intestinal parasitic infection detected by stool examination laboures kaohsiung. kaohsiung j med sci. 26: 136142. jacobsen kh., ribeiro ps., quist bk., rydbeck bv. 2007. prevalence of intestinal parasites in young quichua children in the highlands of rural ecuador. j health popul nutr. 25: 399-405. [pub med] kabatereine nb, brooker s, koukounari a, kazibwe f, tukahebwa em, fleming fm, zhang y, webster jp, strthard jr, fenwick a (2007).: impact of a national helminth control programme on infection and morbidity in ugandan school children. bull world health organ. 85:91-99. khurana s., aggarwal a and malla n. 2005. comparative analysis of intestinal parasitic infections in slum, rural and urban population in and around union territory, chandigarh. j commun dir. 37: 239243. mehraj v, hatcher j, akhtar s, rafique g, beg ma. 2008. prevalence and factors associated with intestinal parasitic infection among children in an urban slum 15 vol 4 (1), january 2013; 11-16 16 vol 4 (1), january 2013; 11-16 of karachi plos one 3: e3680. okyay p, ertug s, gultekin b, onen o, beser e. 2004. intestinal parasites prevalence and related factors of parasitic infections in school children, a western city sample-turkey bmc public health., 4:1-9. savioli l., albonico m. 2004. soil transmitted helminthiasis. nat rev microbiol. 2:618-619 [pub med] shaukat n., bilqees f.m., hadi r., ziadi v.a. and nadia a. 2006. a record of intestinal parasitic infections from ehsanullah diagnostic laboratory, nazimabad karachi. j. baqai med. univ. 9: 29-36. siddiqui, m.i., bilqees f.m., ilyas m, and perveen s. 2002. prevalence of parasites infections in a rural area of karachi pakistan j. pak. med. assoc. 52: 315-320. steinmann p, usubalieva j. imanalieva c, minbaeva g, stefiuk k, jeandron a, utzinger j. 2010. rapid appraisal of human intestinal helminth infections among school children in osh oblast, kyrgyzstan. acta trop, 116:178-184. stephenson ls., latham mc., ottesen ea. 2000. malunutrition and parasitic helminth infections. p a r a s i t o l o g y ; 1 2 1 : s 2 3 3 8 [ p u b m e d ] talat r., farzana i., sharifa g., and sobia m. 2012. prevalence of human intestinal parasites in different areas of karachi pakistan. b: helminth infection. p r o c e e d i n g s o f p a r a s i t o l o g y 5 3 ; 6 7 7 6 . wani s.a., ahmad f., zargar s.a., dar p.a., dar z.a., and jan t.r. 2008. intestinal helminthes in a population of children from the kashmir valley, india. j. helminththal, 82, 313-317. who. 2010. soil-transmitted helminthiasis. number of children treated 2007-2008: updated on 2010 global target. wkly epidemiol rec, 85; 141-148. the biochemical profile of chronic kidney disease patients. afshan zeeshan wasti1*, saba haider2, shabana rashid2, naureen fatima1, sumaira iqbal1, afshan ashiq1, erum khalid1 1department of biochemistry, jinnah university for women, karachi 74600, pakistan. 2department of botany, jinnah university for women, karachi 74600, pakistan. abstract chronic kidney disease-ckd is a major public health problem and cause of morbidity and mortality worldwide. in pakistani population, the prevalence of ckd is unexpectedly high therefore not unexpected since incidence of hypertension and diabetes in pakistani population is one of the highest in the world. ckd is defined as impaired glomerular filtration rate (gfr) or elevated albumin excretion in the urine, and has been recognized as an important risk factor contributing to cardiovascular disease and death. the most common risk factors for ckd includes; diabetes mellitus, hypertension, dyslipidemia and most important older age, but the clinical implications still remain uncertain in elderly persons suffering from ckd. the present study aims to evaluate the biochemical profile in the patients with chronic kidney disease as compared to the healthy controls. analysis of lipid profile (cholesterol, total lipid, hdl-c, ldl-c, and triglycerides), renal profile (urea, creatinine, bun and uric acid), electrolytes (sodium, potassium, chloride and bicarbonate) and hematological indices were carried out in ckd patients (n=50) as compared to the healthy individual (n=50) by using automated and standardized kit methods. we suggests the use of stringent hematological and biochemical testing such as lipid and renal profile in the patients suffering from chronic kidney disease, for correct diagnosis and more accurate treatment strategy to decrease morbidity and mortality related with ckd. keywords: chronic kidney disease (ckd), lipid profile, renal profile, hematological profile. introduction according to the survey conducted in 2011 in karachi a city harboring 9% of pakistan's population (>30 years of age), an astonishing 25.3% had some degree of reduced glomerular filtration rate (gfr), with 5 % h a v i n g m o d e r a t e c k d ( g f r < 6 0 ml/min/1.73m2). even more disturbing was the fact that only 2.3% individuals were aware of having renal disease, most people with lower income falling in to this category (zeb et al., 2012; jafar et al., 2005; couser et al., 2011). ckd is defining as a progressive loss in renal function over a period of months or years, having non-specific symptoms of worsening kidney function. the risk factors for chronic kidney disease include an age more than 60 years, hypertension, diabetes, cardiovascular disease and a family history of ckd (peter, et al., 2007). moreover, the structural and functional changes in the kidneys have been associated with the ageing process, the kidneys atrophy and the cortical thickness decreases by approximately 10% per decade after the age of 30 years may lead to kidney scarring (hsieh, 2009). the diagnosis for ckd is based on the screening of peoples known to be at risk such as those with high blood pressure or diabetes, first degree blood relatives, *corresponding author: a_wasti_76@yahoo.com 05 vol 4 (1), january 2013; 05-10 suffering from chronic kidney disease. it may also be identified in case of cardiovascular disease, anemia or pericarditis, which may be one of its recognized complications (leeann et al., 2012; levin, 2003). the markers of renal function tests such as urea, uric acid, creatinine, bun and electrolytes were used to assess the normal functioning of kidneys. the increase or decrease values of these biomarkers were indicating the glomerular filtration rate and the tubular function (concentrating and diluting capacity) of kidneys or simply the dysfunction of kidney (shivaraj et al., 2010; eiichiro et al., 2013; ian wu and parikh 2008). hsieh et al., (2009) have reported that the electrolyte abnormalities such as hyponatremia, hypernatremia, hyperkalemia and hypokalemia are the most observed changes in the renal and cardiac functions, especially in the advanced age make them prone to chronic kidney disease. the management of electrolyte abnormalities is often complex, especially due to the adverse effects the numerous drugs and co morbidities that often present in ckd patients. it has been suggested that hyperlipidemia could cause renal injury and contribute to the progression of renal disease. there have been a number of observational studies showing that lipid abnormalities are associated with a reduction in kidney function in the general population (wanner and ritz, 2008). although some studies demonstrated that cardiac related cause of death in patient with ckd are not all directly related with atherosclerotic process but most probably related to arrhythmia, are extremely common causes of death in all stages of ckd (hsieh 2011). furthermore, in the general population sudden death is most commonly due to coronary artery disease but there are less clear data on the mechanism of sudden death in ckd. hematological disturbance such as anemia is considered as a frequent complication occurs in chronic kidney disease and is associated with morbidity and mortality and a decline in quality of life. the severity of anemia is directly proportional to the degree of renal function (kalantar et al., 2009). the present study aims to evaluate the biochemical profile in chronic kidney disease patients as compared to normal healthy controls. materials and methods the present study was designed to evaluate the alterations in the biochemical profile in serum samples of chronic kidney disease patients (n=50), who were referred as the out patients from different kidney and urology clinics and hospitals from all over karachi-pakistan. the analysis of lipid profile (cholesterol, total lipid, hdl-c, ldl-c, and triglycerides) and renal profile (urea, creatinine, bun and uric acid) were measured by using chemistry analyzer (stat lab 300 plus). the serum electrolytes (sodium, potassium, chloride and bicarbonate) were estimated by flame photometer (jenway clinical pfp7c) and hematological indices using hemato-analyzer, in chronic kidney disease patients as compared to the apparently healthy and non smoking subjects(n=20) were selected at random. statistical analysis was performed using standard statistical software (spss version 16.0). all data are expressed as mean ± sd. the data were also tested using student’s t-tests; the significance level was set as p < 0.05. results the characteristics lipid profile was reported in table i suggesting significant (p<0.0001) increase in the level of triglyceride (26%) in ckd patient as compared to the normal controls. significant (p>0.001) decrease of about (14%) was observed in cholesterol concentration of patient with ckd however, the levels of hdl-c was observe to be decrease (48%) significantly while no change in the level of ldl-c in the patients suffering from ckd with reference to normal controls. the levels of total lipids were also found to be significantly increased in ckd patients as compared to the normal group. 06 vol 4 (1), january 2013; 05-10 07 vol 4 (1), january 2013; 05-10 table i: lipid profile of ckd patients vs controls. table ii: renal profile of ckd patients vs controls. figure 1: comparison of hematological profile in ckd patients vs normal controls. hbhemoglobin (g/dl), rbcred blood cell concentration (103/mm2), pcv-packed cell volume (%), mcv-mean cell volume (fl), mch-mean cell hemoglobin (pg), mchcmean cell hemoglobin concentration (%),wbc-white blood cell concentration (103/mm2). parameter control (n=20) samples (n=50) p values 199.55+12.8 170.46+51.01* 0.0144 70.75+6.04 36.75+11.5* 0.0001 102.90+21.3 104.02+38.4 0.9027 98.50+7.68 131.0+47.9* 0.0037 506.90+94.7 641.1+200.5* 0.0056 cholesterol mg/dl hdl-c mg/dl ldl-c mg/dl tg mg/dl total-lipid mg/dl 24.46+2.48 18.2+3.93 0.0001 bicarbonste mg/dl parameter control (n=20) samples (n=50) p values 26.5+5.65 72.45+38.50* 0.0001 urea mg/dl 1.053+0.26 4.87+3.642* 0.0001 creatinine mg/dl 138.5+3.39 139.6+5.13 0.2219 sodium mg/dl 4.10+0.411 4.54+0.92* 0.0.0026 potassium mg/dl 104.9+3.54 110.3+6.28* 0.0001 chloride mg/dl table ii illustrates the renal profile comprising the levels of urea, creatinine and electrolytes (sodium, potassium, chloride and bicarbonate) in serum samples of ckd patient as compare to the normal controls. significant (p<0.0001) increase was observed in serum urea, and creatinine (63.43%, 78.48% respectively) in ckd patients as compared to the normal controls. however, slight but significant (p<0.025, p<0.0001respectively) changes was observed in potassium (9.7% ) and chloride (4.91% ) with insignificant slight increase in sodium (0.77 % ) ions. however, significant (p<0.0001) decrease was observed only in the level of serum bicarbonate (34.1 % ) concentration in chronic kidney disease patient as compared to normal controls. figure-1 depicts the comparison of hematological profile in ckd patients as compared to normal controls. significant (p>0.0001) decrease was observed in both hematological indices such as (rbcs count and hb concentration) and in absolute indices such as (pcv, mch and mchc) levels in ckd patients while the mcv did not show any significant difference in ckd patients as compared to healthy controls. discussion in ckd patients, our results shows that they exhibit atherogenic lipid profile which is characterized by an increase serum level of total lipid and triglycerides as compared to the normal controls (table-i) suggesting that the triglyceride concentration is high due to down regulation of lipoprotein lipase (lpl), hepatic lipase, very low density lipoprotein (vldl) and low density lipoprotein receptor (ldl-r) expression, lead to the main dyslipidemia disturbance in these ckd patients (silva ls et al., 2012; khedidja 2009; piecha et al., 2009; keane 2013). in ckd patients (table-ii), the increased concentration of urea, creatinine and electrolytes was observed suggesting their association with inflammatory and malnutrition condition. this increase in the concentration of urea and creatinine could lead to proteinuria, hematuria and renal dysfunction, as observed in high percentage in ckd patients as compared to normal healthy individuals (thomas et al., 2008; amin-ul-haq et al., 2010; alcázar, 2008). in ckd patients, the observed hematological disturbances like decrease in rbc count, hemoglobin concentration, packed cell volume and leukocyte count suggesting anemia (figure-1). changes in red cell indices are due to a number of factors aside erythropoietin productions, deficiencies of iron, vitamin b12 and folate as a result of nutritional insufficiency or due to increased blood loss are contributory factors. the impaired erythropoietin because as high as 90% of erythropoietin is produced in the juxta glomerular apparatus of the kidney while 10% are produced in the liver and other organs. the severity of affects depends on the stage of renal failure (george et al., 2009; guenter et al., 2005) we suggests the use of stringent hematological and biochemical testing such as lipid and renal profile in the patients suffering from chronic kidney disease, for correct diagnosis and more accurate treatment strategy to decrease morbidity and mortality related with ckd. conclusion we conclude that multiple factors are involved in the progression of kidney disease in our population including the most frequent use of contaminated water, unhygienic food stuff (vegetables), rampant lack of awareness, under-detection of earlier stages of ckd, lack of preventive measures etc. all of them inevitably facilitates progression of mild, potentially treatable ckd to full-blown kidney failure. it is not surprising that only 10% of kidney failure patients receive any renal replacement therapy due to the unaffordable treatment cost. there is a dire need to stress on the national and international programs aimed to prevent and control ckd in third-world countries such as pakistan. acknowledgement this work was supported by institutional funds of jinnah university for women, karachipakistan. the authors thank the patients, their families and 08 vol 4 (1), january 2013; 05-10 09 vol 4 (1), january 2013; 05-10 the healthy volunteers for their participation. conflict of interest and funding the authors have not received any funding or benefit from industry to conduct this study. references alcázar a. r. 2008. electrolyte and acid-base balance disorders in advanced chronic kidney disease. nefrologia.,28 suppl 3:87-93. amin-ul-haq, mahmood r, ahmad z, jamil-urrehman, jilani g. 2010. association of serum uric acid with blood urea and serum creatinine. pak j physiol, 6 (2):46-49. couser wg, remuzzi g, mendis s et al. 2011. the contribution of chronic kidney disease to the global burden of major noncommunicable diseases. kidney int, 80: 1258–1270. eiichiro k, masumi a, masayuki y, renjiro k and tatsuo s. 2013. high serum bicarbonate level within the normal range prevents the progression of chronic kidney disease in elderly chronic kidney disease patients. bmc nephrology, 14:4. guenter w and goodnough lt. 2005. anemia of chronic disease. n engl j med., 352:1011-23 ian wu, and parikh cr. 2008. screening for kidney diseases: older measures versus novel biomarkers. clinical journal of the american society of nephrology, 3 (6): 1895-1901. jafar th sc, levey as. 2005. serum creatinine as marker of kidney function in south asians: a study of reduced gfr in adults in pakistan. j am soc nephrol, 16: 1413–1419. kalantarzadeh.k and aronoff g. 2009. hemoglobin variability in anemia of chronic kidney disease california. j am soc nephrol., 20: 479–487. keane wf, tomassini je, neff dr. 2013. lipid abnormalities in patients with chronic kidney disease: i m p l i c a t i o n s f o r t h e p a t h o p h y s i o l o g y o f atherosclerosis. j atheroscler thromb., 20(2):12333. khedidja m, josiane p, mustapha r, jacques b and malika b. 2009. long term hemodialysis aggravates lipolytic activity reduction and very low density, low density lipoproteins composition in chronic renal failure patients. bmc cardiovascular disorders, 9:41. leeann b, vipan s, susan h, bonnie l, and catherine c. 2012. high burden and unmet patient needs in chronic kidney disease int j nephrol renovasc dis., 5: 151–163. levin a. 2003. clinical epidemiology of cardiovascular disease in chronic kidney disease prior to dialysis. semin dial.,16(2):101-5. michael h, david ap. 2009. abnormal renal function and electrolyte disturbances in older p e o p l e . j p h a r m p r a c t r e s . , 3 9 : 2 3 0 4 . ming-fang h, i-wen w, chin-chan l, shun-yin w; mai-szu w. 2011. higher serum potassium level associated with late stage chronic kidney disease. chang gung med j.,34:418-25. peter, wl. 2007. chronic kidney disease: a burgeoning health epidemic. j manag care pharm., 13(9 suppl d): s2-s5. piecha ga, ritz m. 2009. dyslipidemia in chronic kidney disease: pathogenesis and intervention. polskie archiwum medycyny wewn?trznej.,119(7-8):487492. shivaraj g, prakash bd, shruthi sk, vinayak vh, avinash akm, and sonal n v 2010. markers of renal function tests. n am j med sci.,2(4): 170–173. silva ls, oliveira ra, silva gb, lima jw, silva rp, liborio ab, daher ef, sobrinho cr. 2012. cardiovascular disease in patients with end-stage renal disease on hemodialysis in a developing country. saudi j kidney dis transpl., 23(2):262-6. thomas r, kanso a, sedor jr. 2008. chronic kidney 10 vol 4 (1), january 2013; 05-10 disease and its complications. prim care., 35(2):32944. wanner c, ritz e. 2008. reducing lipids for cv protection in ckd patients-current evidence. kidney int suppl., (111):s24-8. zeb is and hussain sa. 2012. chronic kidney disease in pakistan: an under-recognized public health problem. kidney international, 81: 1151. introduction candida albicans is a widespread opportunistic fungal pathogen that grows either as a yeast or as filamentous hyphae, depending on the environmental conditions (pranjape et al.,1990). candida albicans, the causative agent of mycotic infections collectively known as candidiasis, is a convenient organism for studying the regulation of differentiation. antifungals are supplements that make use of their natural ingredient to inhibit harmful fungi. no treatment of candida infection is complete without an effective antifungal. the rising incidence of serious fungal infections has created an increased demand for reliable methods of in vitro testing of antifungal agents that can assist in their clinical use. the national committee for clinical laboratory standards (nccls) has developed a standardized broth macrodilution method for the testing of candida spp. and cryptococcus neoformans which has greatly improved the reproducibility of antifungal susceptibility testing and serves as the "gold standard”. time-kill testing has become an indispensable tool for assessing the activity of antimicrobials against microorganisms. standardized methods providing instruction on the implementation of time-kill methods have been proposed by the national committee for clinical laboratory standards to ensure the reproducibility and accuracy of test results.( klepser et al. 1998). several studies have used spectrophotometric determination of endpoints to eliminate such subjective interpretation and revealed good agreement with the nccls recommended method (blanco, et al. 1992, pfaller, et al. 1995 ). fungistatic agents as azoles (ketoconazole [ktc], fluconazole [flc], and itraconazole [itc]) and flucytosine (5fc) show less*corresponding author: tubabatool@live.com 49 vol 4 (2), july 2013; 49-55 effect of paneer booti during kinetics on candida tuba batool1*, sayyada ghufrana nadeem1 1department of microbiology, jinnah university for women, karachi-74600. abstract candida albicans is a dimorphic fungus and a commensal of skin in humans. it is opportunistic fungal pathogen that may cause localized as well as systemic infections. as the pathogenecity of fungus is increasing, the demand for effective antifungal agent is also increasing. in the present study the macrodilution, microdilution and time kill curve methods were used to evaluate the effect of paneer booti during growth kinetics of candida. in macrodilution the most effective concentrations of paneer booti were found to be 10-1 and 10-2. similarly fluonazole ‘s concentration of 10-1 and 10-2 effect the growth after 4 hours and 10-3 and 10-4 effect the growth after 5 hours. in microdilution, magnesium and calcium enhanced the growth of c. albicans after 7 hours. in 10-3 and 10-4 concentration the inhibition of candida albicans occured after 8 hours and fluconazole and paneer booti were found to be effective on its growth. the time kill curve of candida albicans showed that paneer booti and fluconazole were effected against the candida albicans and clearly showed the four phases of growth (lag, log, stationary and decline ). as paneer booti was found to be effective against the pathogenic specie of candida i.e., candida albicans, therefore, it can be use for the treatment of infections that are caused by this pathogenic agent. keywords: microdilution, growth curve, fluonazole, paneer booti, candida albicans. defined endpoints and introduce significant subjectivity into the reading of results. materials and methods type of sample: candida albicans was used as a sample. equipments: glass wares (pyrex), syringes (mediocre), incubator (mermmet), autoclave (memmert), spectrophotometer (germany), elisa well reader (germany), juster (eppendroff ), oven (dawlance), filter paper (whattman no.1). chemicals and medias: rpmi 1640 medium, sabouraud dextrose agar (sda), potato dextrose agar (pda), magnesium sulphate (mgso4), calcium chloride (cacl2), herb ( paneer booti). antifungal agent: fluconazole was used as a antifungal. the stock solutions of fluconazole were prepared in sterile distilled water. fluconazole we used that was 240 mg, it was dissolved in 20 ml of sterile distilled water and then made the working solution. test isolate: candida albicans was used in this experiment. we made the suspension of candida albicans from 48 hours old culture and inoculated in 20 ml sterile distilled water. compared the suspension with 0.5 mcfarland tube (moore et al., 2003). herb extraction: paneer boti was used to check the effect on kinetics of candida. the herb extraction was prepared in 15ml of sterile distilled water (3:15) dilution, then boiled it into the oven for atleast 5 to 10 minutes. after that passed the whole suspension from whattman filter paper (no.1) in a sterile flask and stored at 0-4 °c for further experiments and analysis (huda-faujan et al., 2007). metals dilution: two dilutions of magnesium sulphate ( mgso4) were made i.e., 0.5m and1m. for 0.5m, 0.6g of mgso4 were dissolved in 10 ml of distilled water, for 1m , 1.2g of mgso4 were dissolved in same quantity of distilled water (pranjape et al.1990). two dilutions of calcium chloride (cacl2)were made i.e., 0.5m and 1m. for 0.5m , 0.5g of cacl2 were dissloved in 10 ml of distilled water. for 1m, 1.1g dissolved in 10ml distilled water (pranjape et al.1990). microtiter method: the method used was a microtitre modification of the nccls m27 (a method5 in flatbottomed microtitre plates with either ynbg or rpmi-g broth). the yeast suspensions used for the macrodilution method were then adjusted further (moore et al., 2003). each well contain 40µl rpmi and same amount of yeast suspension(40µl). the first 2 well contain 40µl of 0.5m and 1m of mgso4 and other 2 wells contain same quantity of cacl2 . 40µl of herb (paneer booti) was taken from other well and serially dilute it, same thing we done with fluconazole. fluconazole was taken as a control. the microtitre plates were incubated in a moist chamber at 37°c for 48 hrs. after incubation, the microtitre plates were shaken for 5 min to obtain a uniform suspension before reading (moore et al., 2003). next day optical density was recorded at 492nm using elisa plate reader at every 2 hours interval. m a c r o b r o t h d i l u t i o n m e t h o d : t h e b r o t h macrodilution method employed was that of the ‘report of a working group of the british society for mycopathology’ (moore et al., 2003). in this method 10 ml rpmi 1640 medium were taken in first test tube and 9 ml in other 3 tubes. in first tube put 1ml of fluconazole and serially diluted (10-1 to 10-4), discarded from last one to get the equal quantity in all 4 tubes. after that add 1ml of yeast suspension in all tubes. the same procedure were performed with paneer booti (herb). optical density(od) was taken from spectrophotometer, first was taken at 0 hr. and then incubate test tubes into shaking water bath. other od readings were taken at every 1 hour of incubation and after taken od put test tubes again into the shaking water bath till next od reading. 50 vol 4 (2), july 2013; 49-55 timekill curve method: time-kill curve studies were performed in standard rpmi by the method described by klepser et al. (klepser, et al.1998). before the tests were performed, the isolates were subcultured at least twice and grown for 24 h at 35°c on sda plates. the inoculum was adjusted spectrophotometrically to the density of a 0.5 mcfarland turbidity standard at 530 nm. the adjusted inoculum suspension was diluted 1:20 in rpmi containing the appropriate concentrations of fluconazole and tubes with the test solution were incubated at 35°c without agitation; the final volume was 5 ml. optical densities were taken at 1 hr. interval. volumes of 0.1 ml, (depending on the dilution and the concentration of the drug) were spread onto sda plates and incubated at 35°c for 24 to 48 hr. to determine the number of cfu per milliliter ( canto´n, et al., 2004.). results and discussion in this study the effect of herb i.e., paneer booti was observed on candida. herbs are found effective against different candida species but here we used the only one specie of candida that was candida albicans. in previous studies, it was reported that methanol was a better solvent for the consistent extraction of antimicrobial substances from medicinal plants compared to other solvents such as water, ethanol and hexane. the extract p. endlicherianum was observed against b. megaterium. e. coli, k.pneumoniae, p. vulgaris, c. albicans, c. tropicalis. their results showed that the methanol extract of v. georgicum has inhibitory effect on the growth of all candida albicans isolate (sengul et al.2013). but in this study, only water extraction method was used and this showed the effective result on candida albicans (table i). in macrodilution method we serially diluted the herb (paneerbooti) from 10-1 to 10-4 and according to our observations all four concentrations were found to be effective on candida albicans growth curve. 10-1 and 10-2 concentration of paneer booti was found to be more effective (figure 1), because the first phase (lag) occurred and remained at 4 hours , the log phase started at 6 hours and after 6 hours the stationary phase started but it did not remained long and decline occurred after 8 hours (figure 1). the concentration 10-3 and 10-4 of paneer booti showed that first phase (lag) started and remained at 2 hours, after 2 hours log phase started and remained at 4 hours and decline occured after 4 hours but slight differences were seen in concentration 10-4 , log phase was started after 2 hours and stayed for 4 hour and then stationary phase started and this phase remained longer and after 6 hours decline phase occurred (figure 2). it means that the first two concentration rapidly affect the candida albicans growth and candida albicans was not be able to survive for longer time. other concentrations of paneer booti effect slowly thats why candida showed stationary phase for long time. as control we used fluconazole (antifungal) to observed the difference between the fluconazole and paneer booti that how they both effect the growth of candida albicans. fluconazole exhibited fungistatic (99.9% decrease in the log10 of the number of cfu/milliliter compared with starting inoculum) activity against each of the test isolates; however, three distinct effects on growth were observed (klepser, et al.1998). according to our result candida albicans was found to be highly sensitive to fluconazole (table i). initially the growth of candida albicans was increased but after some time it showed decrased growth (figure 3). the same macrodilution method was done with fluconazole that was already done with paneer booti. in fluconazole we made serial dilution (10-1 to 10-4) and according to (figure 3 and 4) all concentrations showed the effectiveness on candida albicans growth. at the starting candida growth was high but gradually decrased with time. in the next method that was microdilution 96 wells plate was used. in this method magnesium and calcium was used to checked either they are effecting candida albicans or not. if mg values were to be expressed on a dry weight basis, cell mg would remain relatively constant since the dry weight of c. albicans mass increases by 100% over this period. however, the gradual increase was interrupted by a 51 vol 4 (2), july 2013; 49-55 52 vol 4 (2), july 2013; 49-55 peak of mg content per cell which coincided with the onset of germ-tube formation. this peak represents a three-fold increase indicating a transient, but net, increase in cell mg at this time. in a further test, cells were added in cacl2, (10 mm) and incubated for 3 hrs. about 70% of the cells were able to form germ tubes (pranjape et al.1990). according to our results magnesium and calcium both enhanced the growth of candida albicans. we used two concentrations of magnesium and two concentrations of calcium that is 0.5m and 1m (table ii). this result showed that firstly candida was in lag phase and growth was not increased and later on the magnesium and calcium effected on candida albicans growth, both enhanced the growth of candida albicans. there was little effect of concentration seen on growth i.e., 1m of magnesium and calcium effect was higher than 0.5 m, but there was not a big difference between both concentrations. in this method we used the magnesium and calcium as growth enhancing agent because the results clearly indicated that the candia albicans growth increased after the incubation. in this microtiter method we also used the fluconazole as control. with nccls breakpoints, seven isolates were resistant, one intermediate and the remainder susceptible. with bsmm breakpoints (moore et al., 2003), seven isolates were resistant, five intermediate and the remainder susceptible. the seven isolates were classed as resistant regardless of breakpoint use (moore et al., 2003). according to our results candida albicans showed sensitivity against fluconazole and both the dilutions were found to be effective. in this way, we also checked the effect of paneer booti on candida albicans. the survival of resting cells decreased immediately and rapidly with egc, gc, egcg and gcg, and the survival rate was <1% after 4 h. a few colonies still survived after 24 h of culturing (hirasawa et al., 2003). candida albicans is sensitive to paneer booti in method as well. in (figure 4) show effect of paneer booti on candida. here we diluted the paneer booti in rpmi 1640 broth, the both concentrations clearly indicate that candida albicans were not able to grow further. colonial changes of candida albicans was also futher checked. it was cultured on sda or pda to see the changes appeared on growth. in general, worse reproducibility was observed with visual endpoint determinations than with spectrophotometric readings (cuenca-estrella et al., 2001). when we observed the plates after incubation it shows the colonial changes (figure 1). the colonies were large and white in colour but after several period of time there was gradual changes occurred in colonies. in figure 2 changes were clearly shown and the colonies become small and mucoid. these are herb concentrations that effected the candida albicans. figure 3 and 4 showed the effect of fluconazole on candida albicans. initially the colonies were large in number but later on the number of colonies were decreased and morphologically changed. a time-kill plot of the activity of voriconazole against a representative isolate from each of the fungal species tested is presented in a previous paper in which fungistatic activity was observed with voriconazole against all seven isolates. for the isolates of c. neoformans and c. glabrata and the isolate of c. tropicalis, voriconazole concentrations greater than the mic did not appreciably increase the rate or extent of fungistatic activity. against both isolates of c. albicans, however, the maximal fungistatic activity was observed with voriconazole concentrations greater than or equal to four times the mic (klepser et al., 1998) according to our results all four phase appeared that is lag , log stationary, and decline. the growth curve of candida albicans is obtained by using both methods. the time period given to paneer booti and herb that were from 1 to 9 hours and in between these hours all four phase have observed. at the end we concluded that the herb (paneer booti) we used against the candida albicans, was effective, not only one concentration but all were found to be effective. it means that paneer booti can be use for the treatment of infections caused by candida albicans. 53 vol 4 (2), july 2013; 49-55 time period hours 1 2 3 4 5 6 7 8 9 10 0.400 0.336 0.225 0.153 0.119 0.114 0.102 0.100 0.103 0.102 0.271 0.233 0.189 0.154 0.132 0.118 0.101 0.100 0.102 0.101 0.226 0.196 0.179 0.154 0.144 0.132 0.125 0.111 0.110 0.109 0.366 0.209 0.154 0.132 0.120 0.101 0.099 0.100 0.101 0.100 0.112 0.110 0.102 0.111 0.138 0.273 0.280 0.179 0.098 0.064 0.147 0.155 0.150 0.146 0.164 0.89 0.325 0.305 0.101 0.099 0.103 0.112 0.199 0.254 0.266 0.252 0.211 0.152 0.099 0.045 0.120 0.126 0.122 0.172 0.180 0.179 0.172 0.119 0.089 0.024 1 2 3 4 1 2 3 4 fluconazole different concentrations paneer booti different concentrations table i. compare the effect of fluconazole and paneer booti (herb) on candida albicans at different concentrations by spectrophotometer. table ii. comparison of fluconazole and paneer booti (herb) on candida albicans by microtiter plates method. time period hours 2 4 6 8 10 12 14 16 18 20 22 0.912 0.668 0.975 0.981 1.588 1.033 8.590 8.687 5.980 4.360 2.887 0.933 0.849 0.868 0.846 0.780 0.812 7.386 7.775 4.867 3.287 2.790 2.186 2.097 2.097 2.072 2.071 2.040 11.311 11.214 7.428 5.237 2.891 1.007 0.944 0.953 0.918 0.807 0.842 7.491 7.321 6.428 5.652 3.211 1 2 1 2 fluconazole different concentrations paneer booti different concentrations 54 vol 4 (2), july 2013; 49-55 refrences blanco mt, pérez-giraldo c, blanco j, morán fj, hurtado c, gómez-garci´a ac. 1992. in vitro studies of activities of some antifungal agents against candida albicans atcc 10231 by the turbidimetric method. antimicrob. agents chemother. 36:898–901. canton e, peman j, gobernado m, viudes a and espinel-ingroff a. 2004. patterns of amphotericin b killing kinetics against seven candida species. antimicrob. agents chemother. 48:2477-2482. cuenca-estrella m, díaz-guerra tm, mellado e, rodríguez-tudela jl. 2001. influence of glucose supplementation and inoculum size on growth kinetics and antifungal susceptibility testing of candida spp. j clin microbiol, 39(2):525-32. figure 3. this is the early phase picture which shows the large number of colonies but slightly effected not white in colour. figure 4. this is the late phase picture in which the number of colonies are less in number and the colonies were morphologically changed. figure 1. it is the early stage picture that clearly shows that the herb (paneer booti) affect very slightly on candida albicans growth and due to which the large white colonies appeared. figure 2. it is the late phase picture in which the candida albicans was affected more than in the early phase. it means that paneer booti (herb) effect gradually on candida albicans. figure 3 figure 4 figure 1 figure 2 hirasawa m and takada k. 2003. multiple effects of green tea catechin on the antifungal activity of antimycotics against candida albicans.j antimicrob chemother, 53: 225-229. huda-faujan n, noriham a, norrakiah as and babji, a.s. 2007. antioxidative activities of water extracts of some malaysian herbs. asean food journal 14 (1): 61-68. klepser me, ernst ej, lewis re, ernst me, and pfallerma. 1998. influence of test conditions on antifungal time-kill curve results: proposal for standardized methods. antimicrob agents chemother,42(5):1207-1212. moore cb, walls cm and denning dw. 2003. comparison of three methods for in vitro susceptibility testing of candida species with flucytosine. j antimicrob chemother, 51: 297–304. paranjape v, roy bg and atta a.1990 involvement o f c a l c i u m , c a l m o d u l i n a n d p r o t e i n p h o s p h o r y l a t i o n i n m o r p h o g e n e s i s o f cadidaalbicans. j gen microbiol, 136: 2149-2154. pfaller ma, messer sa and coffmann s. 1995. comparison of visual and spectrophotometric methods of mic endpoint determinations by using broth microdilution methods to test five antifungal agents, including the new triazole d0870. j clin microb, 33(5):1094-7. sengul m,.ogutcu h, adiguzel a, et al. 2013. antimicrobial effects of verbascum georgicum bentham extract. turk j biool, 29: 105-110. 55 vol 4 (2), july 2013; 49-55 phytochemical analysis & antibacterial activity of tobacco plants vol.12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 60 op e n ac c e s s f u l l l e n g t h a r t i c l e phytochemical analysis and antibacterial activity of nicotiana tabacum and nicotiana rustica muhammad tariq1, zubair ahmad1, sher ali shah1, zainab gul2, shahid ali khan1,* 1department of chemistry, university of swabi, khyber pakhtunkhwa, pakistan. 2institute of physical medicine and rehabilitation (ipmr), khyber medical university, khyber pakhtunkhwa, pakistan. a b s t r a c t background: all over the world, natural products containing different secondary metabolites have been used for antibacterial purposes, and as folk medicines with significant effects. amongst many different plants, tobacco plants are cultivated all over the world, but natively belong to america. these plants contain variety of secondary metabolites and possess significant antibacterial activity. objectives: to conduct phytochemical analysis and measure the antibacterial potential of tobacco plants nicotiana tabacum and nicotiana rustica using their extracts. methodology: multiple chemical tests such as wagner’s test and fehling’s test were used to determine the presence of different types of secondary metabolites. both plant species were also screened for their antibacterial activity using agar well diffusion method. results: phytochemical analysis of the extracts from both plant species i.e. nicotiana tabacum and nicotiana rustica indicated the presence of secondary metabolites including tannins, alkaloids, terpenoids, saponins, steroids, and flavonoids. significant antibacterial activity of both plant extracts was observed against staphylococcus aureus, but not against escherichia coli. conclusion: it can be concluded that both plant extracts showed the presence of secondary metabolites, with significant inhibitory effect observed against staphylococcus aureus, and no effect against escherichia coli. keywords antibacterial studies, nicotiana tabacum, nicotiana rustica, phytochemical analysis, secondary metabolites. *address of correspondence shahidsawal007@gmail.com article info. received: august 09, 2020 accepted: december 31, 2020 cite this article:tariq m, ahmad z, shah s a, gul z, khan s a. phytochemical analysis and antibacterial activity of nicotiana tobacum and nicotiana rustica . rads j biol res appl sci. 2021; 12(1):60-65. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n medicinal plants have been used for treatment of various diseases in past and around 5000 species have been identified1 so far; that are used in cosmetics and pharma industries for the development of new products2-3. various kinds of organic compounds can be produced by plants, in which some compounds do not contribute to their own functions and are referred as secondary metabolites4. natural products contain diverse bioactive properties and can lead to the drug discovery5. most of them are categorized as generally recognized as safe, (gras) family of plants that are prominently used as folk medicines6-14 which led to the discovery of new drugs6. medicinal plants are the source of natural products having the properties to cure the disease. the 50% available drugs; including pharmaceutical and cosmetics products in the market7 belongs to the natural products isolated from o r i g n a l a r t i c l e phytochemical analysis & antibacterial activity of tobacco plants vol.12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 61 the medicinal plants8-9. in chinese and egyptian cultures, these natural products are still used as medicine and more than 75% population believes and relies on traditional natural products as reported by who10-11. plants are rich source of antioxidant, anti-fungal, anti-viral and anti-parasitic agents12-14. among the plant family solanaceae, nicotiana tabacum plant is cultivated all over the world but natively belongs to america15.it has been reported to be sensitive to environmental factors including humidity, nature of land and temperature. some species of nicotiana tabacum have also been used for ornamental purposes16. similarly, another plant nicotiana rustica belongs to the family solanaceae, contains nicotine in greater amount that is used for the production of pesticides products. these plants contain variety of secondary metabolites and possess significant antibacterial activity12-14. m a t e r i a l & m e t h o d s distilled water and all the necessary glassware were obtained from department of chemistry, university of swabi, kpk. all the chemicals used in these experiments were purchased from the sigma aldrich. instrumentation the plant leaves were grinded to fine powder by using grinder. similarly, the extract was concentrated using water bath (hh-s6-china). filter paper discs (oxoid usa) were used for the antibacterial assessment while, sterilization was done by using autoclave. collection of plants the whole plants of nicotiana tabacum and nicotiana rustica were collected from the district swabi, kpk. plants were then washed with water in order to remove dust and impurities. after washing, nicotiana tabacum and nicotiana rustica plants were shade dried for one week in order to avoid any phytochemical reaction. after that, the dried plants were grinded into small pieces with the help of electric grinder. the grinded powder then subjected to the extraction procedure. extraction procedure grinded powder from both plants was soaked separately into a mixture of methanol and distilled water in a ratio of 1:2 in conical flask (erlenmeyer flasks), caped with the aluminum foil. these flasks were kept in shelf for 4 days at room temperature. after 4 days, the mixture was subjected to extraction techniques; evaporated in water bath at 70°c and concentrated samples were collected. the procedure was repeated thrice and the extract was stored in clean sterile bottle for further use. s y n t h e s i s o f r e a g e n t s fehling’s solution a for the preparation of fehling solution, 7g of cuso4.5h2o was added to 100ml of distilled water and then 2 drops of dilute h2so4 were added to the solution, that turned the solution blue suggesting the fehling solution a is ready.20 fehling’s solution b for the preparation of fehling solution b, 35g potassium tartrate and 12g naoh was mixed with distilled water, as a result a colorless solution was formed confirming the formation of fehling solution b.20 wagner’s solution a total of 2g of iodine and 6g of ki (potassium iodide) were dissolved in 100l of distilled water to prepare wagner solution.21 b i o l o g i c a l a c t i v i t y agar well diffusion method the biological activity was checked by agar well diffusion method following procedure from murray et al., well diffusion protocol17. antibacterial activity was done using mueller–hinton agar (mha). for the preparation of media, 1.9g mha was dissolved in 50ml of deionized water, the prepared media was autoclaved for 15min. media (15ml) each was poured into sterilized petri dishes to make mha sensitivity testing plates. later, the agar surface was inoculated by spreading 200µl of the microbial inoculum of s. aureus and e. coli, followed by making wells (~8mm) in agar with a sterile cork borer. 100µl nicotiana tabacum and nicotiana rustica extracts were transferred to their respective wells in each plate. the plates were incubated at 37°c for 24h. after 24h, the zones of inhibition were observed surrounding wells, which indicates that extract from both the species possess significant antibacterial activity. phytochemical analysis & antibacterial activity of tobacco plants vol.12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 62 scheme-1. extraction procedure, phytochemical analysis and anti-bacterial activity of nicotiana tabacumand nicotiana rustica. r e s u l t s a n d d i s c u s s i o n phytochemical analysis of the nicotiana tabacum (fig. 1a) and nicotiana rustica (fig. 1b) extracts was performed using different reagents. the following test has been performed for the identification of different secondary metabolites in the extract of nicotiana tabacum and nicotiana rustica species. the entire procedure is depicted in scheme-1. tannins identification for the identification of tannins in the extract of both plant species, specific amount of the selected plant extract was taken and mixed with distilled water. the filtrate was then collected from the reaction mixture after heating. after this, the filtrate was poured in the test tube and ferric chloride drops were added to that filtrate resulting in the formation of new color i.e. dark greenish. the appearance of new color confirms the presence of tannins in both plants as shown in the inset of fig. 1(c)17. alkaloid identification for the identification of alkaloids, 0.3g of each extract was dissolved in 2ml of distilled water separately and warmed up with 2% sulphuric acid for 3min, and then filtered. after that, few drops of mayer’s solution was individually added to each plant extract in the test tube. the creamy white precipitates were observed in both extracts which indicates the presence of alkaloids in fig. 1(d) 18. amidine identification both extracts (0.5g) were separately mixed in 3ml of ammonium hydroxide. benzene was added to the reaction medium in test tubes. lack of reddish color indicated the absence of amidine in the both extracts.19 phlobatannins identification for the detection of phlobatannins, 0.5g of both extracts was mixed with distilled water in the separate test tubes, followed by filtration. after this, 3% hcl was added and boiled. absence of reddish color suggests the absence of phlobutannins in both plants.19 terpenoid identification for this purpose, 0.3g from both extracts was added to 2ml chloroform and filtered into the test tube. after that, 3ml of sulphuric acid was introduced resulting in the layer formation followed by the appearance of reddish brown color, which shows the positive result for terpenoids. we have observed that each plant extract possesses terpenoids see fig. 1(c).19 phytochemical analysis & antibacterial activity of tobacco plants vol.12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 63 fig 1. raw extracts of nicotiana tabacum(a) and nicotiana rustica (b). after the addition of specific reagents, change in color indicates the presence of tannins (c), alkaloides (d), terpenoides (e), saponins (f), reducing sugars (g), steroids (h) and flavonoids (i). saponins identification for the identification of saponins, 0.4g of both extracts was mixed with distilled water separately. after 10min, the formation of froth and change in color in test tube b indicated the presence of saponins as manifested in fig. 1(f).19 reducing sugar identification each plant extract (0.4g) was mixed with 2ml of distilled water in test tube and then filtered. after that, fehling solution a & b were introduced and boiled for 8min. according to the literature, the presence of precipitates in orange-red color is the indication of reducing sugars which was absent in these species see fig. 1(g).19 steroid identification for the confirmation of steroids in the selected plant species, the acetic anhydride (3ml) was mixed with both plant extracts separately and then filtered into the test tubes, followed by the addition of 2ml of conc. sulphuric acid. the blue green color was observed in both species, which confirmed the presence of steroids; see fig. 1(h). 19 flavonoids identification about 0.3g from each extract was mixed with dilute naoh, and then hcl was added in test tubes. appearance of yellow color confirmed the presence of flavonoids in the plant extracts. it was observed that both plant species have flavonoids; see fig. 1(i).19 antibacterial activity both the species were subjected to antibacterial activity using agar well diffusion method against staphylococcus aureus and escherichia coli. results showed that the inhibitory effect against staphylococcus aureus was found in the extract of both nicotiana tabacum and nicotiana rustica see fig. 2(b), while no inhibitory effect was observed against e. coli species; see fig. 2(a).19 fig 2. antibacterial activity of nicotiana tabacum and nicotiana rustica against escherichiacoli (a) and staphylococcus aureus (b). c o n c l u s i o n keeping in view the findings of present research work, it can be concluded that like many different plants, quantitative assessment of nicotiana tabacum and nicotiana phytochemical analysis & antibacterial activity of tobacco plants vol.12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 64 rustica plant extracts also indicates the presence of bioactive phytochemicals like flavonoids, alkaloids, steroids, saponins, terpenoids, alkaloids and tannins. interestingly, both plant extracts also confirmed significant antibacterial effect toward staphylococcus aureus, while no significant effect have been observed against escherichia coli. c o n f l i c t s o f i n t e r e s t none. f u n d i n g s o u r c e none. a c k n o w l e d g e m e n t s the authors greatly acknowledge the management of department of chemistry, university of swabi, pakistan for providing research facilities. l i s t o f a b b r e v i a t i o n s gras ki cuso4 mha hcl generally recognized as safe potassium iodide copper sulphate mueller–hinton agar hydrochloric acid h2so4 sulphuric acid naoh sodium hydroxide who world health organization r e f e r e n c e s 1. chen sl, yu h, luo hm, wu q, li cf, steinmetz a. conservation and sustainable use of medicinal plants: problems, progress, and prospects. chinese med. 2016; 11(1):37-45. 2. huang h. plant diversity and conservation in china: planning a strategic bioresource for a sustainable future. bot j linnean soc. 2011; 166(3):282-300. 3. halberstein ra. medicinal plants: historical and crosscultural usage patterns. ann epidemiol. 2005; 15(9):686-99. 4. croteau r, kutchan tm, lewis ng. natural products (secondary metabolites). biochem mol biol plants. 2000; 24:1250-319. 5. sarker sd, nahar l. an introduction to natural products isolation. in natural products isolation, springer: 2012; 1-25. 6. koehn fe, carter gt. the evolving role of natural products in drug discovery. nature rev drug dis. 2005; 4(3):206-20. 7. gordaliza m. natural products as leads to anticancer drugs. clin transl oncol. 2007; 9(12):767-76. 8. yarnell e, abascal k. dilemmas of traditional botanical research. herbalgram. 2002; 55:46-54. 9. jain sk. medicinal plants. national book trust, india.1968; 18-65. 10. samuelson g, bohlin l. drugs of natural origin. a texboot of pharmacognosy. stockholm: swedish pharmaceutical pres. 1992; 1-25. 11. gibbons s. anti-staphylococcal plant natural products. nat prod rep. 2004; 21(2):263-77. 12. rajaei a, barzegar m, mobarez am, sahari ma, esfahani zh. antioxidant, anti-microbial and antimutagenicity activities of pistachio (pistachia vera) green hull extract. food chem toxicol. 2010; 48(1):107-12. 13. wink m. medicinal plants: a source of anti-parasitic secondary metabolites. mol. 2012; 17(11):12771-91. 14. ruma k, sunil k, prakash h. antioxidant, antiinflammatory, antimicrobial and cytotoxic properties of fungal endophytes from garcinia species. int j pharm pharm sci. 2013; 5(3):889-97. 15. li l, shen qp, liu cb, wang y, yao jj, zhang t, et al. isoflavones from the leaves of nicotiana tabacum and their anti-tobacco mosaic virus activities. phytochem let. 2015; 13:156-9. 16. kenton a, parokonny as, gleba yy, bennett md. characterization of the nicotiana tabacum l. genome by molecular cytogenetics. mol gen genetics. 1993; 240(2):159-69. 17. roux dg. some recent advances in the identification of leuco-anthocyanins and the chemistry of condensed tannins. nature. 1957; 180(4593):973-5. 18. preininger v, antavý f. isolation and chemistry of the alkaloids of the genus papaver. 51st report: isolation of the alkaloids from p. bracteatum, p. fugax and p. triniaefolium, and the identification of several earlier isolated alkaloids from plants of the sections orthorhoeades, mecones and pilosa. pharmazie. 1970: 25:356-60. phytochemical analysis & antibacterial activity of tobacco plants vol.12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 65 19. sokunvary oeung, voleak nov, huykhim ung, koemlin roum and et.al phytochemical analysis of different extracts of leaves of nicotiana tabacum l. of cambodia,2017, 1(3):18-26. 20. prof. robert j. lancashire, 2015, http://wwwchem.uwimona.edu.jm/courses/fehling.ht ml 21. fatima tuz zohra,extraction of secondary metabolites, phytochemical screening and the analysis of antibacterial activity in stevia rebaudiana, 2015,pg 18. sd-ldl as risk factor for atherosclerosis in type 2 dm vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 15 r a d s j . b i o l . r e s . a p p l . s c i . 15 op e n ac c e s s f u l l l e n g t h a r t i c l e small dense low-density lipoprotein as risk factor for atherosclerosis in type 2 diabetes mellitus haji muhammad rashid*, hiza hassan, mudassar khan, jamal khan, hasanat khan department of medical lab technology, university of haripur, khyber pakhtunkhwa, pakistan a b s t r a c t background: dyslipidemia is a common complication of diabetes and a major risk factor for atherosclerosis. small dense lowdensity lipoprotein cholesterol (sdldl-c) is a subclass of low-density lipoprotein cholesterol. sdldl-c is highly atherogenic and has been wildly studied in diabetic dyslipidemia. objectives: the study was carried out to explore the hidden risk of atherosclerosis due to variation of sdldl-c in type 2 diabetic patients, whose lipid profiles were normal. methodology: we enrolled 126 t2dm patients and 126 age and sex-matched normal controls in this study. fasting lipid levels in the normal range were the selection criteria for both groups. hemoglobin a1c (hba1c), sdldl-c, triglycerides (tg), total-cholesterol, low-density lipoprotein (ldl) and high-density lipoprotein (hdl) were performed for both groups. results were compared by two samples t-tests. results: sdldl-c levels (54.6 ± 10.2) in t2dm were significantly higher than the control group (41.6 ± 8.4) with p-value <0.00001. there was a strong positive correlation (r2 = 0.591) between hba1c and sdldl-c of t2dm patients. conclusion: t2dm patients even with normal lipids profile are at risk of atherosclerosis due to a high level of atherogenic sdldl-c. keywords small dense ldl-cholesterol, t2dm, dyslipidemia, cardiovascular diseases, lipids profile. *address of correspondence 4849487@gmail.com article info. received: august 09, 2020 accepted: august 22, 2020 cite this article: rashid hm, hassan h, khan m, khan j, khan h. small dense low-density lipoprotein as risk factor for atherosclerosis in type 2 diabetes mellitus. rads j biol res appl sci. 2020; 11(1):15-18. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n diabetes mellitus (dm) is one of the most prevalent disease which is characterized by hyperglycemia and divided into two broad categories i.e. type 1 dm & type 2 dm. type 1 dm is characterized by an absolute deficiency of insulin, while type 2dm is associated with relative insulin deficiency or insulin resistance1. type 2 dm is the most prevalent form of diabetes in adults and it induces many complications; diabetic dyslipidemia being one of them2, with the prevalence of 95% in t2dm patients3. the trend of increase in the plasma concentration of triglyceride (tg), total cholesterol, lowdensity lipoprotein and decrease in high-density lipoprotein is known as diabetic dyslipidemia4, which is considered to be highly atherogenic than any other type of dyslipidemias due to a predominance of small dense low-density lipoprotein cholesterol (sdldl-c)5. based on their density, low-density lipoprotein cholesterol (ldl-c) is further divided into two subclasses i.e. large buoyant ldl-c (lbldl-c) and small dense ldl-c (sdldl-c). sdldl-c is highly atherogenic than lbldl-c6. elevated levels of sdldl-c are closely associated with coronary artery disease (cad) and it is an accepted risk factor for o r i g i n a l a r t i c l e sd-ldl as risk factor for atherosclerosis in type 2 dm vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 16 r a d s j . b i o l . r e s . a p p l . s c i . 16 cad by national cholesterol education program7. sdldlc has high atherogenic potential due to many various mechanisms like lower affinity for ldl receptors, high penetration effects in arterial walls, high susceptibility for oxidation, and high arterial retention. these sdldl particles are small in size and contain less cholesterol. a high level of sdldl-c provides increased numbers of atherogenic particles8. in t2dm, there is a possibility to develop cad without the increase in ldl-c9. ldl-c is one of the major risk factors for cad as it is the primary target for cad risk reduction therapies10. it has been observed that not only elevated ldl-c is atherogenic, but the changes in its composition and variations in the concentration of its subclasses may also induce atherosclerosis even when ldl-c is in its normal ranges11. the purpose of the present study was to compare sdldlc and lipids parameters between diabetic and control group to explore the hidden risks of atherosclerosis in type 2 diabetic patients with normal lipid profile. m a t e r i a l s a n d m e t h o d s this study was conducted from jan 2018 to jan 2019 at the pathology lab of asia diagnostic, islamabad. a total of 565 t2dm patients were screened for their lipid profile in fasting. among them, 126 t2dm patients with normal lipid profile and 126 age and sex-matched healthy individuals with normal lipid profile were enrolled as a control group. the lipid profile of t2dm and control group was measured by spectrophotometric method, while hba1c was measured with an immunofluorescence point of care testing device. small dense ldl-c was measured with a spectrophotometric method, using the commercial kit12. r e s u l t s after initial screening, a total of 126 t2dm patients and 126 healthy controls were enrolled in this study. gender wise distribution is given in table 1. although the inclusion criteria for both groups were normal lipids profile in fasting, however, to determine the variation of lipid parameters within the normal range, the comparison of all lipids profile parameters (like tg, total cholesterol, hdl, and ldl) between t2dm and healthy controls was performed by two-sample t-test (table 2). comparison of mean of sdldl-c between t2dm and healthy control was also performed by two samples-test (table 2). it was observed that all lipid parameters of t2dm had a significant difference from the lipid parameters of healthy controls. small dense ldl-c was significantly higher in t2dm than in a control group with a p-value <0.0001. table 1. gender-wise frequency distribution of subjects. s. no. subjects males females total 01 diabetic group 72 54 126 02 control group 68 58 126 table 2. comparison of triglyceride, total-cholesterol, hdl, ldl and sdldl-c between diabetic and control groups. s. no. subjects triglyceride total-cholesterol hdl ldl sdldl-c 01 diabetic group 137 ± 11 169 ± 18 43.9 ± 4.5 98 ± 17 54.6 ± 10.2 02 control group 122 ± 17 154 ± 17 47.25 ± 5.5 82 ± 16 41.6 ± 8.4 03 p value <0.00001 <0.00001 <0.00001 <0.00001 <0.00001 table 3. correlation of lipids profile parameters and sdldl-c with hba1c in t2dm. s. no. parameters triglyceride total-cholesterol hdl ldl sdldl-c 01 r2 0.516485 0.46069 0.298692 0.38454 0.591631 sd-ldl as risk factor for atherosclerosis in type 2 dm vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 17 r a d s j . b i o l . r e s . a p p l . s c i . 17 in the t2dm group, correlation of tg, total cholesterol, hdl, ldl and sdldl-c with glycated haemoglobin (hba1c) was performed by pearson correlation as shown in table 3. from lipid profile, total cholesterol, ldl and tg showed a positive correlation while hdl showed a negative correlation with hba1c. small dense ldl-c levels were also positively associated with hba1c, but this correlation (r2= 0.591) was stronger than lipid parameters. d i s c u s s i o n in our study, we observed that sdldl-c and all parameters of lipid profile in t2dm subjects were significantly higher with p-value <0.001 than the normal controls, except that hdl which was markedly low in t2dm with p-value <0.001. we also found a positive correlation between hba1c and lipid parameters except for hdl, which was negatively correlated with hba1c levels of t2dm subjects. a strong positive correlation was observed between hba1c and sdldl-c. although our study was limited for the t2dm subjects with normal lipid profile, within normal ranges, we observed the same pattern of changes in lipid profile parameters as it can be observed in diabetic dyslipidemia, which is associated with high levels of tg, total cholesterol, ldl and low levels of hdl along with predominant sdldlc13,14. high levels of sdldl-c in t2dm is a major complication of diabetes that can induce atherosclerosis in diabetic patients6,15,16. in the present study, high sdldlc levels indicate that t2dm patients may have atherogenic risk despite the lipid profile within a normal range. diabetic dyslipidemia due to high tg, total cholesterol, ldl and low hdl levels were observed in various studies17,18. glycemic status of t2dm patients contributes to the severity of diabetic dyslipidemia19 and tg has a strong positive association with hba1c20,21. within normal ranges, the same type of relationships was found in our study. t2dm with increased tg levels show a greater capacity to induce sdldl-c production, which may contribute to the high incidence of coronary artery disease (cad) in the diabetic population22. sdldl also induces thickness in intima-media of blood vessels and numbers of sdldl particles are associated with insulin resistance in t2dm22. glycation rates are associated with increased atherosclerosis, morbidity and mortality in cardiovascular diseases (cvd)6. in this regard, our study (which did not include any dyslipidemic patient) demonstrated that there is a strong positive correlation between glycemic status and sdldl-c in t2dm patients that may contribute for the induction of atherosclerosis and may lead towards cvd. c o n c l u s i o n form this study, it was concluded that all lipid parameters in t2dm patients with normal lipid profile show their trend in favour of diabetic dyslipidemia. moreover, the presence of high sdldl-c in normal lipids profile of t2dm patients is a strong risk factor for cad. f u t u r e p e r s p e c t i v e lipid-lowering therapy should be given to t2dm patients even having lipid parameters in normal reference ranges and sdldl-c should be incorporated in lipid profile as an integrated parameter of lipid profile as now it is available on commercial diagnostic kits. a c k n o w l e d g e m e n t s i would like to thank ceo of asia diagnostic center islamabad for moral, financial and technical support. l i s t o f a b b r e v i a t i o n cad coronary artery disease cvd cardiovascular disease dm diabetes mellitus hba1c hemoglobin a1c hdl high density lipoprotein ldl low density lipoprotein sdldl small dense low-density lipoprotein tg triglyceride r e f e r e n c e s 1. a ali. determination of plasma fibrinogen level in diabetic patients in shendi town: omkalthoum osman hamad; 2018; 1-59. 2. trikkalinou a, papazafiropoulou ak, melidonis a. type 2 diabetes and quality of life. world j diabetes. 2017; 8(4):120-9. sd-ldl as risk factor for atherosclerosis in type 2 dm vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 18 r a d s j . b i o l . r e s . a p p l . s c i . 18 3. abdel-aal nm, ahmad at, froelicher es, batieha a, hamza mm, ajlouni km. prevalence of dyslipidemia in patients with type 2 diabetes in jordan. saudi med j. 2008; 29(10):1423-8. 4. pontrelli l, parris w, adeli k, cheung rc. atorvastatin treatment beneficially alters the lipoprotein profile and increases low-density lipoprotein particle diameter in patients with combined dyslipidemia and impaired fasting glucose/type 2 diabetes. met cli exp sci. 2002; 51(3):334-42. 5. bando y, toyama h, kanehara h, hisada a, okafuji k, toya d, et al. switching from atorvastatin to rosuvastatin lowers small, dense low-density lipoprotein cholesterol levels in japanese hypercholesterolemic patients 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atherosclerosis rep. 2016; 18(1):2-14. 11. bandyopadhyay d, qureshi a, ghosh s, ashish k, heise lr, hajra a, et al. safety and efficacy of extremely low ldl-cholesterol levels and its prospects in hyperlipidemia management. j lip. 2018; 4:13-23. 12. renjith r, jayakumari n. a simple economical method for assay of atherogenic small dense lowdensity lipoprotein-cholesterol (sd-ldl-c). indian j clin biochem. 2011; 26(4):385-8. 13. joshi sr. saroglitazar for the treatment of dyslipidemia in diabetic patients. exp opin pharmacoth. 2015; 16(4):597-606. 14. zuhroiyyah sf, sukandar h, sastradinanja sb. the relationship between physical activity and total cholesterol levels, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol in the jatinangor community. j health sys. 2017; 2(3):2332. 15. khalil rmaz, al-azab dam, akl oa. is sd-ldl a valuable screening tool for cardiovascular disease in patients with metabolic syndrome? alex j med. 2017; 53(4):299-305. 16. khan m. small dense ldl: new marker for cardiovascular risk assessment and its therapeutic inflection. biochem anal biochem. 2012;1(6):1-4. 17. rawal jr, mehelli irani p. atherogenic dyslipidemia. indian j endocrinol metabol. 2013; 17(6): 969-76. 18. mooradian ad. dyslipidemia in type 2 diabetes mellitus. nature rev endocrinol. 2009; 5(3):150-9. 19. taskinen m-r. diabetic dyslipidaemia: from basic research to clinical practice. diabetol. 2003; 46(6):733-49. 20. alzahrani sh, baig m, aashi mm, al-shaibi fk, alqarni da, bakhamees wh. association between glycated hemoglobin (hba1c) and the lipid profile in patients with type 2 diabetes mellitus at a tertiary care hospital: a retrospective study. diabetes, metabolic syndrome and obesity: targets and therapy. 2019; 12:1639-48. 21. rodriguez-gutierrez r, mancillas-adame lg, rodríguez-tamez g, diaz gonzalez-colmenero a, solis-pacheco rc, elizondo-plazas as, et al. hypertriglyceridemia and its association with hba1c test: a prospective in vivo controlled study. int j endocrinol. 2019; 1-7. 22. shen h, xu l, lu j, hao t, ma c, yang h, et al. correlation between small dense low-density lipoprotein cholesterol and carotid artery intima-media thickness in a healthy chinese population. lipids health dis. 2015; 14(1):137-43. review effect of caffeine overdose vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 154 op e n ac c e s s f u l l l e n g t h a r t i c l e review effect of caffeine overdose zaheer ahmed chandio1,*, aaisha sidiqua1, mazhar iqbal khaskheli2, amrat waghani1, wazir ali metlo3 1department of chemistry, shaheed benazir bhutto university, shaheed benazirabad, pakistan. 2m. a. kazi institute of chemistry, university of sindh, jamshoro, pakistan. 3department of molecular biology and genetics, shaheed benazir bhutto university, shaheed benazirabad, pakistan. a b s t r a c t background: caffeine is from methylxanthine class which mainly stimulates the central nervous system. caffeine is one of the most widely used psychoactive drugs in the world. it is commonly found in beverages, chocolates, cocoa containing products and in medications. caffeine is recognized as legal and its consumption is unregulated all over the world. objectives: the objective of this study is to summarize the impact of caffeine on behavioral and health alterations, in both controlled and overdose conditions. moreover, the specifics of caffeine withdrawal and a number of guidelines on how to handle reducing or quitting caffeine intake altogether is also highlighted. methodology: to compose this review, more than seventy research and review articles were overviewed that were published over a period of the last twenty years, using google scholar search engines. results: caffeine regular use causes physical dependence which may become the caffeine withdrawal sign that can consequently harm normal working. the most important function of caffeine is that it can reversibly block the adenosine performance on its receptor and as a result which prevents the beginning of drowsiness encouraged by adenosine. caffeine also stimulates certain portions of the autonomic nervous system symptom including fatigue, drowsiness, depressed mood, headache, difficulty concentrating, decreased energy, decreased contentedness, decreased alertness, irritability and unclear headed. conclusion: it is concluded that utilization of caffeine in a prescribed dose can have a good impact on health and may decrease addictive symptoms. additionally, reducing caffeine dosage over a six-week period guide to successful, long-term caffeine cessation with very few side effects. keywords caffeine, beverages, headache, fatigue, insomnia, anxiety *address of correspondence zaheerchandio1@yahoo.com article info. received: january 5, 2020 accepted: november 1, 2020 cite this article: chandio za, sidiqua a, khaskheli mi, waghani a, metlo wa. review effect of caffeine overdose. rads j biol res appl sci. 2020; 11(2):154-158. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n caffeine is considered to be the most widely used psychostimulant drugs throughout the world. chemically, it is an alkaloid belonging to family methylxanthine. its chemical and systematic name is 1,3,7-trimethylxanthine. in its pure form, caffeine is white glistening needle or powder, intensely bitter and fleecy1. caffeine can be found in various products, but a majority of caffeine is available in caffeinated drinks, tea, coffee and energy drinks. caffeine is naturally found in chocolate but it is also added by the manufacturers to some foods and candies. medications like flu and cold tablets may also contain some amount of caffeine. its supplements are easily available in markets in the form of powder or tablets. this form contains far more amount of caffeine as compared to coffee and therefore, it becomes difficult for people to measure how much caffeine they have already taken. caffeine is also naturally present r e v i e w a r t i c l e review effect of caffeine overdose vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 155 in variable amounts in the leaves, beans and fruits of over sixty plants. caffeine has turned the entire world to it and is on the top of the list of beverages which are sold regularly2. apart from this regular intake of caffeine, it is considered to be dangerous for humans. taking a cup of coffee daily all through the year i.e., for 365 days and testing your blood on the 366th day will measure 3.03% of caffeine toxins or poisons that could, kill a person if its percentile reaches above 10%3,4. caffeine may have both positive and negative health effects. literature reveals that caffeine plays a very important role in protecting the liver from cancer when consumed in moderate amount5. another review indicated that taking 2-4 cups of coffee regularly reduces the risk of death from almost all the causes. recent studies have connected caffeine use with good effects even on the brain6. a study published in 2013, suggested that taking 24 cups of coffee every day can enhance long term memory, furthermore, it can also decrease suicide risk in adults who consume a moderate amount of caffeine7,8. caffeine containing beverage consumption has been reported to be associated with reduced bone mass and increased fracture risk in some, but not most, observational studies9. a study also claims that regular caffeine intake can protect an individual against type 2 diabetes, cardiovascular disease, stroke and parkinson's disease10. on the other hand, ingesting a high amount of caffeine may lead to more adverse health effects. the results of a study suggested that using 300mg of caffeine per day during pregnancy can raise the risk of a baby having a low birth weight11. an article identified lots of unfavourable effects when consumption of caffeine is over 400 mg per day, including anxiety, sleeping problems, fast heartbeat, restlessness, agitation and tremors12. the lethal dose of caffeine for adults has long been estimated to be approximately 10 g. characteristic symptoms preceding death in confirmed cases of caffeine fatality include abdominal pain, vomiting, markedly increased heart rate, cardiac arrhythmia, agitation, and seizures. risk of fatal and non-fatal harm due to caffeine poisoning is increased by several characteristics of the drug and the circumstances surrounding its use, including its generally unregulated availability to children and adults alike, and rapid absorption and distribution within the body. additionally, caffeine effects are dose-dependent when ingested in amounts that exceed typical dietary levels, and its effects may be additive or synergistic when ingested concomitantly with a range of other compounds including pharmaceuticals and recreational drugs13. the reports of death caused by caffeine high dose are usually ventricular fibrillation. this situation occurs if lower chambers of heart vibrate rather than contracting frequently. during ventricular fibrillation, the heart stops beating from average, which results in cardiac arrest14. in another review, a researcher reported 92 deaths from caffeine overdose15. it was believed that one-third of these deaths were probably suicidal. usually, caffeine affects the body when its concentration is more than 15mg/l in the blood. however, if the amount exceeds 80 to 100 mg/l it could be deadly16. overdose of caffeine usually occurs by taking caffeine tablets or dietary supplement rather than taking coffee; mainly people combine these products with coffee, energy drinks or sodas17. a supplement raises the risk because caffeine levels become higher than foods and drinks. consumption of purified caffeine is highly dangerous and is likely to cause the increase in blood caffeine level. the us fda warns that one teaspoon of powdered caffeine is almost equivalent to twenty-eight cups of coffee. the fda stresses that highly concentrated and pure caffeine products can cause serious health hazards. a question arises why do people usually get addicted to caffeine? caffeine belongs to methylxanthines which is structurally similar to tryptophan (try). tryptophan is considered to be a neurotransmitter secreted in cases such as orgasm, when a person is in love, hallucinated to be happy or in coitus18, 19. therefore, when a person generally takes chocolate or caffeine, they also usually contain some amount of tryptophan and which acts as a precursor to serotonin. serotonin is a known neurotransmitter which has the mood-enhancing property whilst high serotonin levels cause the elation effects. phenylethylamine is a derivative of amphetamine, which is identified for love and attraction, therefore, these are also released when chocolates and caffeine are ingested20. https://www.medicalnewstoday.com/articles/172408.php https://www.tandfonline.com/doi/abs/10.3109/15622975.2013.795243 https://www.tandfonline.com/doi/abs/10.3109/15622975.2013.795243 https://www.medicalnewstoday.com/info/diabetes/type2diabetes.php https://www.medicalnewstoday.com/articles/7624.php https://www.medicalnewstoday.com/info/parkinsons-disease/ https://www.medicalnewstoday.com/info/anxiety/ review effect of caffeine overdose vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 156 s y m p t o m s o f c a f f e i n e o v e r d o s e headache people have been using caffeine for the treatment of migraine and common pain from a long time because of its unique ability to decrease blood flow, particularly in the brain. though, consuming caffeine frequently may also cause a headache. quitting or reducing caffeine after long term use can cause severe migraine-like headache in some persons. because it is both soluble in water and fat caffeine quickly crosses the blood-brain obstruction where it contracts the blood vessels. narrowing the blood vessels may cause a reduction in blood flow, which is very helpful to overcome migraine pain. quitting or reducing caffeine dose rapidly will let the blood vessels to grow unexpectedly, which an increase in the blood flow. this sudden striking increase in blood flow may cause severe pain, throbbing headaches just like in the case of migraine. such headaches are caused because of the withdrawal of caffeine and can vary in time length and severity. usually, people use caffeine for the treatment of such headaches; however, it should be kept in mind that they do not use more caffeine than previously consumed. headaches will be subsided gradually as the brain gets used to the change in the blood flow. fatigue mostly in the morning, people use caffeine to improve their alertness levels. by blocking adenosine receptors in the brain, caffeine prevents fatigue and enhances alertness. adenosine is a neurotransmitter which slows down the central nervous system when the body gets ready to sleep. nevertheless, when someone rapidly stops or lowers the caffeine dosage, they can have temporarily reverse effect and which feels more tiredness throughout the day. a person can overcome this feeling of tiredness during the day by enhancing his/her sleep throughout the night. changes in mood regularly, low consumption of caffeine may also cause alertness in mood and also diminishes feelings like anxiety. however, using reasonable to high level dosage of caffeine may cause feelings like jitteriness, nervousness and anxiety21. such mood changes are caused because of the effect which caffeine has on a variety of neurotransmitters. these include norepinephrine, glutamate and dopamine. dopamine has the characteristics of activating pleasure centers which are present in the brain and responsible for controlling behaviours and emotions. a study showed that caffeine use is not only stimulating dopamine production, but it can increase the available number of dopamine receptors present in the brain, which overall enhance dopamine's effect on the brain22. glutamate supports communication between nerve cells and plays a very important role during learning and memory. the norepinephrine is produced in the brain when a person feels stress or danger in a process known as "fight-orflight" reaction. norepinephrine boosts breathing rate, heart rate and blood glucose levels. quickly leaving caffeine may cause drastic effects on the chemical composition in the brain, results in the feelings like depression, anxiety or irritability. difficulty in concentrating caffeine can interact with certain chemicals present in the brain therefore, it can affect concentration level and memory. a study reveals that taking just 80 mg of caffeine dose results in the up-gradation in working memory and provides quick response time in humans23. findings recommend that regular use of caffeine can lower the risk level of dementia or cognitive impairment in old aged women24. people usually experience obscurity in concentration when suddenly quitting caffeine intake. in unavailability of caffeine, molecules of adenosine can encourage feelings like fatigue which may affect the capacity to concentrate. constipation caffeine stimulates contractions in the intestines and colon. these contractions assist the food to move fast and waste substances through the gastrointestinal tract. the regular consumption of caffeine may face mild constipation when they reduce the caffeine dose. people can prevent this problem by regularly taking fiber rich foods and staying hydrated maximum level25. review effect of caffeine overdose vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 157 c o n c l u s i o n people can be able to prevent caffeine withdrawal symptoms by slowly reducing their caffeine dose over time. progressively reducing caffeine dosage over a six-weeks period guides to successful, long-term caffeine cessation with very few side effects. caffeine quitting may cause dramatic changes to brain chemistry, which may affect a cognitive ability and a person's mood. people who regularly drink coffee can slowly decrease their caffeine dosage by mixing some amount of decaf into their coffee. those people who take multiple cups can aim to replace one or more with decaf. maximum sleep also supports to fight against fatigue and feeling well-rested will also support to minimize the body's reliance on caffeine. the remaining hydrated entire day is necessary since headaches and fatigue are also experienced due to dehydration. c o n f l i c t s o f i n t e r e s t none. f u n d i n g s o u r c e none. a c k n o w 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stimulation of defecation: effects of coffee use and nicotine on rectal tone and visceral sensitivity. scan j gastroenterol. 2005; 40: 808-13. https://www.nature.com/articles/tp201546#auth-1 https://www.nature.com/articles/tp201546#auth-2 https://www.nature.com/articles/tp201546#auth-3 https://www.nature.com/articles/tp201546#auth-4 https://www.nature.com/articles/tp201546#auth-5 https://www.nature.com/articles/tp201546#auth-6 https://www.nature.com/tp https://www.nature.com/tp https://www.ncbi.nlm.nih.gov/pubmed/?term=benson%20s%5bauthor%5d&cauthor=true&cauthor_uid=30625179 https://www.ncbi.nlm.nih.gov/pubmed/?term=tiplady%20b%5bauthor%5d&cauthor=true&cauthor_uid=30625179 https://www.ncbi.nlm.nih.gov/pubmed/?term=scholey%20a%5bauthor%5d&cauthor=true&cauthor_uid=30625179 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6326409/ https://www.ncbi.nlm.nih.gov/pubmed/?term=driscoll%20i%5bauthor%5d&cauthor=true&cauthor_uid=27678290 https://www.ncbi.nlm.nih.gov/pubmed/?term=shumaker%20sa%5bauthor%5d&cauthor=true&cauthor_uid=27678290 https://www.ncbi.nlm.nih.gov/pubmed/?term=snively%20bm%5bauthor%5d&cauthor=true&cauthor_uid=27678290 https://www.ncbi.nlm.nih.gov/pubmed/?term=margolis%20kl%5bauthor%5d&cauthor=true&cauthor_uid=27678290 https://www.ncbi.nlm.nih.gov/pubmed/?term=manson%20je%5bauthor%5d&cauthor=true&cauthor_uid=27678290 https://www.ncbi.nlm.nih.gov/pubmed/?term=manson%20je%5bauthor%5d&cauthor=true&cauthor_uid=27678290 https://www.ncbi.nlm.nih.gov/pubmed/?term=vitolins%20mz%5bauthor%5d&cauthor=true&cauthor_uid=27678290 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5106861/ introduction traditional method of teaching is prone to inefficient delivery. it is practiced by teaching the context without any meaningful learning outcome. the learner would not be able to relate the existing knowledge with the previous information on the subject and thus would be unable to apply the knowledge in a purposeful way. it is therefore desired to deliver the context through connectivity in a meaningful, easier and thoughtful way. concept mapping is a methodology for the delivery of facts, concepts and skills in one package. it makes teaching and learning; easier and purposeful. teaching and learning through connectivity is a basic idea of teaching by an arrangement of concepts or issues through interacting systems in which all relationships between concepts and issues are made clear, up front, to the leaner using a concept map-like representation. it involves establishing a hierarchy of concepts striving for underscoring a more or less closed system of concepts to clarify the interrelationships among concepts. lesson modeled on the basis of connectivity diagrams help to overcome the traditional snags. to ausubel (ausubel, 1963), meaningful learning is a process in which new information is related to an existing relevant aspect of an individual’s knowledge structure and which, correspondingly, must be the result of an overt action by the learner. teachers can encourage this choice by using tools such as concept maps. some theories postulate that continued learning of new information relevant to the previous information produces constructive changes. michael (michael, 2001), stated that meaningful learning occurs when the learner interprets, relates and incorporates new information with existing knowledge and applies the new information to solve novel problems. meaningful learning presupposes that the learner has a disposition to relate the new materials to his or her cognitive structure and that new material will be potentially helpful for the learner. concept mapping is a device that can be used to communicate to the learner as well as providing a vehicle to help the learner with meaningful learning tasks. it provides the basis of relating new knowledge to previously assimilated knowledge in a systemic way (naqvi, et al., 2012). concept mapping also incorporates a strong element of constructivism; in the sense that a student can teaching and learning through connectivity part-iv: a model lesson for teaching metabolism iftikhar imam naqvi1, shaima hasnat2* 1 department of chemistry, jinnah university for women, karachi-74600 pakistan. 2 department of biochemistry, jinnah university for women, karachi-74600 pakistan. abstract teaching without learning and inculcating creativity is a source of boredom for the students and ends up with traditional rote learning without any knowledge enhancement to the student. through our this effort we aim at changing the scenario of teaching entirety. concept mapping or teaching through connectivity is one of such steps that help the teacher and the learner in making the lectures easier, motivating and constructive. in this model lesson, we tried to make metabolic process and its interrelated variables simple and understandable. keywords: teaching, students, chemistry, conectivity. *corresponding author: shaima.hasnat@gmail.com 17 vol 4 (1), january 2013; 17-19 build his/her understanding of new concepts on that which he/she already has a deep familiarity. a number of issues pertaining to chemistry have been thus addressed in our previous discourses (nazir and naqvi, 2011; shazia and naqvi, 2002). a lesson model for teaching biochemistry has been developed and presented herein. “metabolism” is one of the most difficult and conceptually hard contexts of biochemistry which often causes the student to get confused or withdraws them. it involves different terms which themselves have to have a detailed mapping of their own like organs, enzymes, biomolecules, energy, nutrition or diet and nutritional states (fed/starvation) etc. figure 1 outlays the related material in a linear way. from here we redirect our presentation such that teaching metabolism through concept mapping like that in figure 2 makes it easier for the students and it will provide them meaningful and purposeful outlays. figure 2 highlights the connectivity of metabolism with the each of the stake holders that contribute to the thought process behind this subject of vital importance. students usually have previous knowledge regarding organs, diet, and energy. however, they may or may not have concepts developed enough to approach enzymes and their functions in the body, formation of biomolecules and the response of body in different biological states (fed/starved). in figure 2, these vital contributors towards the life process are indicated and highlighted for ensuring a lively classroom discussion. through this connectivity diagram the student understand metabolism better and let him/her delve into the beneficial effects leading towards a better comprehension. we expect that when taught this way, student will be able to appreciate the role of each of the identified components that controls metabolic processes. the important issues related to metabolism are expressed through a concept diagram as shown in figure 2. he/she will be able to understand how our body organs work, where different enzymes are located and how they perform their actions, what are biomolecules and what are their nutritional importance in the diet, they will also appreciate the route through which the body can get necessary energy when taking particular amount of biomolecules in the diet, further to that student will be able to understand effects of nutritional status (fed/starvation) on our body organs. through the 18 vol 4 (1), january 2013; 17-19 figure 1: linear inputs figure 2 discussion involving interconnectivity of the individual unfamiliar contents on the face value, students will be able to grasp metabolism. following the discussion on this pattern the connectivity diagram (figure 2) is modified as in figure 3. this amply illustrates the correlation of salient variables that relate to the metabolism and their interrelationship. clarifying all the individual contributors to the metabolism by the teacher through this connectivity diagram will let the students to correlate each concept with the other and hence they will be able to understand and value each of the individual parameter that is important for the regulation of metabolic processes in the body. this will also help the student to recognize that when a person takes in diet or in the fed state; organs will respond by activating particular enzymes which act on different biomolecules present in the diet and provide energy & also initiate anabolic process of metabolism. the students also grasp the fact that in starvation condition the organs of our body respond differently by activating different enzymes leading to the catabolic processes of biomolecules to provide energy to the body. summary the implementation of teaching through connectivity assists to deliver the concepts of underlying metabolic process in an effective and meaningful way. concept mapping provides a better understanding of the basics of metabolic reactions, their importance in the regulation of body’s function and in understanding the associated diseases developing out of improper metabolic reaction. this teaching technique will open new thinking approach and develop interest in students, they will not get confused and worried of learning. teaching through connectivity will let the comprehension of basic concepts in an excellent way and students will be able to correlate it to the related issues and will be able to clarify them at a glance. references ausubel, d. p. (1963). the psychology of meaningful verbal learning; grune and stratton: new york. michael, j. (2001). adv. physiol. educ. 25, 145–158. naqvi i i., kanwal g. and shafi a. (2012). teaching undergraduate students through connectivity: part 3, rads j. biol. res. appl. sci., 3 (2): 18-20. nazir, m. and naqvi, i i. (2011). teaching undergraduate students through connectivity, rads j. biol. res. appl. sci., 2 (2); 07-10. shazia, s and naqvi, i i. (2012). teaching undergraduate students through connectivity: part 2, rads j. biol. res. appl. sci., 3 (1): 07-08. 19 vol 4 (1), january 2013; 17-19 figure 3 study of the modulating interactions of multitrait rhizobacteria vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 44 r a d s j . b i o l . r e s . a p p l . s c i . 44 op e n ac c e s s f u l l l e n g t h a r t i c l e study of the modulating interactions of multitrait rhizobacteria using zea mays l. as the host plant sana shakeel, ambreen ahmed*, ifrah javaid department of botany, university of the punjab, quaid-e-azam campus, lahore 54590, pakistan a b s t r a c t background: rhizosphere is a soil region closest to roots of the plants inhabiting different types of microorganisms including rhizobacteria. chemical fertilizers which are conventionally used for increasing crop production are dangerous in terms of minimizing the nutritional value of crops and may also be hazardous for biological agents. therefore, the use of plant growth promoting rhizobacteria (pgpr) are favorable for improved crop production over chemical fertilizers. objectives: the current study highlights the growth promoting traits of bacterial isolates through isolation of rhizospheric bacterial strains from different plants. methodology: in this study, ten rhizospheric bacterial isolates were used, which were morpho-physiologically characterized and then tested for plant growth-promoting traits i.e., hcn production, ammonification and auxin production. most of the bacterial strains gave positive results for these plant growth-promoting traits. to study the beneficial effects of these bacteria on plants, plantmicrobial interaction assay was conducted using zea mays. results: results revealed that these bacteria enhanced the growth as compared to control plants. bacterial isolates streptomyces lydicus (cn6), staphylococcus aureus (cn7) and bacillus pumilus (pp3) showed strong ammonia producing effects. the isolates bacillus subtilis (cn2), pp2 and pp5 exhibited strong potential of hcn production whereas only streptomyces lydicus (cn6) and bacillus pumilus (pp3) were observed to be auxin producers. a maximum increase in fresh weight of the plants was observed in treatment with pp2 showing 94.36% increase over controls. cn1 showed an increase (26.12%) in shoot length while cn5 revealed a prominent increase (64.95%) in root length compared to the control plant. the isolates cn5 and cn4 showed improvement in the total chlorophyll content of the treated plants with a percentage increase of 100% and 99.82%, respectively compared to the control. conclusion: in conclusion, these pgpr may be further used in agriculture research for growth improvement. keywords rhizobacteria, pgp, plant-microbial interaction assay, pgpr, zea mays. *address of correspondence ambreenahmed1@hotmail.com article info. received: october 10, 2018 accepted: october 30, 2019 cite this article: shakeel s, ahmed a, javaid i. study of the modulating interactions of multitrait rhizobacteria using zea mays l. as the host plant. rads j biol res appl sci. 2019; 10(2):44-53. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n the relationship between species is frequently defined via the influence of associations and interactions among species. plants live in an association with a rich variety of microorganisms throughout their development. these associations relate to plant-microbe interactions from mutualism to parasitism, on account of the role of symbionts1. different groups of bacteria and fungi interact with higher plants. while studying plants and microbes, rhizosphere is a critical segment of soil. in plants, the rhizosphere is a multifarious and diverse environment occupied by numerous microbes2. the root colonizing bacteria are known as rhizospheric bacteria3. different species of rhizobacteria that flourish in the rhizosphere are able to stimulate plant growth by different o r i g i n a l a r t i c l e study of the modulating interactions of multitrait rhizobacteria vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 45 r a d s j . b i o l . r e s . a p p l . s c i . 45 mechanisms and are collectively known as plant growthpromoting rhizobacteria/rhizospheric bacteria (pgpr)4. in the past few years, the number of pgpr that has been recognized has shown a significant increase, largely because the part of rhizosphere in an ecosystem has attained prominence in the working of the biosphere. many species of bacteria like azospirillum, azotobacter, pseudomonas, enterobacter, serratia, klebsiella, burkholderia, alcaligenes, arthrobacter and bacillus species have been testified to increase plant growth5. whenever the roots are inoculated with a pgpr, reduced growth rate of the primary roots and increased number and/or length of lateral roots may be observed6. accordingly, through improved uptake of water and minerals, the growth of the whole plant can be augmented. a number of these effects, including augmented root and shoot biomass, are also acknowledged for pgpr-inoculated plants growing in soil7,8. pgpr amend rsa and the assembly of roots, chiefly via their capability to meddle with the plant hormonal balance9. pgpr utilization meets the current need to lessen nitrogen input to achieve better sustainability in the production of crops, predominantly cereals10. maize (zea mays l.) is one of the major crops all over the globe reserving the mounting claim for food and cultivated animals feed11. exhaustive corn production requires broad consumption of pesticides as well as chemical fertilizers, which have harmful effects on the soil, water and on the food chain. it has been verified that although chemical fertilizers improve the crop yield, they cause toxic effects too, for instance soil acidification as well as production of the greenhouse gas, nitrous oxide (n2o) by denitrification on ecosystems12,13. it has been reported that one possible mode to reduce the harmful environmental impacts ensuing from continuous utilization of chemical fertilizers is inoculation with biofertilizers such as pgpr14. several findings have revealed that pgpr are latent growth enhancers in numerous crops15. the present study deals with four already isolated and identified bacterial strains [bacillus subtilis (cn2), streptomyces lydicus (cn6), staphylococcus aureus (cn7) and bacillus pumilus (pp3)] and isolation of six rhizospheric bacterial strains from plants convolvulus arvensis and polygonum plebium collected from balochistan, highlighting the growth promoting traits of bacterial isolates. these bacterial isolates were then used to inoculate the seeds of zea mays and various parameters of treated and non-treated plants were studied regarding plant growth improvement. m a t e r i a l s a n d m e t h o d s sample collection for the isolation of bacteria, convolvulus arvensis and polygonum plebeium plants were collected from the zhob district of balochistan (table 1). isolation and characterization of bacteria six bacterial strains were isolated from the rhizosphere of the collected plant using the serial dilution method (table 1). the isolates were routinely grown on l-agar (luria agar) or l-broth (luria bertani broth). the cultures were purified by repeated streaking on l-agar medium. all the bacterial strains including four already isolated and identified strains [bacillus subtilis (cn2), streptomyces lydicus (cn6), staphylococcus aureus (cn7) and bacillus pumilus (pp3)] were characterized morphologically and physiologically. culture characteristics as well as cell morphology was observed. to examine bacterial physiology, growth curve, temperature and ph, the studies were conducted following the research of fatima and ahmed16. plant growth-promoting traits to determinate plant growth-promoting traits, tests for ammonification, hcn production and auxin production were performed following the research of cappuccino and sherman17; lorck18 and ahmed and hasnain19. plant-microbe interactions ten strains were used in the plant-microbe interaction experiment and their effect on plant growth was observed by recording various growth parameters following the study by fatima and ahmed16. the experiment was repeated thrice. statistical analysis the data obtained were statistically analyzed using the software spss v. 16. study of the modulating interactions of multitrait rhizobacteria vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 46 r a d s j . b i o l . r e s . a p p l . s c i . 46 r e s u l t s isolation and characterization of bacteria for rhizospheric soil samples, convolvulus arvensis and polygonum plebeium plants were collected from the zhob district of balochistan. six bacterial strains were isolated from the rhizosphere of the selected plants through the serial dilution method and were used for further study including four already isolated strains [bacillus subtilis (cn2), streptomyces lydicus (cn6), staphylococcus aureus (cn7) and bacillus pumilus (pp3)]. different bacterial isolates showed different colony size, shape, color, texture, elevation, margin and growth. bacterial isolates cn1, bacillus subtilis (cn2), cn4, staphylococcus aureus (cn7) and pp5 showed irregular shape; cn5, streptomyces lydicus (cn6), pp2 and bacillus pumilus (pp3) were round in shape, while only pp4 possessed filamentous shape. all the bacterial isolates were gram +ve and except for cn1, bacillus pumilus (pp3), pp4 and pp5, all were cocci (table 1). for physiological characterization, growth curve, temperature test and ph studies were performed. the growth curve revealed that all the strains gave optimal growth after 24 and 48 hours of incubation whereas a significant decline was observed after 72 and 96 hours of incubation. bacterial strains bacillus subtilis (cn2), cn5, staphylococcus aureus (cn7) and pp5 showed maximum growth after 24 hours of incubation while maximum growth of strains cn1, streptomyces lydicus (cn6), cn4, pp2, bacillus pumilus (pp3) and pp4 was observed after 48 hours of incubation (figure 1). the temperature test showed that all the strains gave optimal growth at 37ºc temperature while bacterial growth was observed to decrease at 25 ºc and 45 ºc, respectively (figure 2). all the strains showed different behavior towards different ph ranges from 5, 6, 7, 8 and 9. optimum ph for growth of bacterial isolate pp2 was ph5. on the other hand, the bacterial strains cn1, bacillus subtilis (cn2), cn5 and streptomyces lydicus (cn6) showed maximum growth at ph7, staphylococcus aureus (cn7) at ph8, while cn4 and pp5 showed maximum growth at ph9 (figure 3). plant growth-promoting traits all the bacterial isolates showed positive ammonification test except for cn5 and pp4. only streptomyces lydicus (cn6) bacterial strain gave negative results for hcn production whereas all other strains were hcn producers. on the other hand, only bacterial strains streptomyces lydicus (cn6) and bacillus pumilus (pp3) were auxin producers while rest of the strains showed negative results for auxin production (table 1). figure 1. growth curve of bacterial strains in l-broth medium for different intervals of time (24, 48, 72 and 96 hours). study of the modulating interactions of multitrait rhizobacteria vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 47 r a d s j . b i o l . r e s . a p p l . s c i . 47 figure 2. growth of bacterial strains grown in l-broth medium at different temperatures (25, 37 and 45oc). figure 3. growth of bacterial strains in l-broth medium with ph range (5, 6, 7, 8 and 9). study of the modulating interactions of multitrait rhizobacteria vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 48 r a d s j . b i o l . r e s . a p p l . s c i . 48 table 1. morphological characterization of bacterial isolates. 1 c n 1 1 ir re g u la r w h ite s m o o th r a is e d e n tir e m o d e ra te g ra m + ve r o d s c h a in s + + + 2 c n 2 0 .2 -0 .3 ir re g u la r o ra n g e s m o o th s lig h tly r a is e d ir re g u la r m o d e ra te g ra m + ve c o c c i c h a in s a n d s in g le + + + + + 3 c n 4 0 .2 -0 .3 ir re g u la r o ra n g e s m o o th f la t ir re g u la r m o d e ra te g ra m + ve c o c c i c h a in s a n d s in g le + + + 4 c n 5 1 .5 r o u n d w h ite s m o o th r a is e d e n tir e m o d e ra te g ra m + ve c o c c i c h a in s a n d c lu st e rs + 5 c n 6 0 .1 -0 .2 r o u n d t ra n sl u c e n t s m o o th r a is e d e n tir e m ild g ra m + ve c o c c i c h a in s + + + + + + 6 c n 7 5 ir re g u la r o ff w h ite s p re a d f la t ir re g u la r y ie ld e d g ra m + ve c o c c i c h a in s + + + + 7 p p 2 1 r o u n d p a le y e llo w s p re a d r a is e d e n tir e m o d e ra te g ra m + ve c o c c i s in g le + + + + 8 p p 3 5 r o u n d o ff w h ite s m o o th e le va te d e n tir e y ie ld e d g ra m + ve r o d s c h a in s o r s in g le + + + + + + + g ra m + ve r o d s + + + + + s o u rc e c h a in s c h a in s m a rg in / e d g e 1 0 p p 5 0 .2 -0 .3 ir re g u la r o ra n g e s m o o th f la t ir re g u la r m ild e n tir e y ie ld e d g ra m + ve r o d s + a u xi n convolvulus arvensis polygonum plebium 9 p p 4 5 f ila m e n to u s b ro w n m a tt f la t g ro w th m ic ro s c o p y c e ll m o rp h o lo g y a rr a n g e m e n t a m m o n ia h c n c u lt u re m o rp h o lo g y c e ll m o rp h o lo g y p g p t ra it s s r# s tr a in s c o lo n y s iz e ( m m ) s h a p e c o lo r t e xt u re e le v a ti o n study of the modulating interactions of multitrait rhizobacteria vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 49 r a d s j . b i o l . r e s . a p p l . s c i . 49 plant-microbe interactions to determine beneficial interactions between plants and microbes, pmi assay was done. zea mays seeds were inoculated with the bacterial strains (cn1, bacillus subtilis (cn2), cn4, cn5, streptomyces lydicus (cn6), staphylococcus aureus (cn7), pp2, bacillus pumilus (pp3), pp4 and pp5) and growth parameters of plants e.g., fresh weight, shoot length and root length were noted to estimate the growth promotional potential of these strains. considerable increase in fresh weight of plants was observed in strains pp2, cn1, cn5, cn4 and streptomyces lydicus (cn6) showing 94.36, 74.50, 74.21, 70.41, and 65.03% increase respectively as compared to control (figure 4). treatment with strains namely cn1, cn5, bacillus subtilis (cn2) and pp2 showed significant percentage increase in shoot length of up to 26.12, 23.23, 21.37 and 21.0% respectively over control. but the two strains bacillus pumilus (pp3) and pp5 showed percentage decrease in shoot length over control (figure 5). a careful study demonstrated that some bacterial strains namely cn5, streptomyces lydicus (cn6), bacillus subtilis (cn2), cn4 and pp4 showed significant percentage increase in root length of upto 64.95, 64.60, 53.22, 50.74 and 50.39% respectively, compared to control (figure 5). analysis of chlorophyll ‘a’, ‘b’ and total chlorophyll content was done. few strains namely cn5, cn4, streptomyces lydicus (cn6) and pp2 showed significant percentage increase in chlorophyll ‘a’ of upto 100, 99.9, 82.80 and 80.99% respectively, then non-inoculated control plants. four strains namely cn5, cn4, streptomyces lydicus (cn6) and pp2 showed maximum chlorophyll ‘b’ concentration in treated plants with percentage increase of 100, 99.72, 82.84 and 80.95% respectively over control. bacterial strains namely cn5, cn4, streptomyces lydicus (cn6) and pp2 showed maximum total chlorophyll content in treated plants with a percentage increase of 100, 99.82, 82.81 and 80.96% respectively over control. exceptionally, bacillus subtilis (cn2) showed percentage decrease in chlorophyll (a, b and total chlorophyll) concentration over control (figure 6). figure 4. effect of bacterial inoculations on the fresh weight of zea mays under laboratory conditions. data represent mean of twelve replicates. different letters indicate significant differences between treatments using duncan’s multiple range test (p=0.05) (c=control). study of the modulating interactions of multitrait rhizobacteria vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 50 r a d s j . b i o l . r e s . a p p l . s c i . 50 figure 5. effect of bacterial inoculations on shoot/root length of zea mays under laboratory conditions. data represent mean of twelve replicates. different letters indicate significant differences between treatments using duncan’s multiple range test (p=0.05) (c=control). figure 6. effect of bacterial inoculations on chlorophyll ‘a’ of zea mays under laboratory conditions. data represent the mean of three replicates. different letters indicate significant differences between treatments using duncan’s multiple range test (p=0.05) (c=control). study of the modulating interactions of multitrait rhizobacteria vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 51 r a d s j . b i o l . r e s . a p p l . s c i . 51 d i s c u s s i o n the current study focused on determining the plant growth-promoting potential of rhizobacteria isolated from the rhizosphere of convolvulus arvensis and polygonum plebeium collected from mountainous region of the zhob district of balochistan including four already isolated and identified strains. as balochistan is not an agricultural land so our purpose was to develop methods using rhizobacteria from balochistan native plants to enhance the productivity of that region. in pmi assay, fresh weight, root length and shoot length of inoculated and control plants were observed. a maximum increase in fresh weight of plants was observed in strains pp2 (94.36%), which was observed to be a strong hcn producer and ammonia producer, both of which directly and indirectly contributed to an increase in growth parameters. in addition, all the treated plants showed percentage increase in fresh weight relative to control plant (figure 4). this was due to individual or synergistic effects of plant growth-promoting traits of rhizobacteria, which modified the uptake of minerals by plants8. similarly the inoculation of zea mays seeds with rhizobacterial strains showed a noteworthy increase in shoot length and root length. cn1, cn5 and few other strains showed a significant percentage increase in shoot length. pgpr may help plant growth promotion by using their hormones producing ability, nitrogen fixation or even phosphate solubilization. they may also affect plant metabolism to increase water and minerals uptake, by suppressing plant pathogens i.e. through hcn production or even by collaborating with other beneficial microorganisms4. balseiro-romero et al. (2017) and many other researchers working on the effect of pgpr on growth parameters of various plants reported that shoot length increased in their experiments20. the present study demonstrated that out of the ten bacterial strains, five bacterial strains namely cn5, streptomyces lydicus (cn6), bacillus subtilis (cn2), cn4 and pp4 showed considerable percentage increase of more than 50% in root length over control. cn5 and pp4 neither produced ammonia nor auxin but these were hcn producers and showed considerable increase in root length because hcn provides indirect mechanism of protection by controlling other pathogens. moreover, the presence of other plant growth-promoting traits may be one of the causes, as these may also be siderophore producers and phosphate solubilizers21 (figure 5). zahid et al. (2015) reported similar results of increase in root length in zea mays after bacterial treatment22. all the bacterial strains showed increased chlorophyll content in inoculated plants than control plants except for bacillus subtilis (cn2). actually, the bacterial inoculated plants which showed a significant increase in fresh weight, and shoot/root length were also rich in chlorophyll content over control. pgpr inoculated plants showed dark greenish leaves, which were due to an increase in chlorophyll content23. it can be assumed that the increase in chlorophyll content was due to increased leaf area and balanced nutritional uptake from the soil24. uzair et al. (2018) worked on 18 different strains of p. aeruginosa isolated from balochistan and tested these for their plant growth-promoting potential and obtained significant results25. sarwar et al. (2014) also worked on pgpr isolated from rhizosphere of rice plants of jafferabad district of balochistan and revealed significant increase in plant parameters26. our results are in accordance with the findings of kamran et al. (2016) who worked on pgpr strain i.e., pseudomonas putida and reported significant increase in chlorophyll content of eruca sativa. they revealed that plant growth-promoting bacteria supported the defense mechanism to produce various enzymes that ultimately increase the chlorophyll content of treated plants27,28. c o n c l u s i o n from the so forth experimental work, it is deduced that pgpr are beneficial for the growth promotion of plants. our results revealed that bacteria isolated from plant samples, collected from unproductive areas of baluchistan were ammonia producing and possessed the ability to produce hcn, an important plant growth promoter, while only two bacteria were auxin producers. these bacteria were then treated with zea mays l., which showed significant enhancement in plant growth parameters including chlorophyll content of plants exhibiting beneficial behavior of bacteria towards plant traits. hence, the present study gives an idea to enhance plant productivity on such barren lands that can be utilized for agricultural purposes. study of the modulating interactions of multitrait rhizobacteria vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 52 r a d s j . b i o l . r e s . a p p l . s c i . 52 a c k n o w l e d g e m e n t s this work was funded by the grant from the university of the punjab, quaid-e-azam campus, lahore. l i s t o f a b b r e v i a t i o n s pgpr plant growth promoting rhizobacteria n2o nitrous oxide lagar luria agar lbroth luria broth r e f e r e n c e s 1. bulgarelli d, schlaeppi k, spaepen s, van themaat ev, schulze-lefert p. structure and functions of the bacterial microbiota of plants. annu rev plant biol. 2013; 64:807-38. 2. haldar s, sengupta s. plant-microbe cross-talk in the rhizosphere: insight and biotechnological potential. open microbiol j. 2015; 9:(1):1-7. 3. hardoim pr, van overbeek ls, berg g, pirttilä am, compant s, campisano a, et al. the hidden world within plants: ecological and 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p p l . s c i . 53 21. souza rd, ambrosini a, passaglia lm. plant growthpromoting bacteria as inoculants in agricultural soils. genet mol biol. 2015; 38(4): 401-19. 22. zahid m, abbasi mk, hameed s, rahim n. isolation and identification of indigenous plant growth promoting rhizobacteria from himalayan region of kashmir and their effect on improving growth and nutrient contents of maize (zea mays l.). front microbiol. 2015; 6: 207-17. 23. habib sh, kausar h, saud hm. plant growthpromoting rhizobacteria enhance salinity stress tolerance in okra through ros-scavenging enzymes. biomed res int. 2016; 2016: 6284547. 24. seema k, mehta k, singh n. studies on the effect of plant growth promoting rhizobacteria (pgpr) on growth, physiological parameters, yield and fruit quality of strawberry cv. chandler. 2018; 7(2): 383-7. 25. uzair b, kausar r, bano sa, fatima s, badshah m, habiba u, et al. isolation and molecular characterization of a model antagonistic pseudomonas aeruginosa divulging in vitro plant growth promoting characteristics. bio med res int. 2018; 2018: 6147380-7. 26. sarwar a, sajid i, riaz r, urrehman f, ahmed ss, shahwani n, et al. bacterial community profiles in rhizosphere of paddy rice based on 16s rrna sequence analysis. int j biol biotech. 2014; 12(1):119. 27. kamran ma, bibi s, xu rk, hussain s, mehmood k, chaudhary hj. phyto-extraction of chromium and influence of plant growth promoting bacteria to enhance plant growth. j geochem explor. 2017; 182: 269-74. 28. habib s, fatima h, ahmed a. comparative analysis of pre-germination and post-germination inoculation treatments of zea mays l. to mitigate chromium toxicity in cr-contaminated soils. pol j environ stud. 2019; 28(2): 597-607. the response of pakistan to covid-19 vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 159 r a d s j . b i o l . r e s . a p p l . s c i . 159 op e n ac c e s s f u l l l e n g t h a r t i c l e the response of pakistan to covid-19 and current situation of disease in the country rizwan ullah1, ibrar khan1, aneela rehman1,*, azam hayyat1, mujaddad ur rehman1, salvatore massa2 1depatment of microbiology, abbottabad university of science and technology, pakistan. 2faculty of medicine, university of foggia, italy. a b s t r a c t background: coronavirus 2019 (covid-19) was reported for the first time in china and then quickly spread to other countries. in pakistan, the first case appeared in sindh province on february 26, 2020, and within a few weeks, more than 100 cases were reported across the country. currently, all provinces and territories of the country are affected and cases are growing faster every day. pakistan is among those countries that have problems with health and diagnostic facilities due to poor economic conditions. objectives: the present review aims to highlight the emergence, spread and control of the disease in pakistan. furthermore, it also highlights the response of pakistan in the management of covid-19 outbreak, and the country’s contribution in the field of science and technology for the betterment of diagnostic and treatment capabilities against the disease. methodology: collection of data was done through national and international forums like covid-who report and covid-19 health advisory platform by ministry of national health services regulation. results: the important mean for prevention is the proper use of standard operating procedure (sops). quarantine and isolation centers help in limiting the spread of this deadly disease. conclusion: it is concluded that no vaccine is available for the treatment of this emerging disease so far. the only prevention is to properly and strictly follow the sops guidelines. keywords coronavirus, covid-19, pakistan, outbreak, death cases *address of correspondence aneelarehman88@gmail.com article info. received: august 21, 2020 accepted: december 19, 2020 cite this article: ullah r, khan i, rehman a, hayyat a, rehman mu, massa s. the response of pakistan to covid-19 and current situation of disease in the country. rads j biol res appl sci. 2020; 11(2):159-163. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. i n t r o d u c t i o n viral-disease-outbreak’s emergence has caused many serious public health issues in the past, and is still expected to occur in future. epidemics of severe acute respiratory syndrome coronavirus (sars-cov) in 20022003 and middle east respiratory syndrome coronavirus (mers-cov) in 2012 have been recorded previously. it was december 2019 when another viral outbreak occurred in wuhan city, hubei province, china and has affected more than 210 countries across the world. the world health organization (who) declared the covid 19 outbreak as sixth public health of emergency services (sphec) on january 3, 20201. initially, the infected individuals exposed respiratory disease symptoms with unknown aetiology, hence called pneumonia of unknown aetiology, and later on novel coronavirus pneumonia (ncp)2. world health organization (who) and chinese centre for disease control and prevention (china cdc) started an investigation of the disease and they found the severe r e v i e w a r t i c l e the response of pakistan to covid-19 vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 160 r a d s j . b i o l . r e s . a p p l . s c i . 160 acute respiratory coronavirus-2 (sars-cov-2) as the causative agent of coronavirus disease-2019 (covid-19). initially, the disease remained limited to wuhan city and china, but due to contagious nature of the virus, cases were also reported in other countries, hence who declared it a pandemic on march 11, 20203. the current outbreak has so far resulted in more than 32730945 confirmed cases and 991224 deaths worldwide and it is continuously increasing. the highest number of cases and deaths were seen in united states of america (usa) and as of september 27, 2020, there were more than 16233110 cases and 546864 deaths4. in pakistan, the two confirmed cases of covid-19 appeared in sindh, province of pakistan and islamabad capital on february 26, 2020. following the two cases’ history, it was found that they had travelled from taftan, iran border. later on, within 15 days the coronavirus disease-19 positive cases in pakistan reached more than 40, highest in sindh province. about 75% cases had the history of travelling from iran, and remaining were from uae and other countries5. in the current situation, number of cases and deaths have been increasing day by day in pakistan. according to the ministry of national health services of pakistan, confirmed cases of covid-19 in pakistan as of september 29, 2020, were 311,516 and deaths were 6,474. the highest number of cases (136,395) and deaths (2,495) were seen in sindh province6. importantly, the reported incubation period of virus is from 2 to 14 days and common symptoms of the disease include fever, cough, sneezing, breathlessness and headache. some people remained asymptomatic but they have been a source of disease transmission to others7. diagnostic procedures include isolation of suspected persons and collection of the sample through sterile swab from throat or nose. sometimes, blood samples are also taken for pathological determination. samples are properly processed for identification of virus through reverse transcriptionpolymerase chain reaction (rrt-pcr) or nucleic acid amplification test (naat), serological tests, detection of igm and igg antibodies and lungs ct scan. according to the ministry of science and technology, pakistan, scientists of national university of science and technology (nust) have developed more reliable and accurate diagnostic kits that were approved from drug regulatory authority of pakistan (drap) on june 12, 20208. there is no proper treatment for covid-19 but symptomatic treatment has played an important role in the betterment of the diseased person’s health. a number of drugs like remdesivir, chloroquine, hydroxychloroquine, corticosteroids and product of immunoglobulins are recommended by the health professionals9. it has been recommended by the government of pakistan to follow sops and avoid unnecessary gatherings in order to limit further spread of disease to others. e p i d e m i o l o g y the transmission of infectious agent is primarily linked to seafood wholesale market. the researchers detected ratg13 gene from bats and supposed that pangolin and pigs are the possible intermediate host of covid-19 researchers supposed that bat is an initial host. following studies suggested pig or pangolin as a possible intermediate host. it is due to the similarity between the sars-cov-2 and bat-cov ratg13 gene. when the sea food market were closed the transmission of covid-19 continued and rate of infected people increased day by day and later found out that zoonotic transmission of virus was the cause of spread10. the human-to-human transmission sars cov-2 was also reported. this contagious infection is now widely distributed throughout the world affecting more than 210 countries and territories. initially, china and iran were the most affected ones in a very short period of time. pakistan is the neighbor of both countries and has border and trade with both china and iran, therefore, appearance of initial cases in the country had the history of travelling from iran. it was also noted that mostly those people who had a recent history of travelling from foreign countries were detected positive for covid-19. since the beginning, government of pakistan had established quarantine centers in different areas of the country to limit the spread of the disease but due to lack of diagnostic facilities and resources, cases soared all over the country. all the provinces and territories of the country were put under lockdown for more than two months but the asymptomatic spread of disease was on peak due to which disease’s spread occurred all over the country. the response of pakistan to covid-19 vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 161 r a d s j . b i o l . r e s . a p p l . s c i . 161 table 1. the number of cases and deaths from covid-19 in different provinces and regions of pakistan5. s. no. province / regions no. of cases no. of deaths references 1 sindh 136,395 2,495 5 2 punjab 99,292 2,234 5 3 kpk 37,727 1,259 5 4 islamabad 16,532 181 5 5 balochistan 15,177 145 5 6 gb 3,730 88 5 7 ajk 2,636 72 5 table 2. different diagnostic facilities and their location in pakistan provinces and regions. s. no. province/ location no. of centres address / facilities 1 sindh 05 1) agha khan university hospital, karachi 2) dow hospital, ojha campus, karachi 3) civil hospital, karachi 4) indus hospital, karachi 5) south city hospital, karachi 2 punjab 04 1) public health lab, punjab aids control program, lahore 2) shaukat khanum hospital, lahore 3) nishtar hospital, multan 4) armed forces institute of pathology (afip), rawalpindi 3 islamabad 01 1) national institute of health (nih), islamabad 4 kpk 01 1) public health lab, khyber medical university, peshawar 5 baluchistan 01 1) public health lab, fatima jinnah general and chest/tb hospital, quetta 6 ajk 01 1) abbas institute of medical sciences (aims), muzaffarabad 7 gb 01 1) district headquarters hospital, gilgit 8 nih mobile testing lab 01 1) deployed at taftan, iran international border crossing as of september 29, 2020, there were more than 311,516 cases and 6,474 deaths in pakistan. highest number of cases were from sindh followed by punjab, khyber pakhtunkhwa (kpk), islamabad, balochistan gilgit baltistan (gb) and azad jammu and kashmir (ajk). sindh province was on top with more than 136,395 confirmed cases and 2,495 deaths. the number of cases in other provinces and regions of pakistan was as punjab 99,292, kpk 37,727, islamabad 16,532, balochistan 15,177, gb 3,730 and ajk 2,636. numbers of deaths were: sindh 2,495, punjab 2,234, kpk 1,259, islamabad 181, balochistan 145, gb 88 and ajk 72 (table 1)5. people of every age had acquired covid-19, but most severe outcomes of the disease were observed in asthmatic patients, people of older age, cancer patients the response of pakistan to covid-19 vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 162 r a d s j . b i o l . r e s . a p p l . s c i . 162 and other immunodeficient persons. it was also seen that males were more susceptible to infection as compared to females. health care workers including doctors, nurses and others also suffered from disease and many of them have lost their lives. strategies for diagnosis, treatment and prevention in pakistan pakistan, is a developing country usually facing a financial crisis due to which outbreak like the current covid-19 has more negative impacts on the economy of country. due to lack of resources and health facilities, it is difficult to screen the whole population for disease, however, few diagnostic centers have been established by the government in different regions of the country, and are working to approach for further covid-19 health advisory platform by ministry of national health services regulation coordination (table 2). scientists have also started working on designing different reliable diagnostic kits and producing its own ventilators which are approved by drap and will be soon handed over to the national disaster management agency (ndma)8. strategies for viral diagnosis in pakistan include swab (mouth or nose) sampling, serological testing of blood samples, molecular detection of viral particles by rt-pcr, lungs scans and detection of immunoglobulin. more severe cases are admitted to different quarantine centers and teaching hospitals where they are placed in isolation wards and properly cared and treated. use of some drugs like remdesivir, chloroquine, hydroxychloroquine, corticosteroids and passive immunization has been observed for the treatment of severe covid-19 patients11. due to economic crisis of the country and limited resources of health, it is difficult to manage such a high number of cases, therefore, the government has advised people to stay isolated at home if someone has a mild infection. for the management of severe cases, authorities in pakistan has taken precautionary measures in the form of quarantine centers and isolation wards12. as there is no proper treatment for current covid-19 hence, it is recommended by the government of pakistan to follow who guidelines and sops for covid-19. on a community level, every individual should wear a surgical mask, keep social distance (6 feet), use sanitizers, frequently wash hands with soap, and avoid crowded gatherings and unnecessary travelling until the pandemic is controlled13. c o n c l u s i o n pakistan is a developing country in the neighborhood of china and iran which were initially affected by covid-19. unfortunately, the country is facing an economic crisis due to which it becomes difficult to manage and face the fast-growing pandemic effectively. however, the initial steps that were taken by the government such as screening the passengers at airports and borders, partial or complete lockdown, following sops and establishment of quarantine centers for isolation of confirmed and suspected persons remained helpful for the country. due to lack of health facilities, number of cases and deaths were seen all over the country and resulted in more than 311,516 cases and 6,474 deaths up to september 29, 2020. with such limited resources, the country is struggling for the invention of their diagnostic kits, ventilators and therapeutic drugs and vaccines. since there is no proper treatment for covid-19 yet therefore, it is recommended by the government of pakistan to properly follow sops and prevent others from deadly virus infection spread. c o n f l i c t s o f i n t e r e s t none. f u n d i n g s o u r c e none. a c k n o w l e d g e m e n t s i am thankful all my colleges for data collection and analysis throughout this work. l i s t o f a b b r e v i a t i o n s covid-19 coronavirus disease-2019 drap drug regulatory authority of pakistan ncp novel coronavirus pneumonia ndma national disaster management agency naat nucleic acid amplification test the response of pakistan to covid-19 vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 163 r a d s j . b i o l . r e s . a p p l . s c i . 163 r e f e r e n c e s 1. s. bilgin o kurtkulagi, g.b. kahveci, t.t. duman, b. m.a tel. millennium pendemic: a review of coronavirus disease (covid-19) exp bioned res. 3 (2) (220), pp. 117-125. 2. huang c, wang y, li x, ren l, zhao j, hu y, et al. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet. 2020; 395(10223): 497-506. 3. hui ds, zumla a. severe acute respiratory syndrome: historical, epidemiologic, and clinical features. inf dis clin. 2019; 33(4):869-89. 4. waris a, khan au, ali m, ali a, baset a. covid-19 outbreak: current scenario of pakistan. new mic new inf. 2020; 35: 681-7. 5. saqlain m, munir mm, ahmed a, tahir ah, kamran s. is pakistan prepared to tackle the coronavirus epidemic? drug ther pers. 2020; 36:213-4. 6. covid-19 health advisory platform by ministry of national health services regulation coordination. http://covid.gov.pk/stats/pakistan. 7. velavan tp, meyer, cg. the covid‐19 epidemic. trop med int health. 2020; 25(3):278-84. 8. drug regulatory authority of pakistan (drap). https://www.dra.gov.pk/. 9. lu r, zhao x, li j, niu p, yang b, wu h et al. genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. lancet 2020; 395(10224):565-74. 10. sun p, qie s, liu z, ren j, xi j. clinical characteristics of 5732 patients with 2019-ncov infection. 2020. available at: ssrn 3539664. 11. ilyas n, azuine re, tamiz a. covid-19 pandemic in pakistan. int j trans med pub heal. 2020; 4(1): 3749. 12. mansoor sn, gill za, rathore fa, uttra km. establishing and managing a quarantine and isolation centre in covid-19 pandemic. j pak med ass 2020; 70(5):11-4. 13. uyeki tm, bundesmann m, alhazzani w. clinical management of critically ill adults with coronavirus disease 2019 (covid-19). https://www.cdc.gov/coronavirus/2019ncov/index.html. isolation of gram positive and gram negative organisms from pus samples: one center study naheed afshan1*, madiha shahid1 1department of microbiology, jinnah university for women, karachi-74600 pakistan. abstract the contamination of pus organisms in infected wounds, ears, brain abscesses and post surgical infection is a common problem for many patients which causes great distress in terms of discomfort, delayed healing and significantly increased healthcare cost by creating systemic infection and complication. the aim of the study was to find out the frequency as well as antimicrobial susceptibility of gram positive and gram negative organisms isolated from pus samples. the study was conducted at the microbiology department of jinnah university for women karachi, pakistan during september to november 2012. for this purpose pus samples collected from infected wounds, ears and post surgical infection, from different health care hospitals. out of 35 positive cultures 11(31%) of gram positive followed by staphylococcus aureus was identified 8(22.85%) and streptococcus pyogenes 2(5.71%), streptococcus epidermidis 1(2%) on the basis of colony morphology, gram’s stain, catalase and dnase test. 3(8.57%) were gram negative like pseudomonas spp 3(8.57), klebsiella spp 4(11.42%), e.coli 9(25.71%) and candida spp 1(2.85%) were identified on the basis of microbiological and biochemical test. keywords: abscesses, gram positive, gram negative, post surgical infection. introduction pus is a thick, whitish to yellowish material composed primarily of dead cells that generally forms as a by-product of bacterial infections. the inflammatory cells that participate in the body’s immune response at the site of an infection eventually degrade and die, creating the substance known as pus. one of the most common types of bacteria that cause pus formation is staphylococcus aureus, although any bacterial infection may produce pus. an infection that leads to the production of pus is called purulent infection. when pus forms within enclosed spaces in the tissues, it causes abscesses. when it forms on the skin surface, it causes lumps known as pustules or pimples. pus can also form when infections develop in internal organs, such as the bones, brain, lungs, and gastrointestinal tract. because the formation of pus usually indicates a bacterial infection, people with conditions that weaken the immune system have a higher risk of infection and subsequent pus formation. the formation of pus generally signals a bacterial infection, which may be a serious condition (bowler et al., 2001). pus is caused by the breakdown of neutrophils, which are inflammatory cells produced by the body to fight infection. typically, pus forms during the course of a bacterial infection. although neutrophils initially engulf and kill bacteria, they themselves are eventually broken down and become a major constituent of pus. all types of bacteria that cause disease are capable of producing infections that lead to pus. pus formation is usually caused due to inflammation which is a response of body tissues to injury or irritation which is what produces the pus during a bacterial infection. pus is caused by the breakdown of neutrophils which are inflammatory cells produced by the body to fight infection. an accumulation of pus in an enclosed tissue space is known as an abscess, whereas a visible collection of pus within or beneath the*corresponding author: naheedafshan7@hotmail.com 20 vol 4 (1), january 2013; 20-22 epidermis is known as a pustule or pimple (anguzu and olila, 2007). pus consists of a thin, protein-rich fluid, known as liquor puris, and dead leukocytes from the body (mostly neutrophils). during infection, macrophages release cytokines which trigger neutrophils to seek the site of infection by chemotaxis. there, the neutrophils engulf and destroy the bacteria and the bacteria resist the immune response by releasing toxins called leukocidins. as the neutrophils die off from toxins and old age, they are destroyed by macrophages, forming the viscous pus. bacteria that cause pus are called suppurative, pyogenic or purulent. if the agent also creates mucus, it is called mucopurulent. purulent infections can be treated with an antiseptic. despite normally being of a whitish-yellow hue, changes in the color of pus can be observed under certain circumstances. pus is sometimes green because of the presence of myeloperoxidase, an intensely green antibacterial protein produced by some types of white blood cells. green, foul-smelling pus is found in certain infections of pseudomonas aeruginosa. the greenish color is a result of the pyocyanin bacterial pigment it produces .amoebic abscesses of the liver produce brownish pus, which is described as looking like "anchovy paste". pus can also have a foul odor in almost all cases when there is a collection of pus in the body, the clinician will try to create an opening for it to evacuate this principle has been distilled into the famous latin aphorism "ubi pus, ibi evacua!"some common disease processes caused by pyogenic infections are impetigo, osteomyelitis, septic arthritis, and necrotizing fasciitis (adegoke et al., 2010). materials and methods pus collection: take a swab and gently but firmly rotate it on the surface directly where infection is suspected. media: blood agar, mac conkey agar, nutrient agar, msa (mannitol salt agar) procedure: 1. pus specimens inoculated on nutrient, msa, blood and mac conkey agar. 2. streaked plates inoculated at 37 degree centigrade for 24 hours. 3. identification of isolates were done based on cultural, morphological and biochemical characteristics. results and discussion among 35 positive pus samples in which most common isolates are found to be staphylococcus aureus 8(22.85%), pseudomonas aerogenosa 3(8.57%), klebsiella 4(11.42%), e. coli 9(25.71%), streptococcus pneumonia 2(5.71%), proteus mirabilis 2(5.71%), streptococcus pyogenes 1(2.85%), pseudomonas specie 3(8.57%), candida albicans 1(2.85%). according to this result e. coli is the most significant specie (figure 1), important pathogen in human and was found to be the most prevalent gram negative bacilli in male and female and also staphylococcus aureus causes a variety of suppurative (pus forming) infection in human and also causes superficial skin lesions such as boil, carbuncle and in post surgical infection pus and drainage a very serious problem. pus formation may indicate a serious bacterial infection. conclusion the study concludes that the incidence of pus infection and contamination with other nosocomal infections is expected to be highest in our community. especially staphylococcus aureus and e. coli was 21 vol 4 (1), january 2013; 20-22 figure 1: prevalence rate of bacteria. reported to be the most common pathogens. so we should take preventive measurements. references adegoke, a. a., tom, m., okoh, a.i.1 and jacob, s. (2010). studies on multiple antibiotic resistant bacteria isolated from surgical site infection scientific r e s e a r c h a n d e s s a y s , 5 ( 2 4 ) : 3 8 7 6 3 8 8 1 . anguzu, j.r. and olila, d. (2007). drug sensitivity patterns of bacterial isolates from septic post operative wounds in a re-gional referral hospital in uganda. afr. health sci., 7(3): 148-154. bowler, c., chigbu, o. c. and giacometti, h. (2001). emergence of antimicrobial resistance bacteria. journal of antimicrobial and chemotherapy, 23:12 – 23. 22 vol 4 (1), january 2013; 20-22 call for papers publication is free of cost rads journal of biological research and applied sciences welcomes the submission of research papers, review articles and short communications in the following subject fields: t biochemistry t biotechnology t botany t chemistry t environmental science t medical & health sciences t microbiology t molecular biology t pharmacy t physics t zoology t clinical research note:all manuscripts should be sent by e-mail to the editor with your mobile number : rads@juw.edu.pk or deanresearch@juw.edu.pk our aim is to provide a platform for budding scientists researchers, research scholars, academicians etc. to present their research findings in the field of biological and applied sciences. evaluation of selected pakistani honeys vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 54 r a d s j . b i o l . r e s . a p p l . s c i . 54 op e n ac c e s s f u l l l e n g t h a r t i c l e evaluation of selected pakistani honeys in comparison with manuka honey against vibrio cholerae muhammad barkaat hussain* department of microbiology, faculty of medicine, king abdulaziz university, rabigh branch 21589, saudi arabia a b s t r a c t background: antibacterial resistance in vibrio cholerae has been reported in many parts of the world. therefore, it is important to explore novel therapies which stand less chances of developing antimicrobial resistance. in this regard honey is getting worldwide attention because antibacterial resistance against honey is unlikely. objectives: to determine the antibacterial activity of locally produced sidr (ziziphus jujuba), kalonji (nigella sativa) and eucalyptus (eucalyptus spp) honey against twenty-six clinical isolates of vibrio cholerae to compare the antibacterial activity of indigenous honey with medically graded manuka honey. methodology: identification of vibrio cholerae was done by standard cultural, biochemical and serological methods. susceptibility pattern of vibrio cholerae was also determined. minimum inhibitory concentrations (mic) of locally produced sidr, kalonji and eucalyptus honey, and medically graded manuka honey was determined by agar dilution and kirby bauer test. american type culture collections (atcc) escherichia coli 25922, staphylococcus aureus 25923 and acinetobacter baumannii 29213 were used as standard control strains. results: manuka and eucalyptus honey have comparable antibacterial activity against both sensitive and resistant clinical isolates of vibrio cholerae. the lowest mics were between 3.7 to 4% for medically graded manuka honey, whereas eucalyptus honey inhibited between the range 4 to 4.3%. kalonji and sidr honey inhibited these isolates between 6.7 to 7.0% and 6.3 to 7.0%, respectively. conclusion: it is concluded that manuka and eucalyptus honey could be evaluated in a clinical trial for the treatment of gastroenteritis caused by vibrio cholerae. keywords honey, vibrio cholerae, antibacterial activity, antibacterial resistance, eucalyptus honey *address of correspondence mbhussain1@kau.edu.sa article info. received: november 22, 2018 accepted: october 25, 2019 cite this article: hussain mb. evaluation of selected pakistani honeys in comparison with manuka honey against vibrio cholerae. rads j biol res appl sci. 2019;10(2):54-62. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: this project was funded by the university of health sciences, lahore, pakistan. conflict of interest: nil i n t r o d u c t i o n cholera is a major life-threatening diarrheal disease confronting developing countries, particularly where proper sanitation, hygienic water and food are not available1. it affects approximately 2.86 million people and accounts for 95000 deaths annually in developing countries2. however, these figures do not reflect the true global burden of the disease because the majority of cholera cases are not reported due to various reasons3. according to who, only small fractions of cases are actually reported4. medical advice for mild to moderate cases is usually not sought. stool samples are not routinely cultured for identification of vibrio cholerae5. therefore, without laboratory isolation of the bacteria, the symptoms of cholera are difficult to distinguish from other causes of diarrhea. moreover, poor epidemiological surveillance and economic disincentives for reporting also contribute to low reporting rate2. in pakistan, cholera remains one of the major cause of morbidity and mortality among susceptible individuals6. o r i g i n a l a r t i c l e evaluation of selected pakistani honeys vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 55 r a d s j . b i o l . r e s . a p p l . s c i . 55 various reports suggest that vibrio cholerae el tor, ogawa is endemic in pakistan7. contaminated water or food is responsible for the spread of infection. the bacterium colonizes the small intestine after passage through the gastric barrier and produces massive secretory diarrhoea and vomiting8. fluid and electrolytes replacement is the primary treatment, however, in severe cholera, antibiotics are recommended to reduce the duration of illness and excretion of vibrio cholerae8. recently multidrug-resistant vibrio cholerae has been reported in many parts of the world9. moreover, prolonged antibiotic therapy inhibits the growth of beneficial flora of the gut10. the rise in multi-drug resistant vibrio cholerae and associated side effects of antibiotics has led to the development of new therapeutic agents effective against vibrio cholerae. honey compared to antibiotics has multiple advantages. antibacterial resistance to honey has not been reported so far because it contains multiple bees and plants derived antibacterial substances11. hydrogen peroxide is one of the important bio-active antibacterial compounds produced by oxidase enzyme (bee origin), which converts water and sugar into hydrogen peroxide and gluconic acid12. the enzyme remains inactive in undiluted honey and becomes maximally active when honey is diluted between 40%-60%13. therefore, diluted honey may be more effective in the treatment of diarrhoea caused by vibrio cholerae. besides, there are numerous plants and bees derived antibacterial substances present in honey like flavonoids, phenolic acids, methylglyoxal, bee defensin-1, etc.14 these substances target different sites of bacterial structure and generate synergistic effects15. this might be the reason that bacterial resistance to honey has never been reported. because of these unique characteristics, honey could serve as a potential therapeutic agent for multi-drug resistant vibrio cholerae. the benefits of honey are not just limited to antibacterial activity, rather it also contains beneficial lactic acid bacteria originated from honey bee stomach which includes lactobacilli and bifidobacteria16. the beneficial flora inhibits the attachment of pathogenic bacteria on epithelial lining of the intestinal tract and secretes antibacterial substances which have inhibitory growth effects on pathogenic bacteria17. honey also contains undigested oligo polysaccharides knows as prebiotic which enhances the growth of normal flora (probiotics) of gut18. the level of antibacterial activity and composition of honey varies greatly and depends on the type of plant species, geographical areas, soil composition, climatic conditions and processing of honey19. this could also be a disadvantage of using honey. it is important to evaluate the antibacterial activity of indigenous honey because they are easily available and affordable to the local population. in this study, the minimum inhibitory concentrations of three common locally produced honeys have been determined and comparison of their antibacterial activity with medically graded manuka honey have been conducted. m a t e r i a l s a n d m e t h o d s in this study, twenty-six vibrio cholerae were isolated from stool cultures. the isolates were obtained from the national institute of health (nih), islamabad (n=15), civil hospital, mirpur khas (n=6) and combined military hospital, lahore (n=5). identification of clinical isolates was re-confirmed by growing them on mcconkey’s agar, thiosulphate citrate bile salt sucrose (tcbs), deoxycholate citrate agar (dca) and blood agar. pale coloured colonies from macconkey agar, β-hemolytic colonies from blood agar and yellow sucrose fermenting colonies from tcbs were selected and gram stained. distilled water immobilization was performed and their motility was observed by hanging drop preparation. oxidase and catalase tests were performed for identification. biochemical identification was done by api 20ne (biomerieux). polyvalent and monovalent antisera (bd difco®) were used for serological confirmation. biotype was determined on the basis of sensitivity to polymyxin b (300 iu), beta-hemolysis on sheep blood agar, voges-proskauer test, string test, camp test and agglutination of chicken red cells as bergey’s manual of determinative bacteriology. antimicrobial susceptibility test antibiotic-resistant profile of clinical isolates were calibrated with 0.5 macfarland index tube was determined by kirby-bauer disc diffusion method on mueller-hinton (mh) agar. the antibiotic tested were amikacin, ampicillin, evaluation of selected pakistani honeys vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 56 r a d s j . b i o l . r e s . a p p l . s c i . 56 aztreonam, ceftazidime, ceftriaxone, cefuroxime, chloramphenicol, cotrimoxazole, imipenem, levofloxacin, moxifloxacin, nalidixic acid, ofloxacin and tetracycline. atcc 25922 escherichia coli was used as a quality control strain20. honey samples locally produced kalonji, eucalyptus and sidr honey were obtained from local apiarists. the floral identification was performed by the beekeepers based on their geographical areas, floral availability at the location of beehives (foraging radius) and season19. medically graded manuka honey (unique manuka factor-21+) derived from manuka tree indigenous to new zealand was included in the study for comparison. agar dilution assay susceptibility of vibrio cholerae and atcc reference strains to honey was evaluated by agar dilution assay as adopted by french et al. (2005)21. a stock solution of honey was prepared at 20% (v/v) and 50% (v/v) in sterile distilled water. appropriate volumes of the honey stock solutions, double strength mueller-hinton (mh) agar (thomas scientific) and sterile distilled water were mixed to obtain 1-20% incremental dilutions. these dilutions were kept at 50ºc for 20 minutes and vortex vigorously to achieve uniform homogenization. the different dilutions were dispensed into petri dishes (thomas scientific) in triplicate and allowed to dry for 20 minutes. working bacterial culture of each clinical isolates and reference strains was prepared in tsb and adjusted to 0.5 mcfarland’s standard (1 × 108 cfu/ml). a volume of 3μl from each adjusted culture was inoculated onto the agar plates with multi-point inoculator (akribis scientific limited). the inoculated agar plates were incubated for 18 hours at 37ºc. the mic was considered to be the lowest concentration of honey at which visible growth of bacteria was inhibited. r e s u l t s statistical analysis the data were analyzed by the statistical package for social sciences (spss 23.0). the arithmetic mean of minimum inhibitory concentrations (mics) and sds of mean values of honey was calculated. the variances of mean mics among tested honey samples were detected by applying the kruskal-wallis test. bonferroni post hoc test is applied for pairwise comparison between different kinds of honey. differences were considered significant at p < 0.05. all clinical isolates of vibrio cholerae were identified as vibrio cholerae o1 biotype el tor serotype ogawa. table 1 & 2 show the susceptibility pattern of twenty-six clinical isolates of vibrio cholerae. twenty isolates were resistant to cotrimoxazole and nalidixic acid, two isolates were nalidixic acid-resistant and four were sensitive to all antibiotics tested. manuka and eucalyptus honey have comparable antibacterial activity against both sensitive and resistant clinical isolates of vibrio cholerae (table 2). the lowest mics were 3.7 to 4% for medically graded manuka honey against clinical isolates of vibrio cholerae, whereas eucalyptus honey inhibited between the range 4 to 4.3%. kalonji and sidr honey inhibited these isolates between 6.7 to 7.0% and 6.3 to 7.0%, respectively. there is variation between the levels of antibacterial activity of different tested honeys. manuka honey also inhibited atcc 25923 staph aureus at lowest mic 4%, whereas eucalyptus honey had more inhibitory against atcc 29213 a. baumannii and atcc 25922 e. coli in comparison with manuka and other honey (table 3). generally, the most susceptible organisms were vibrio cholerae and staph aureus and the least susceptible organisms were e. coli (table 3). there is a significant difference (kruskal-wallis test, p=0.000) among the mean minimum inhibitory concentrations (mics) (%v/v) of kalonji, eucalyptus, sidr and manuka honey against clinical isolates of vibrio cholerae. there is a significant difference (p=0.00, bonferroni post hoc test) between mean mics of manuka and kalonji, manuka and sidr honey against vibrio cholerae. similarly, there is a significant difference (p=0.00, bonferroni post hoc test) between mics of eucalyptus and kalonji, manuka and sidr honey. however, there is no difference (p=0.061) between the mics of manuka and eucalyptus honey (table 4 & figure 1). regarding atcc reference strains significant difference (p=0.039) was recorded among mean mics of manuka, eucalyptus, sidr and kalonji honey. there is a significant difference (p=0.040) between mean mics of eucalyptus and sidr honey against atcc reference evaluation of selected pakistani honeys vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 57 r a d s j . b i o l . r e s . a p p l . s c i . 57 strains. there is also a significant difference (p=0.014) between the mean mic of eucalyptus and kalonji honey. against atcc reference strains (table 5 & figure 2). table 1. summary of antimicrobial resistance profile of vibrio cholerae isolates. number of isolates (n) antimicrobial resistance pattern 4 s 20 sxtr, nar 2 nar sxt: cotrimaxazole, na: nalidixic acid, s: sensitive, r: resistant. table 2. susceptibility pattern of vibrio cholerae and minimum inhibitory concentrations mics (%v/v) of honeys. species resistant profile mics* kalonji eucalyptus sidr manuka-21 vibrio cholerae-1 sxtr, nar 6.7±0.5 4.3±0.5 6.7±0.5 manuka-21 vibrio cholerae-3 s 7.0±0.0 4.0±0.0 7.0±0.0 4.0±0.0 vibrio cholerae-4 sxtr, nar 6.7±0.5 4.3±0.5 7.0±0.0 4.0±0.0 vibrio cholerae-7 sxtr, nar 7.0±0.0 4.0±0.0 7.0±0.0 4.0±0.0 vibrio cholerae-9 sxtr, nar 6.7±0.5 4.0±0.0 7.0±0.0 3.7±0.5 vibrio cholerae-10 sxtr, nar 6.7±0.5 4.0±0.0 7.0±0.0 4.0±0.0 vibrio cholerae-13 sxtr, nar 7.0±0.0 4.3±0.5 7.0±0.0 4.0±0.0 vibrio cholerae-15 sxtr, nar 7.0±0.0 4.0±0.0 7.0±0.0 4.0±0.0 vibrio cholerae-16 sxtr, nar 6.7±0.5 4.3±0.5 6.3±0.5 4.0±0.0 vibrio cholerae-17 s 7.0±0.0 4.0±0.0 7.0±0.0 4.0±0.0 vibrio cholerae-18 sxtr, nar 6.7±0.5 4.3±0.5 7.0±0.0 4.0±0.0 vibrio cholerae-19 sxtr, nar 7.0±0.0 4.0±0.0 7.0±0.0 4.0±0.0 vibrio cholerae-20 s 6.7±0.5 4.3±0.5 7.0±0.0 4.0±0.0 vibrio cholerae-21 nar 7.0±0.0 4.0±0.0 7.0±0.0 4.0±0.0 vibrio cholerae-23 sxtr, nar 6.7±0.5 4.3±0.5 7.0±0.0 4.0±0.0 vibrio cholerae-31 nar 7.0±0.0 4.0±0.0 7.0±0.0 4.0±0.0 vibrio cholerae-32 sxtr, nar 6.7±0.5 4.3±0.5 7.0±0.0 4.0±0.0 vibrio cholerae-25 sxtr, nar 7.0±0.0 4.3±0.5 7.0±0.0 4.0±0.0 vibrio cholerae-33 sxtr, nar 7.0±0.0 4.0±0.0 7.0±0.0 4.0±0.0 vibrio cholerae-27 sxtr, nar 6.7±0.5 4.3±0.5 7.0±0.0 3.7±0.5 vibrio cholerae-30 sxtr, nar 7.0±0.0 4.0±0.0 7.0±0.0 4.0±0.0 vibrio cholerae-27 s 7.0±0.0 4.3±0.5 7.0±0.0 4.0±0.0 vibrio cholerae-26 sxtr, nar 7.0±0.0 4.3±0.5 7.0±0.0 4.0±0.0 vibrio cholerae-28 sxtr, nar 6.7±0.5 4.3±0.5 7.0±0.0 4.0±0.0 vibrio cholerae-24 sxtr, nar 6.7±0.5 4.0±0.0 6.3±0.5 3.7±0.5 vibrio cholerae-24 sxtr, nar 6.7±0.5 4.0±0.0 7.0±0.0 4.0±0.0 *mic values are the mean of triplicate determinations, and shown as mean ± sd. s: sensitive, r: resistant, sxt: cotrimaxazole, na: nalidixic acid evaluation of selected pakistani honeys vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 58 r a d s j . b i o l . r e s . a p p l . s c i . 58 table 3. minimum inhibitory concentrations (mics) (%v/v) of honeys against atcc reference strains. s.no. species mics kalonji eucalyptus sidr manuka 1 staphylococcus aureus 25923 9.0±0.0 4.3±0.5 7.7±0.5 4.0±0.0 2 acinetobacter baumannii 29213 8.0±0.0 4.0±0.0 7.0±0.0 7.0±0.0 3 escherichia coli 25922 9.0±0.0 6.0±0.0 10±0.0 6.7±0.0 table 4. pairwise comparison of mean mics of manuka, sidr, kalonji and eucalyptus by bonferroni post hoc test against clinical isolates of vibrio cholerae. s.no. honey types p-value 1 manuka versus eucalyptus 0.061 2 manuka versus kalonji 0.000 ⃰ 3 manuka versus sidr 0.000 ⃰ 4 eucalyptus versus kalonji 0.000 ⃰ 5 eucalyptus versus sidr 0.000 ⃰ 6 kalonji versus sidr 0.261 *denotes significant p-value table 5. pairwise comparison of mean mics of manuka, sidr, kalonji and eucalyptus by bonferroni post hoc test against atcc reference strains. (staphylococcus aureus 25923, acinetpbector baumannii 29213 and escherichia coli 25922) s. no. honey types p-value 1 eucalyptus versus manuka 0.608 2 eucalyptus versus sidr 0.040 ⃰ 3 eucalyptus versus kalonji 0.014 ⃰ 4 manuka versus sidr 0.124 5 manuka versus kalonji 0.053 6 sidr versus kalonji 0.690 *denotes significant p-value evaluation of selected pakistani honeys vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 59 r a d s j . b i o l . r e s . a p p l . s c i . 59 figure 1. minimum inhibitory concentrations mics (%v/v) of honeys against vibrio cholerae. figure 2. minimum inhibitory concentrations mics (%v/v) of honeys against atcc reference strains. staphylococcus aureus 25923, acinetpbector baumannii 29213 and escherichia coli 25922 (mean value) d i s c u s s i o n multidrug-resistant strains of vibrio cholerae has been increasingly recognized around the world22. new resistance phenotypes of vibrio cholerae have been emerged recently23. both sensitive and resistant strains of vibrio cholerae were inhibited by honey at the same mics (table 2). this means that tested honey have different and unique mechanism of action against bacterial pathogens. recently, studies have identified the bacterial cellular targets and underlying mechanism of action of honey24,25. since, honey contains multiple antibacterial evaluation of selected pakistani honeys vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 60 r a d s j . b i o l . r e s . a p p l . s c i . 60 bioactive compounds and they act synergistically on multiple targets of the bacterial cell, therefore, antibacterial resistance to honey is less likely11. the present study revealed that locally produced honey has significant antibacterial activity against all tested pathogens and atcc reference strains. however, the level of antibacterial activity generated by different types of honey is quite variable (table 2 & 3, figure 1 & 2). the antibacterial activity of eucalyptus honey is superior to that of medically graded manuka honey (table 2). eucalyptus honey is more bactericidal than manuka honey against a. baumannii and e. coli (table 3). previously, lusby et al. (2005) have shown that honey other than the medically graded honey may have similar antibacterial effects26. therefore, it is important to evaluate the antibacterial activity of untested and locally produced honey. it also demands standardization of honey for medical exploitataion of local honeys. in this study, we used agar dilution assay for evaluation of the antibacterial activity of honey because it provides quantifiable and more accurate results as compared to diffusion assay27. honey is uniformly distributed in agar dilution assay and is direct contact with testing organisms, whereas in agar well diffusion assay there are many factors which affect the rate of diffusion of active constituents of honey. it is likely that large size active substances present in honey may not diffuse in agar well diffusion assay28. in one of the clinical trials, haffejee and moosa (1985) have demonstrated that honey is effective in treating bacterial gastroenteritis29. in the present study, all clinical isolates of vibrio cholerae were inhibited by eucalyptus honey at quite low concentrations (4 to 4.3%) (v/v), which is comparable to medically graded manuka honey (3.7 to 4% v/v). thus, eucalyptus honey taken orally can shorten the duration of cholera-like antibiotic and at the same time unlike antibiotic may not disrupt the growth of beneficial git flora. moreover, it can enhance the growth of normal flora because honey contains prebiotics which has positive growth effect on probiotics microorganism11. previously pal et al. (2016) determined the antibacterial activity of four different types of honey against vibrio cholerae and found that all isolates are susceptible to honey30. however, the authors used the disc diffusion method which is unable to provide precise and quantitative results. secondly, no comparison was made between standardized honey. in our study, we determined the mics of tested honey by agar dilution assay which offers precise and quantitative results and compared the results with medically graded honey. one of the important constituents of oral rehydration solution (ors) for the treatment of diarrhoea is glucose (2g / 100ml), based on the recommendations of who31. honey also contains glucose and fructose. unlike glucose, the fructose is absorbed in the intestine by diffusion instead of active transport; therefore, sodium ion is not coupled with this process32. as a result, water is absorbed without augmenting the absorption of sodium. it has been shown in one of the clinical trials that orally given honey supplemented with electrolytes reduces the duration of bacterial diarrhoea in comparison with ors33. another study compared the effect of honey and ors in the treatment of diarrhoea and found that the honey treatment group had fewer bowel movements and a shorter diarrhoea period compared to the control group34. recently shariatpanahi et al. (2018), evaluated the efficacy of honey in diarrhoea patients in a double-blind, randomized controlled trial comprising of 32 patients and found that honey altered the gut microflora and reduced the occurrence of diarrhoea in these patients35. these studies highlight the importance and effectiveness of honey in the treatment of diarrhoea. c o n c l u s i o n locally produced honey exhibited variable antibacterial activity against vibrio cholerae and atcc reference strains. eucalyptus honey showed comparable or in some cases better activity than well-known new zealand manuka honey. eucalyptus honey may be used as a potential alternative therapy against diarrhoea caused by vibrio cholerae in future studies. a c k n o w l e d g e m e n t s i am highly obliged to university of health sciences for funding this project and dr. nadeem shafique butt for data analysis. evaluation of selected pakistani honeys vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 61 r a d s j . b i o l . r e s . a p p l . s c i . 61 l i s t o f a b b r e v i a t i o n s sd standard deviations na nalidixic acid r resistant s sensitive sxt cotrimaxazole r e f e r e n c e s 1. reidl j, klose ke. vibrio cholerae and cholera: out of the water and into the host. fems microbiol rev. 2002; 26(2):125-39. 2. ali m, nelson ar, lopez al, sack da. updated global burden of cholera in endemic countries. plos negl trop dis. 2015; 9(6):e0003832. 3. zuckerman jn, rombo l, fisch a. the true burden and risk of cholera: implications for prevention and control. lancet infect dis. 2007; 7(8):521-30. 4. legros d. global cholera epidemiology: opportunities to reduce the burden of cholera by 2030. j infect dis. 2018; 218(suppl_3):s137-40. 5. syafirah een, najian an, foo pc, ali mrm, mohamed m, yean cy. an ambient temperature stable and ready-to-use loop-mediated isothermal amplification assay for detection of toxigenic vibrio cholerae in outbreak settings. acta tropica. 2018; 182:223-31. 6. naseer m, jamali t. epidemiology, determinants and dynamics of cholera in pakistan: gaps and prospects for future research. j coll physicians surg pak. 2014; 24(11):855-60. 7. hannan a, raja m, arshad mu, absar m. gastro’of mirpur khas (sindh) is resolved. j ayub med coll abbottabad. 2008; 20(1):10-2. 8. silva aj, benitez ja. vibrio cholerae biofilms and cholera pathogenesis. plos neglected tropical diseases. 2016;10(2):e0004330. 9. rashed sm, hasan na, alam m, sadique a, sultana m, hoq m, et al. vibrio cholerae o1 with reduced susceptibility to ciprofloxacin and azithromycin isolated from a rural coastal area of bangladesh. front microbiol. 2017; 8:252-60. 10. angelakis e, million m, kankoe s, lagier jc, armougom f, giorgi r, raoult d. abnormal weight gain and gut microbiota modifications are side effects of long-term doxycycline and hydroxychloroquine treatment. antimicrob agents chemother. 2014; 58(6):3342-7. 11. hussain mb. role of honey in topical and systemic bacterial infections. j altern complement med. 2018; 24(1):15-24. 12. da silva pm, gauche c, gonzaga lv, costa aco, fett r. honey: chemical composition, stability and authenticity. food chem. 2016; 196:309-23. 13. chaudhary a, bag s, barui a, banerjee p, chatterjee j. honey dilution impact on in vitro wound healing: normoxic and hypoxic condition. wound repair regen. 2015; 23(3):412-22. 14. roshan n, rippers t, locher c, hammer ka. antibacterial activity and chemical characteristics of several western australian honeys compared to manuka honey and pasture honey. arch microbiol. 2017; 199(2):347-55. 15. khan ru, naz s, abudabos am. towards a better understanding of the therapeutic applications and corresponding mechanisms of action of honey. environ sci pollut res int. 2017; 24(36):27755-66. 16. olofsson t, vásquez a. honeybee-specific bifidobacteria and lactobacilli. the bifidobacteria and related organisms: elsevier; 2018. 235-41. 17. liévin-le moal v, servin al. anti-infective activities of lactobacillus strains in the human intestinal microbiota: from probiotics to gastrointestinal antiinfectious biotherapeutic agents. clin microbiol rev. 2014; 27(2):167-99. 18. aly h, said rn, wali ie, elwakkad a, soliman y, awad ar, et al. medically graded honey supplementation formula to preterm infants as a prebiotic: a randomized controlled trial. j pediatr gastroenterol nutr. 2017; 64(6):966-70. 19. hussain mb, hannan a, akhtar n, fayyaz gq, imran m, saleem s, et al. evaluation of the antibacterial activity of selected pakistani honeys against multidrug resistant salmonella typhi. bmc complement altern med. 2015; 15(1):32-40. 20. wikler ma. performance standards for antimicrobial susceptibility testing: seventeenth informational supplement: clinical and laboratory standards institute; 2007. 21. french v, cooper ra, molan pc. the antibacterial activity of honey against coagulase-negative staphylococci. j antimicrob chemother. 2005; 56(1):228-31. 22. miwanda b, moore s, muyembe j-j, nguefacktsague g, kabangwa ik, ndjakani dy, et al. antimicrobial drug resistance of vibrio cholerae, democratic republic of the congo. emerg infect dis. 2015; 21(5):847-51. 23. vinothkumar k, rajpara n, nair m, bhardwaj ak. multidrug resistance in vibrio spp. and shigella spp.: emergence of untreatable pathogens. resistance to antibiotics: are we prepared to handle this growing ghost? 2017:9. 24. brudzynski k, sjaarda c. antibacterial compounds of canadian honeys target bacterial cell wall inducing evaluation of selected pakistani honeys vol. 10 (2), december 2019 issn (print): 2305 – 8722 issn (online): 2521 – 8573 62 r a d s j . b i o l . r e s . a p p l . s c i . 62 phenotype changes, growth inhibition and cell lysis that resemble action of β-lactam antibiotics. plos one. 2014; 9(9):e106967. 25. tsang as, dart aj, dart cm, jeffcott lb. mechanisms of action of manuka honey in an equine model of second intention wound healing: current thoughts and future directions. med res arch. 2018; 6(7). 26. lusby pe, coombes al, wilkinson jm. bactericidal activity of different honeys against pathogenic bacteria. arch med res. 2005; 36(5):464-7. 27. jorgensen jh, turnidge jd. susceptibility test methods: dilution and disk diffusion methods. manual of clinical microbiology, eleventh edition: american society of microbiology; 2015. 1253-73. 28. balouiri m, sadiki m, ibnsouda sk. methods for in vitro evaluating antimicrobial activity: a review. j pharm anal. 2016; 6(2):71-9. 29. haffejee i, moosa a. honey in the treatment of infantile gastroenteritis. br med j (clin res ed). 1985; 290(6485):1866-7. 30. pal bb, badaik s, swalsingh g, khuntia hk, sahoo p, pal sb. in vitro vibriocidal efficacy of monofloral honey against different sero groups of vibrio cholerae. j pure appl microbiol. 2016; 10(1):593-9. 31. duggan c, fontaine o, pierce nf, glass ri, mahalanabis d, alam nh, et al. scientific rationale for a change in the composition of oral rehydration solution. jama. 2004; 291(21):2628-31. 32. wright em, martı ́n mng, turk e. intestinal absorption in health and disease—sugars. best pract res clin gastroenterol. 2003; 17(6):943-56. 33. abdulrhman ma, mekawy ma, awadalla mm, mohamed ah. bee honey added to the oral rehydration solution in treatment of gastroenteritis in infants and children. j med food. 2010; 13(3):605-9. 34. sharif a, noorian a, sharif mr, ardakani at, zahedi a, kheirkhah d. a randomized clinical trial on the effect of honey in the acute gastroenteritis. journal of research in medical and dental science. 2017; 5(6):144-8. 35. shariatpanahi zv, jamshidi f, nasrollahzadeh j, amiri z, teymourian h. effect of honey on diarrhea and fecal microbiotain in critically ill tube-fed patients: a single center randomized controlled study. anesthesiology and pain medicine. 2018; 8(1): e62889. anti-proliferative effect of arsenic, cadmium and lead on human placental cells aftab ahmad1*, aamina shahm2, abdul rauf shakoori1 1school of biological sciences, university of the punjab, quaid-e-azam campus, lahore. pakistan. 2institute of biochemistry and biotechnology, university of the punjab, lahore, pakistan. abstract heavy metals are ubiquitously distributed in the environment and can effect human health. some of these heavy metals can even cross placenta and cause harm to developing fetus. in the present study we investigated the anti-proliferative effects of arsenic, cadmium and lead on human placental cells (pcs). pcs were isolated from placental tissue and cell line was developed. anti-proliferative effects of arsenic, cadmium and lead were tested by neutral red uptake assay. both arsenic and cadmium proved to be very toxic for pcs. there was marked decrease in cells proliferation when cells were exposed to metal concentrations for a period of 24 hrs. reduction in proliferation was recorded on exposure to leadbut the effect of lead was not as severe as arsenic and cadmium. arsenic, cadmium and lead are very toxic for pcs. proper measures should be taken for the disposal of heavy metals to protect the environment and humans from exposure. k e y w o r d s : p l a c e n t a l c e l l s , h e a v y m e t a l s , p r o l i f e r a t i o n , c y t o t o x i c i t y, m o r p h o l o g y. introduction during pregnancy placenta,amniotic fluids and fetal membranes help in growth and development of fetus by facilitating exchange of gases, nutrients, and waste products between mother’s circulation and fetus (gude et al., 2004; kippler et al., 2010). placenta also prevents pollutants and harmful substances from entering into fetus with the help of macromolecular protein complexes present on its cell membrane (osman et al., 2000; kuhnert et al., 1987). placenta is also known as dual marker in case of certain heavy metals that can effect the fetus (iyengar and rapp, 2001). cadmium exposure to human occurs by cigrette smoking, consumption of polluted food including cereals, vegetables, nuts, pulses, starchy roots or potatoes, meat, meat products and contaminated water (zenzes et al., 199; jarup et al., 1998) and retains in placenta with the help of metalothionin and doesnot enter in fetus (brambila et al., 2002). but cadmium altersplacental transport of calcium and zinc, causes early decidualization of human endometrial stroma cells, inhibits trophoblast cell migration by affecting actin cytoskeletal organization, reduces leptin synthesis, enhances corticosterone concentrations and interferes with placental progesterone production (ronco et al., 2009; lin et al., 1997; tsutsumi et al., 2009; alvarez and chakraborty, 2011; stasenko et al., 2010; kawai et al., 2002). endoplasmic reticulum (er) is essential for survival and normal functioning of cells and cadmium also has an effect on er (ferri and kroemer, 2001; rao et al., 2004). er chaperons causes the folding of nascent proteins but if these client proteins exceed the chaperons, er undergoes stress which in turn reduces the placental development and fetal growth (iwawaki et al., 2009; lian et al.,*corresponding author: aftabac@yahoo.com 67 vol 4 (2), july 2013; 67-74 2011; yung et al., 2008). if the fetus is exposed to cadmium then it can impair child’s iq, birth weight, birth length and head circumference (tian et al., 2009; gonzalez-cossio et al., 1997; salpietro et al, 2002; osman, 2000; ronco et al, 2009; gundacker et al., 2010; zhu et al, 2010; lin et al., 2011). cadmium toxicity is found to be responsible for this stress related damage to placenta (wang et al., 2012). since cadmium has a long half-life in the human body; the placental burden is affected by the length of gestation (kantola et al., 2000). arsenic is a metalloid that occurs in environment both naturally and due to human activities.it is present in glasses, pesticides, food prservatives and pigments etc.human are exposed to arsenic via contaminated drinking water, rice, other grains and fish etc. (acsh, 2002). the two oxidation states of inorganic arsenic,arsenite (asiii) and arsenate(asv)can cross and accumulate in placenta (desesso et al., 1998; lindgren et al., 1984) upto a level five folds more than control (concha et al., 1998a). its accumulation can have deleterious vascular effects leading to placental abnormalities and less flow of blood which eventually lead to retarded fetal growth (hopenhayn et al., 2003). higher exposure to arsenic causes increased miscarraige rates, premature deliveries and lower birth rates(aschengrau et al., 1989). if the pregnant lady is exposed to lead contaminated air or food then lead may cross the placenta to enter into fetus by passive diffusion (goyer, 1990) and accumulates in syncytiotrophoblast cells of placenta and minimise cytochrome oxidase activity (reichrtova et al., 1998), thus may cause neurodevelopmental disorders and subclinical brain dysfunction in neonates. transportation of lead and cadmium through placenta occurs by binding to glutathione (gsh) forming a complex which is then transferd with the helpof abc family such as mrp1, mrp2 and pglycoprotein (gundacker et al., 2010; thevenod, 2010, unecb). in the present study placental cells were used to investigate the effect of heavy metals such as arsenic, cadmium and lead on proliferation and morphology of cells. materials and methods heavy metals: three heavy metals were used in this study including lead, cadmium and arsenic. stock solutions were prepared and filter sterilized with 0.2 µm filter (orange scientific). heavy metals solutions were stored at room temperature. isolation of placental cells (pcs): the placental tissue was collected after normal delivery. the tissue was cut into small pieces and added in sterile phosphate buffer saline (pbs) containing penicillin and streptomycin (20 ml pbs with 1 ml penicillin/streptomycin). the samples was immediately transferred to cell culture lab. the tissue pieces was washed multiples time with pbs and chorion layer of placenta was seprated by surgical knife. the placental tissue was cut into small pieces and added the piece on tissue culture dish. let the tissues to adhere for short time (5min) and completed medium (dmem containing glutamine, 10% fbs, 100 u/ml penicillin, and 100 µg/ml streptomycin) was added. the dish was incubated in co2 incubator at37oc with 5% co2 in humidified environment. observe the dish on daily basis to see migrating cells from tissue. cell culture and cv staining: when enough cells migrated out from tissue explants, the explants were removed and cells were treated with trypsin-edta (gibco, usa). the numbers of cells were counted and cultured them into new culture flasks under standard culturing conditions. when cells became confluent, the cells were stained with crystal violet (cv) (sigma). for staining, cells were washed twice by pbs, stained with 0.5% cv solution for 5-10 min. the stain was removed from plate with dh2o, until no stain come out. images were taken by inverted microscope. cytotoxicity assay: pcs were cultured in 75 cm2 68 vol 4 (2), july 2013; 67-74 69 vol 4 (2), july 2013; 67-74 tissue culture flask. the cells were incubated for 24 hrs at 37oc in a humidified environment with 5% co2 to grow the cells in monolayer. when cells grew to 90% confluency, they were washed with pbs, trypsinized with 1x trypsin-edta. the cells were counted with hemocytometer and 5 x 103 cells were added in each well of 96 wells plate with a total volume of 200 µl of complete dmem medium. cells were incubated for 24 hrs at 37 oc in a 5% co2 incubator with humidified environment. the old medium was replaced by 200 µl of fresh medium containing 0-10 µg/ml cadmium, 0-10 µg/ml arsenic and 0-100 µg/ml lead respectively and incubated the plates under the same culture conditions for 24 hrs. cytotoxic effects were tested by neutral red uptake method. aspirate treatment medium and incubated cells with neutral red medium for 3 h at 37oc. cells were washed twice with pbs and 150 µl of neutral red destain solution was added in each well and put the plate on shaker at 100 rpm for 10 min. the supernatant was taken and measured the differential absorbance at 492nm and 630 nm using elisa reader (humareader plus, human). all assays were done in triplicate. results isolation and crystal violet staining of pcs: when explant were cultured under standard culture condition, cells started moving out from explant after 3 days and in 5 days they covered the surface of whole plate. cells were trypsinized and cultured again. initially the cells were showing spindle shaped morphology and also these were very rapidly dividing cells. when cells were stained with cv to observe the morphology of cells after 2nd passage, the cells were appearing in between the spindle and flattened shape (fig. 2). cytotoxic effect of arsenic, cadmium and lead on pcs: pcs were exposed to different concentration of arsenic, cadmium and lead for 24 hrs but all the metals have different effect on proliferation of pcs. the maximum effect was observed by arsenic followed by cadmium and least was observed in case of lead. when lc50 was calculated for arsenic and cadmium, it was 2.52 and 6.2 µg/ml respectively. the percentage reduction in growth in case of lead on 24 hrs of exposure was 46% only and lc50 could not be calculated as lead did not have drastic effects on proliferation of pcs compared to arsenic and lead. discussion in the present study we investigated in vitro effect of three heavy metals on human placental cells. a significant difference in arsenic content in the human placenta under exposed (5.9 ng/g) and non-exposed (2.6ng/g) environmental conditions have been demonstrated (iyengar and rapp, 2001). it was observed in this study that when pcs were exposed to arsenic for 24 hrs, its effect was most severe compared to other heavy metals used in this study a b figure 1. growth of pcs. (a) the cells are migrating out from placental explant tissue. (b) morphology of pcs after confluent growth. the cells are appearing spindle shaped and showing similar morphology to that of mesenchymal stem cells (mscs). figure 2. morphology of pcs. the morphology of cells is in between spindle and flattened shape as clearly visible in both the images after cv staining. 70 vol 4 (2), july 2013; 67-74 on proliferation as indicated with the measurement of lc50 which came out to be 2.52µg/ml. when inside the cells arsenic mainly effects mitochondria, this result in changes in the trans-membrane potential and ultimately leads to death of cells (haga et al., 2005). cadmium also proved to be fatal for pcs but to a lesser extent than arsenic. on exposing pcs to cadmium for a period of 24 hrs the lc50 was calculated to be 6.2µg/ml. according to a studythe average concentration of cadmium under nonexposed environmental conditions was4 ng/g, with a range of 1–6 ng/g based on the wet weight of placenta (iyengar and rapp,2001).the placental cells causes interruption in cadmium passage either completelyor partially(baranowska, 1995; kuhnert et al., 1982; roels et al., 1978; schramel et al., 1988; korpela et al., 1986; needham et al., 2011) so there are chances that fetus get exposed to cadmium when higher level of cadmium is present inside the maternal body. according to different studies increase in double-stranded rna-activated kinase (pkr)-like er kinase (perk)-peif2a signaling is associated with decreased cellular proliferation in placenta (lian et al., 2011) which is enhanced due to cadmium exposure (wang et al., 2012). effect of lead was found to be much different than that of arsenic and cadmium and that could be due to permeability of lead to pcs. on exposure to lead for 24 hrs, the percentage reduction in proliferation of cells was calculated to be 46%.the average concentration of lead in placental tissue is approximately 34 ng/g, (normal range5–60 ng/g) (iyengar and rapp, 2001). there does not exist maternal-fetal barrier for lead and thus level of lead in fetus blood is nearly equal to maternal blood lead level (goyer, 1990), so lead exposure to mother during fetal development could be very deleterious for the health of developing fetus especially on nervous system. heavy metals are present in environment and it is very hard to avoid exposure of heavy metals. the exposure of heavy metals during pregnancy is great concern because it is not only dangerous for the life of mother but it could be lethal for developing fetus. the present study was conducted to show the antiproliferative activity of arsenic, cadmium and lead on human pcs. it was concluded that among these heavy metals arsenic is the most toxic heavy metal for pcs as it had maximum anti-proliferation effect, followed by cadmium and least anti-proliferative effect was observed in the case of lead. there is an alarming increase of heavy metals in environment in developing countries and it can result in many abnormalities and malignancies in human and this is also a serious danger to our future generations. figure 3. graphical representation of effect of arsenic and cadmium on pcs. there is gradual reduction in proliferation of cells with increase in concentration of arsenic and lead but arsenic has more severe effect even at lower concentration on proliferation of cells as is clearly shown in figure. there was gradual decrease in proliferation of cells on increase in concentration of cadmium. figure 4. graphical representation of effect of lead on pcs. lead did not have severe effect on proliferation of pcs and even at 100 µg/ml concentration there is just 46% reduction in proliferation of cells. 71 vol 4 (2), july 2013; 67-74 references acsh. 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(2010). maternal low-level lead exposure and fetal growth. e n v i r o n h e a l t h p e r s p e c t 11 8 : 1 4 7 1 – 1 4 7 5 . ca l l f o r pa p e r s publication is free of cost t biochemistry t biotechnology t botany t chemistry t environmental science t medical & health sciences t microbiology t molecular biology t pharmacy t physics t zoology t clinical research note:all manuscripts should be sent by an e-mail to the editor with your mobile number rads@juw.edu.pk or deanresearch@juw.edu.pk our aim is to provide a platform for budding scientists researchers, research scholars, academicians etc. to present their research findings in the field of biological and applied sciences. rads journal of biological research and applied sciences welcomes the submission of research papers, review articles and short communications in the following subject fields: the action of cmv promoter on expression of insulin in rat hepatocytes vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 1 r a d s j . b i o l . r e s . a p p l . s c i . 1 op e n ac c e s s f u l l l e n g t h a r t i c l e the action of cytomegalovirus (cmv) promoter on expression of genetically engineered insulin in rat hepatocytes gehad elsayed mahmoud department of genetics, faculty of science, alexandria, egypt a b s t r a c t background: background: insulin is hormone production in the pancreas that stimulates glucose uptake from blood to enter the body's cells, where it is converted into energy needed by muscles and tissues to function. the pancreatic cells are responsible for the secretion of insulin. in diabetes, the body cannot produce enough insulin or cannot use insulin effectively. objectives: in this study, we attempted to activate the hepatic cells to secrete insulin instead of the pancreatic cells. methodology: this was done by gene therapy; an excellent strategy to treat diabetes by supplying the correct wild type copy of a furin cleavable sites preproinsulin. the preproinsulin was extracted from the rat dna, cloned and mutated to generate the two furin cleavable sites responsible for the removal of c-peptide to form the two chains (a and b) for mature insulin production. this mutated insulin was derived by cytomegalovirus (cmv) promoter to express insulin by its transfection inside the primary rat hepatocytes using a non-viral vehicle to keep the hepatic cells healthier against the transfection. in vitro, the rat hepatocytes could not divide well as in vivo, but special hormones like insulin and dexamethasone lived longer and kept their function. results: the cmv promoter is strong and lead to overexpression of mature insulin inside rat hepatocytes leading to deterioration, the toxicity of hepatocytes and finally cell death. hepatocytes in vitro were more fragile and needed some modification to adapt to the secretion of insulin. conclusion: glucokinase or glucose transporter promoters were much more perfect than cmv. they can activate the hepatocytes to modulate the glucose level and so limiting the amount of insulin secreted. these promoters are weaker than cmv but much more perfect for hepatocytes. keywords cloning, cmv promoter, gene therapy, hepatocytes, mutation, non-viral vector, preproinsulin, transfection. *address of correspondence gehadelsayedkg@gmail.com article info. received: september 15, 2020 accepted: august 16, 2020 cite this article: mahmoud ge. the action of cytomegalovirus (cmv) promoter on expression of genetically engineered insulin in rat hepatocytes. rads j biol res appl sci. 2020; 11(1):1-8. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n the intra-cellular conversion for all known mammalian proinsulin to insulin involves cleavage at two paired basic sites present at either end of the c-peptide1. in both rat and human proinsulins, these are arg31-arg32 at the bchain/c-peptide junction and lys64-arg65 at the cpeptide/a-chain junction. the cleavage is carried out by the endopeptidases pc2 and pc3/12-4. most cell engineering approaches to target the non neuroendocrine cells that lack the specific endopeptidases (pc2 and pc3/1) required to process proinsulin into active mature insulin. to overcome this o r i g i n a l a r t i c l e the action of cmv promoter on expression of insulin in rat hepatocytes vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 2 r a d s j . b i o l . r e s . a p p l . s c i . 2 problem, many researchers used site-directed mutations to engineer proinsulin to be a substrate for furin5-7. the enzyme paired basic amino acid cleaving enzyme also known as (pace) is a golgi-associated propeptide endoprotease that is present in the constitutive secretory pathway of virtually all cells8. the most chosen target cells for insulin gene therapy are hepatocytes. although hepatocytes do not have the machinery to store insulin within secretory vesicles and secrete it in a regulated fashion, hepatocytes are attractive targets for insulin expression because they are closely related to the pancreatic β cells developmentally, play a very important role in glucose homeostasis, and are relatively easy to target9-11. m a t e r i a l s a n d m e t h o d s amplification of the preproinsulin gene from the rat spleen we extracted the rat genomic dna from the rat spleen by genomic extraction kit. amplification of the preproinsulin gene by pcr was performed using the designed primers: f: 5’catggccctgtggatgcgcttcctgcccctg3’ r: 5’gagttgcagtagttctccagttggtagagga3’ the pcr cycle step was done as: initial denaturation (94°c, 3min, and 1cycle), denaturation (94°c, 30sec), annealing (55°c, 30sec), extension (72°c, 1min, 35 cycles), final extension (72°c, 5min, 1cycle). the amplified gene was desalted and purified, then cloned into a cloning neb® pcr cloning vector (cat. no. e1202). further, the gene was mutated by substitution using site directed mutagenesis (cat. no. e0554s) to substitute lysine instead of glutamic acid in a chain, arginine instead of valine in the connected c peptide, and arginine instead of glutamine in the b chain. translated protein malwmrflpllallvlwepkpaqafvkqhlcgphlvea lylvcgergffytpksrrevedpqvpqlelgggpeag dlqtlalevarqkrgivdqcctsicslyqlenycnstop c-peptide wild type insulin r evedpqvpqlelgggpeagdlqtlalevarq k mutant type r kredpqvpqlelgggpeagdlqtlalevarr k cpeptide with tetrabasic furin cleavage site according to the nebase changer, the primers are at the b-chain/c-peptide: f:5'caagtcccgtcgtaaacgggaggacccgcaag3' r: 5'ggtgtgtagaagaaaccacgttccc3' at the c-peptide/a-chain: f:5’gaggttgcccggcggaagcgtggcattg3' r:5’cagtgccaaggtctgaag a tccc3' the vector used for mutation now contains the preproinsulin with the new three amino acids. after mutation, transformation into bacteria and isolation of the pure vector has proceeded. then digested the vector by one restriction enzyme (nrui), amplify the gene of interest by: f:ttggatccaccatggccctgtggatgcgcttcctg cccctg, r:gtgaattcgttgcagtagttctccagttggtagag gga insertion of preproinsulin gene into the expressing plasmid the expressing plasmid (#13031) with cmv promoter was received from the addgene, isolated, purified, and digested by the two restriction enzymes (ecori & bamhi), then purified and desalted by using purification kit (extraction kit cat. no. 20021), then by the same restriction enzymes we digested the amplified preproinsulin gene, the ligation step was later proceeded for the digested plasmid and digested gene. digested expressing vector (0.3µg/ul) 1.5µl digested purified preproinsulin gene (1µg/ul) 3µl ligase buffer 2µl distilled h2o 13µl (mix well) ligase 1µl culture of hepatocytes the culture of hepatocytes was performed according to the protocol by ling et al., with some modifications12. rat hepatic cells were purchased from cell biologics, shipped in suspension (cat. no. ra-6224f), centrifuged, then the action of cmv promoter on expression of insulin in rat hepatocytes vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 3 r a d s j . b i o l . r e s . a p p l . s c i . 3 suspended in 30ml warm william's complete medium (add the following to williams' medium e: l-glutamine 2mm, fetal calf serum (fcs) to 5%, dexamethasone to 100nm, cadmium chloride to 9µg/l, dimethyl sulfoxide (dmso) to 0.5%, penicillin to 100iu/ml and streptomycin to 100mg/ml), and then centrifuged and resuspended again in 20ml warm william's complete medium. cells were counted within the cell suspension using a hemocytometer and cell viability was determined by trypan blue staining. cells were plated in collagen coated plates, cultured at 37°c in 95% air and 5% co2. after 4hrs of culture, the cells were replaced with another medium that is serum free to maintain the morphology of the cells. after 24hrs of culture images, every 2hrs on the first day of culture and every 2 days was captured by olympus inverted microscope. after optimization the cell culture condition, transfection takes place using 1.5µl turbofect transfection reagent (cat. no. r0531) in 1ml culture medium after 24hrs of culture. examination of the released green fluorescent protein the expressed green fluorescent protein was detected under the inverted green fluorescent microscope. measurement of the amount of insulin released the insulin release was measured according to the concentration of glucose added by adding different concentrations of glucose from 5 to 25mm glucose to the media of the transfected cells. we measured the amount of insulin released according to the concentration of glucose added by the cohesion rat insulin elisa kit (cat. no. cek1622). sequencing the designing plasmid containing the genetically engineered insulin was forward and reverse sequenced using the abi prism big dyetm terminator cycle sequencing ready reaction kit (applied biosystems, germany). r e s u l t s to get the rat preproinsulin gene, we extracted the genomic dna from the rat spleen. then we amplified it by pcr amplification technique using the specific primer designed for the rat preproinsulin gene. the amplified preproinsulin gene was inserted into the cloning vector, transformed to get high yield and isolated by midiprep kit. the mutation proceeded in the two chains a & b. then gene of interest was amplified by digesting the vector and inserted into the expressing plasmid. so, we obtained a vector containing the preproinsulin gene with sites of cleavage derived by the cmv promoter. this vector was measured for its concentration to detect its purity (table 1) and sequenced for both forward and reverse sequence to be sure from the location of mutated preproinsulin gene (fig. 1 & 2). table 1. measurement of the concentration and purity of the extracted plasmid using uv spectrophotometer. (the dilution factor was x300). s. no. plasmid a260 a280 concentration (µg/ml) ratio 01 designing cmv expressin g plasmid 0.270 0.140 4050 1.92 fig 1. the forward sequence for designing cmv expressing plasmid showing the area between cmv promoter and the enhanced green fluorescent protein (egfp). fig 2. the reverse sequence for designing cmv expressing plasmid showing the area between egfp and cmv promoter. the action of cmv promoter on expression of insulin in rat hepatocytes vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 4 r a d s j . b i o l . r e s . a p p l . s c i . 4 the expressing plasmid was now ready to transfect into the primary rat hepatic cells. primary rat hepatocytes were washed, diluted, counted (table 2), then cultured in 6 well coated collagen plates with complete williams’ e medium in a humified atmosphere incubator for 2hrs and after that demonstrate the morphology of the cells (fig. 3). fig 3. the rat hepatocytes immediately after plating on 6 well collagen coated plate with williams’ e complete medium under an inverted microscope. shows the cells x200. table 2. the count of live and dead cells on haemocytometer to detect the viability and cell density of the rat hepatic cells. s. no. no. of squares live cells dead cells total count 01 1 71 8 79 02 2 52 0 52 03 3 85 4 89 04 4 68 6 74 05 average 69 5 74 viable cells = 69 x 104 x 2 = 138 x 104 cells/ml. total count = 74 x104 x 2 = 148 x104 cells/ml. viability % = viable/total =138/148 = 93% cell density = total count the rat hepatic cells were investigated after plating under the inverted microscope every 48hrs to detect the cell viability, division, and morphology (fig. 4). the cells show high viability already after plating, the viable cells count decreases day after day to reach about 32% at day 19 after plating. after optimization of the cell culture condition, transfection takes place using 1.5µl turbofect transfection reagent in 1ml culture medium after 24hrs of culture, in a 12 well collagen coated culture plate. then transgenic expression was analyzed 24-48hrs later. we will transfect the cells with the prepared expressing plasmid then examine the cell morphology and viability after transfection (fig. 5). the rat hepatocytes after transfection with the construct containing cmv promoter showed a high change in the morphology and viability, hepatocytes cannot stand more than 5 or 6 days, the viability decreases fast and day after day to reach about 5% on day 5 from transfection. the morphology high changed; the cells appeared very tired. the cmv promoter is strong which resulted overexpression for the gene controlled by it, the rat hepatocytes are fragile and cannot divide well in vitro to stand with this over expression. under the inverted fluorescent microscope, we examined the fluorescent cells which released the fluorescent fused insulin-egfp (fig. 6). the media around the hepatocytes showed no fluorescent even under addition different concentrations of glucose, but the cells show high fluorescent from the first day but, this fluorescence decreases every day till reach day 6 in which almost no fluorescence found. the over expression of the fused protein trapped inside the cells lead to the failure of hepatocytes to stand. fig 4. x100 rat hepatic cells after plating. (a) after 24hrs of plating. (b) after 5 days of plating. (c) after 7 days of plating. (d) after 11 days of plating. (e) after 17 days of plating. (f) after 19 days of plating. the action of cmv promoter on expression of insulin in rat hepatocytes vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 5 r a d s j . b i o l . r e s . a p p l . s c i . 5 fig 5. the rat hepatic cells after the transfection with the designing plasmid with cmv promoter x100. (a) the rat hepatocytes after 7hrs from transfection. (b) the rat hepatocytes after 24hrs from transfection. (c) the rat hepatocytes after 2 days from transfection. (d) the rat hepatocytes after 3 days from transfection. (e) the rat hepatocytes after 4 days from transfection. (f) the rat hepatocytes after 5 days from transfection. cmv promoter has not the ability to make balance out and in the cells, has not the ability to transport glucose from media to cells and vice versa. so, the insulin still trapped inside. glucose was measured in cell lysates and cell media of transfected culture cells 3 times by colorimetric glucose assay (table 3), after the addition of different concentrations of glucose (0-25mm) to the media and obtained the average. fig 6. the transfected rat hepatocytes with the designing plasmid with cmv promoter under the green fluorescent microscope x200. (a) the media after stimulation with 25mm glucose. (b) the media of free glucose. (c) the transfected hepatocytes after 24hrs from transfection. (d) the transfected hepatocytes after 2 days from transfection. (e) the transfected hepatocytes after 3 days from transfection. (f) the transfected hepatocytes after 5 days from transfection. (g) the transfected hepatocytes after 6 days from transfection. the action of cmv promoter on expression of insulin in rat hepatocytes vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 6 r a d s j . b i o l . r e s . a p p l . s c i . 6 table 3. the colorimetric method for the detection of the amount of glucose. s. no. parameters calorimetric glucose assay in mg/dl after 24hrs from the addition of different doses of glucose in the transfected primary hepatocyte culture medium 01 different doses of glucose added in mm free glucose 5mm 10mm 15mm 25mm 02 the designing plasmid with cmv promoter in media nd 81 210 240 301 in cell lysate nd nd nd nd nd note: present in both cell media & lysates after the addition of different amount concentration of glucose to the media of transfected cells with (the designing plasmid with cmv promoter) no detected amount of glucose can be found inside the cells, as the cmv promoter has not the ability to transport glucose in and out the cells. due to the amount of mature insulin released from the designing plasmid with cmv promoter, we measured it 3 times by cohesion rat insulin kit cat. no. cek1622). and get the standard curve for rat insulin (fig. 7). from this standard curve, we can obtain the different amounts of insulin released from the construct in both cell lysate and media in pg/ml from its relative o.d. and so make a relation between the concentration of glucose in mm and the concentration of insulin in pg/ml. fig 7. standard curve for rat insulin. for the designing of a plasmid with cmv promoter, this construct releases overdose for insulin only inside the cells not outside the medium, as this promoter has not the ability to transport the glucose in and out the cell and the insulin remain trapped inside the cells and not out to the media. so, the relation between the glucose and insulin inside the cell be constant and overexpression of insulin found in case of low dose or high dose glucose, and outside the cells (in media), no insulin found to make a relation (fig. 8). fig. 8. the relation between the concentrations of insulin released corresponding to different doses of glucose in cell lysate using the designing plasmid with cmv promoter. no response to glucose appeared even in high expression of insulin. d i s c u s s i o n many alternative approaches to treat type 1 diabetes mellitus (t1dm) using the correct type of gene or genetically engineered gene, without using pancreas transplantation, have been attempted. examples of such attempts include insulin production from various native cells, such as liver cells13-15. in our study, we engineered insulin to be correctly secreted from the liver cells. the mammalian cmv promoter is strong, constitutive transgene expression and would drive consistent, high level expression of insulin even during using low concentration of glucose. thus, a weaker promoter must be used to maintain low levels of insulin production to gain a successful insulin gene therapy in treating t1dm16. the action of cmv promoter on expression of insulin in rat hepatocytes vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 7 r a d s j . b i o l . r e s . a p p l . s c i . 7 c o n c l u s i o n our current study aimed to express insulin in vitro in rat hepatocytes, a strategy to try to adapt hepatic cells to secrete insulin instead of pancreatic cells, using a construct containing the furin cleavable sites preproinsulin derived by a cmv promoter. the mature insulin secreted successfully but the usage of this promoter was a bad choice, as the cmv promoter lead the insulin trapped inside the cells and no passage for the glucose in and out the media. the rat hepatocytes in vitro are fragile and not divide to stand with this insulin overexpression. the usage of the weaker promoter will work better like glucose transporter 2 promoter (glut2) or glucokinase promoter which can transport glucose in and out the cells. a c k n o w l e d g e m e n t s i am gratefully acknowledged mr karim morsi, eng. mohammed abdalla, dr. necolas and the research and medical technology institute, smouha, alexandria, egypt and the research lab of faculty of science, alexandria, egypt. l i s t o f a b b r e v i a t i o n s cmv cytomegalovirus dmso dimethyl sulfoxide fcs fetal calf serum glut2 glucose transporter 2 pace paired basic amino acid cleaving enzyme r e f e r e n c e s 1. steiner df. the proprotein convertases. curr opin chem bio. 1998: 2(1);31-9. 2. seidah ng, gaspar l, mion p, marcinkiewicz m, mbikay m, chretien m. cdna sequence of two distinct pituitary proteins homologous to kex2 and furin gene products: tissue-specific mrnas encoding candidates for pro-hormone processing proteinases. dna cell bio. 1990; 9(6):415-24. 3. smeekens sp, steiner df. identification of a human insulinoma cdna encoding a novel mammalian protein structurally related to the yeast dibasic processing protease kex2. j biol chem. 1990; 265(6):2997-3000. 4. smeekens sp, avruch as, lamendola j, chan sj, steiner df. identification of a cdna encoding a second putative prohormone convertase related to pc2 in att20 cells and islets of langerhans. proceedings of the nat acad sci. 1991; 88(2):340-4. 5. yanagita m, nakayama k, takeuchi t. processing of mutated proinsulin with tetrabasic cleavage sites to bioactive insulin in the non‐endocrine cell line, cos‐7. febs lett. 1992; 311(1):55-9. 6. groskreutz dj, sliwkowski mx & gorman cm. genetically engineered proinsulin constitutively processed and secreted as mature, active insulin. j bio chem. 1994. 269; 6241-5. 7. muzzin p, eisensmith rc, copeland kc, woo sl. hepatic insulin gene expression as treatment for type 1 diabetes mellitus in rats. mol endocrinol. 1997; 11(6):833-7. 8. van de ven wj, voorberg j, fontijn r, pannekoek h, van den ouweland am, van duijnhoven hl, roebroek aj, siezen rj. furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes. mol bio rep. 1990; 14(4):265-75. 9. hovorka r. closed-loop insulin delivery: from bench to clinical practice. nature reviews endocrinol. 2011; 7(7):385-95. 10. vehik k, ajami nj, hadley d, petrosino jf, burkhardt br. the changing landscape of type 1 diabetes: recent developments and future frontiers. curr diab rep. 2013; 13(5):642-50. 11. diabetes control and complications trial research group. the effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. new england j med. 1993; 329(14):977-86. 12. shen l, hillebrand a, wang dq, liu m. isolation, and primary culture of rat hepatic cells. jove. 2012; (64):e3917. 13. auricchio a, gao gp, yu qc, raper s, rivera vm, clackson t, wilson jm. constitutive and regulated expression of processed insulin following in vivo hepatic gene transfer. gene ther. 2002; 9(14):96371. 14. dong h, altomonte j, morral n, meseck m, thung sn, woo sl. basal insulin gene expression significantly improves conventional insulin therapy in type 1 diabetic rats. diab. 2002; 51(1):130-8. the action of cmv promoter on expression of insulin in rat hepatocytes vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 8 r a d s j . b i o l . r e s . a p p l . s c i . 8 15. burkhardt br, parker mj, zhang yc, song s, wasserfall ch, atkinson ma. glucose transporter-2 (glut2) promoter mediated transgenic insulin production reduces hyperglycemia in diabetic mice. febs lett. 2005; 579(25):5759-64. 16. roep bo, peakman m. antigen targets of type 1 diabetes autoimmunity. cold spring harbor perspectives in medicine. 2012; 2(4):a007781. prevalence of depression, anxiety and stress vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 27 r a d s j . b i o l . r e s . a p p l . s c i . 27 op e n ac c e s s f u l l l e n g t h a r t i c l e meta-analysis of prevalence of depression, anxiety and stress among university students shifa shaffique1,*, sehar shahzad farooq2, haseeb anwer1, hafiz muhammad asif3, muhammad akram4, soon ki jung2 1department of physiology, faculty of life sciences, government college university faisalabad, pakistan 2school of computer science and engineering, kyungpook national university, daegu, korea 3university college of conventional medicine, faculty of pharmacy and alternative medicine, the islamia university of bahawalpur, pakistan 4department of eastern medicine, government college university, faisalabad, pakistan a b s t r a c t background: the global burden on mental disorders have become too advanced with serious consequences on health and society. over the years, depression, stress, and anxiety have become more common symptoms that affect all overpopulation pyramid irrespective of gender. mental disorders are the second-most health indicator that causes morbidity. objectives: mental disorders affect the entire population and both genders equally. present study was conducted to carry out metaanalysis study to highlight the prevalence of anxiety, depression, and stress among university students. methodology: various search engines i.e. google scholar, duckduckgo, aol, baidu, yahoo as well as books and newsletter were used to collect the data. results: despite tremendous development in the era of health and education, there is significant prevalence (79.5%) of anxiety, depression, and stress in medical students that leads to morbidity and poor mental health disorders among these students. conclusion: it is concluded from meta-analysis study that prevalence of depression and anxiety is alarmingly high. there is a need of the hour to develop and design the mental health prevention programs and health education programs to overcome this issue. keywords anxiety, community, depression, medical student, stress. *address of correspondence shifa.shafiquee@gmail.com article info. received: july 23, 2020 accepted: august 21, 2020 cite this article: shaffique s, farooq ss, anwer h, asif hm, akram m, jung sk. meta-analysis of prevalence of depression, anxiety and stress among university students. rads j biol res appl sci. 2020; 11(1):27-32. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n according to the world health organization (who), mental disorders are the leading cause of morbidity and it affects the quality of life. studies regarding the emotional health of university students have revealed that depression symptoms are common among this population, and often are more than the general population for a reason that it is repository time of changing from adolescence to adulthood, thus, more prone to getting mental disorders1-3. various prospective and retrospective studies suggested that university students are highly liable to developmental disorders4. stress is an unavoidable relationship between the person and their threatening environment while depression and anxiety are the response of an individual which relates to spectrum of mood disturbance5-7. stress, depression, and anxiety are harmful for community and person as it can o r i g i n a l a r t i c l e prevalence of depression, anxiety and stress vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 28 r a d s j . b i o l . r e s . a p p l . s c i . 28 lead to the poor quality of life, effect on academic performance, negative behavior, and 4% chances of mental and physical ailments. stress can be defined as the unavoidable relationship between a society and a person or an event of physical and emotional tension8, 9. there are various emotional responses to stress i.e. feeling overwhelmed, agitation, sadness, and general unhappiness5, 10. however, depression is a serious medical disorder that is persistent feeling of unhappiness and sadness. it varies from mild to severe ranges of symptoms and can lasts for longer time period without any apparent etiological factor11, 12. depression and other associate disorders affect the student’s mental, physical and interpersonal functioning13, negative academic performance, causes clinical disorders like insomnia, anorexia nervosa etc.14, 15. in a research on depression at university, students had been assessed for mental and neurological disorders that accounted for significant proportion of the total global liability16. depression alone accounts for over 40% of the mental debilities. people with depression have a 40-60% chance of dying prematurely compared to the general population. it can cause many diseases like diabetes, myocardial infection, hypertension, and death due to suicides17-19. furthermore, anxiety can be defined as a natural response to stress. it may be accompanied by a feeling of fear or apprehension. it is a normal reaction with altered physiological response i.e. raised blood pressure, alteration in cardiac activity and respiratory response20-22. therefore, the present study was designed to evaluate and compare the prevalence of depression, stress, and anxiety among university students. m a t e r i a l s & m e t h o d s the comparative analysis was made by using various search engines i.e. google scholar, duckduckgo, aol, baidu, yahoo as well as books, print media (newsletters) and news bulletin to collect the data to access the prevalence of depression, stress, and anxiety among university students. data collection format to provide the most up to date information, the available epidemiological literature on prevalence of depression, stress, and anxiety have been collected since the beginning to the latest published data in 2020 including review papers, journal articles, conference publications and related blogs on the web. r e s u l t s comparative studies a study was conducted during initial phase of covid-19 to explore the psychological impact on college students of china. a total of n=7,143 students had participated in the study. among all student’s mental condition/illness, 75.1% showed normal mental status, followed by mild (21.3%), moderate (2.7%) and severe (0.9%)23. another webbased study was conducted in china to explore the level of stress and anxiety among students. the study was conducted on 1442 college students during first wave of pandemic and showed the raised level (26.63%) of anxiety and depression among students. the covid-19 is an event-related psychological phase. in this period, alarmingly high levels of mental health problems were reported24. various studies during the phase of covid-19 suggest the significant level of depression, stress and anxiety among students regarding their carrier, health, mental health, change in sleep pattern, use of social media, social disconnection and different attributes that causes negativity of thoughts and leads towards the regression25-27. the rate of depression in medical students is high because they face many difficulties during their medical curriculum28. another study conducted in brazil showed that the prevalence of mental disorders among university students was 79.5%, from which 40.5% were males29. the study suggested that mental health problems are more prone to be developed in males rather than females. the franciscan university counseling center reports that the consulting requests to psychiatrists are higher almost after every mid-term session at university, which suggests that mental disorders are alarmingly raised due to numbers of reasons i.e. post-graduation plans, academic performance and pressure to succeed which become three top concerns for depression in graduate students. these factors are demographically identified in various students and also indicates that depressive symptoms in students may also be associated with academic impairment30, 31. many universities are running the mental health resources programs for betterment of students and eliminate depressive symptoms32, 33. different studies prevalence of depression, anxiety and stress vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 29 r a d s j . b i o l . r e s . a p p l . s c i . 29 highlight that college students need more mental health services34-36. since, depressive disorder is an illness that involves body, mood, and thoughts, therefore, continuous stress and depression directly affects the mental health of students. anxiety takes several forms i.e. phobias, social anxiety, obsessive-compulsive, and post-traumatic stress disorders 37-40. quality of life (qol) and training efficiency of students affect due to professional challenges, demands and responsibilities directly or indirectly41, 42. general stress, depression and anxiety symptoms are introduced with irrational beliefs43, 44. furthermore, stress relating to professional and student lif e is very important for learning, finding and attempting to force out of life45. a web-based longitudinal study was conducted in japan to estimate correlation of stress with acculturative and connectedness among international and domestic students. they found a high rate of stress among international students (37.8%) as compared to domestic students (29.85%)46. another study was conducted on 236 students enrolled in a nursing course to investigate the relationship between adverse childhood experience and post-traumatic stress. they revealed a higher level of stress in individuals who had adverse childhood experience47. a study was accompanied in the u.s during the election-2016 documented an eventrelated stress among students. the study revealed that one out of four students met the criteria of the eventrelated stress48. a china-based longitudinal study (1892) discussed the effect of stress, depression and anxiety in males and females. a total of 1888 students were taken from 15 different universities of equal gender. the study revealed that averagely both male and female were suffering from mild anxiety illness. the level of anxiety is more prevalent in females than males, while depression is more prone to be present in females49. another study was conducted to correlate the adverse childhood experience with the prevalence of stress and showed a positive correlation of the adverse childhood experience with the level of stress. however, parents counselling and moral support among 321 individuals reported no mental illness50. in a study, anxiety, and depression with the mental status of a person was correlated. these two indicators are mostly found in the students of the pharmaceutical and medical field. to observe depression and anxiety, two types of tests i.e. beck anxiety inventory and beck depression inventory are used51. the anxiety and stress are two major factors that drive students or individuals to take their live and commit suicide. students with learning disabilities have higher rates of depression and anxiety52. research work was published in china to notify the effect of uncertainty stress on mental disorders. this study consists of 11,594 individuals from 50 different universities. the student daily stress questionnaire (sdsq) were used as a gold standard to find the correlation effect. they showed 22.8% mental disorders in medical students and positive correlation between the uncertainty stress and mental disorders53. various other factors such as culture, values, environment and sex difference can also determine the level of depression and anxiety among students54, 55. a study was conducted to find out the level of stress and depression in the crossculture model of asian, americans and european americans. a total of 414 participants were taken to elucidate the effect of cross-culture. they observed a positive relationship between stress and depression with a cross-cultural model55. women are more likely to depress as compare to males at any stage of the life56. a study was conducted in 2002 at medical university lahore, pakistan which showed 43.7% and 19.5% of females to be reported for anxiety and depression, respectively. they concluded that females were more prone to mental illness than males57. study evidence from the different universities in 37 countries suggested the emotional factors that may cause depression and stress. the unclear vision also leads to stress and anxiety. the students who come from other countries also face these problems. they get tension and anxiety as they live in very different societies, traditions, culture, and values. they are having a tough time adapting to the new climate 58-60. therefore, they usually go into depression easily. sleep disturbances are also a specific and significant variable of depression. moreover, the anxiety also leads towards depression in the first regression30, 61, 62. the medical field is highly significant in terms of a burden for the students. the medical students encounter increased depression and anxiety especially during their transition toward clinical settings. the relentless burden of anxiety and depression is taking them to the point of burn-out. prevalence of depression, anxiety and stress vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 30 r a d s j . b i o l . r e s . a p p l . s c i . 30 mental, social, and physical wellbeing have different dysfunctions, and psychosocial issues in students are often revealed because of anxiety63. a study by shams uddin (2013) revealed that stress, depression, and anxiety are very common in students. those are also linked to the difficulties of living professionally and academically. the working environment, performance pressure and culture of the academic institutes also affect the mental health of the students. the students belonging to the low socioeconomic background and financial vulnerability comparatively express depression, anxiety, and stress more64. the study also showed that depression or stress is less prevalent in students as compared to anxiety. age is also contributing as an important factor with these three variables. however, there is need for further exploration to develop an appropriate support services and better intervention programs49, 65, keeping the fact in mind that university students are at high risk of anxiety and depression. c o n c l u s i o n in the present study, cumulative, in-depth analysis of vast data from previously conducted researches validates that university students are at high risk of developing stress, anxiety, and depression. among university students, medical students are more prone to become affected. it mainly affects academic performance, quality of life, serious health disorders and in several cases, lead to suicidal attempts. certain measure such as primary and secondary health education programs and strategies to cope the certain unfavorable circumstances should be adopted to overcome and reduce the prevalence of depression, stress, and anxiety in overall population. a c k n o w l e d g e m e n t s this research was supported by basic science research program through the national research foundation of korea (nrf) funded by the ministry of education, science and technology (nrf-2019r1a2c1010786). this study was also supported by the bk21 plus project (sw human resource development program for supporting smart life) funded by the ministry of education, school of computer science and engineering, kyungpook national university, korea (21a20131600005). l i s t o f a b b r e v i a t i o n covid-19 corona virus disease qol quality of life sdsq student daily stress questionnaire who world health organization r e f e r e n c e s 1. crow g. social solidarities: theories, identities and social change: open university; 2002. 2. ross se, niebling bc, heckert tm. sources of stress among college students. social psychol. 1999; 61(5):841-6. 3. yakel e, rieh s, st. jean 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frequency of depression, anxiety and stress among university students. pak j med sci. 2020; 36(5): 4-12. prevalence of transfusion transmitted infection in replacement and voluntary blood donor syeda kanwal zehra1*, saira bano1, shazia tabassum hakim1, sayyada ghufrana nadeem1 1department of microbiology, jinnah university for women, karachi-74600, pakistan. abstract the aim of this study is to present the status of transfusion –transmitted infections among the apparently healthy donors so as to increase the awareness of complications of blood transfusion and make the clinicians more vigilant with regard to judicious use of blood. screening of blood is mandatory for providing safe blood. the magnitude of transfusion transmitted infections (tti) varies from country to country depending on tti’s load in that particular population. transfusion transmitted infections create significant burden on health care system. donor selection is of paramount importance because infected individuals serve as an asymptomatic reservoir and a potential source of transmission. this retrospective study was carried out in healthy blood donors in the age group of 18-60 years; study was done on blood units collected from replacement and voluntary donors. the serum samples were screened for hepatitis b surface antigen (hbsag); antibodies against hiv i and ii, hepatitis c virus (hcv) by chemiluminescent microparticle immuno assay (cmia) method. screening for syphilis was carried out by rpr rapid plasma reagent. seropositivity of transfusion transmitted disease in replacement donors was 1.93% in hepatitis b surface antigen, 2.41% in hepatitis c virus, 0.09% in hiv and 1.15% in syphilis. voluntary donors had low infectivity rate as compare to replacement donor. k e y w o r d s : c m i a , h b s a g , r e p l a c e m e n t d o n o r s , s y p h i l i s , t t i , vo l u n t a r y d o n o r s . introduction blood is life. blood transfusion is the transfer of biological material from man to man. blood has been used in 1930s for the various indications (zafar, 2000). blood transfusion, a fundamental part of medicine and surgery, also carries the risk of transfusion-related infections like hepatitis b and c, hiv and syphilis, malaria and infrequently toxoplasmosis, brucellosis and viral infections like cmv, epstein barr virus and herpes (widmann fk, 1985). transfusion of blood and blood products is a lifesaving fact. measuring their sternness, who has recommended pre-transfusion blood test for hiv, hbv, hcv and syphilis as obligatory (world health organization). all these diseases are competent of causing major mortality, morbidity along with an economic load for both the affected person and the country. with every one unit of blood transfusion there is 1% chance of transfusion related complications including transfusion transmitted infections (widmann fk, 1985) the safety of blood and its products has gained tremendous importance since the documentation of blood born viral infections in regularly transfused patients. these may be thalasemic children, oncology patients or aplasticanemia patients (naseem and haq, 2009). detection of hepatitis b surface antigen (hbsag) in blood is diagnostic for infectivity with hbv and in blood bank screening for hbsag is approved routinely to detect hbv infection (bhattacharya et al., 2007). similarly antibodies to hepatitis c virus (anti-hcv) are used to detect hcv infectivity*corresponding author: juskz_110@hotmail.com 56 vol 4 (2), july 2013; 56-63 (olokoba et al., 2008). safe blood transfusion is the expression which refers to legal and coherent healing use of blood and blood products. world health organization (who) recommendation of safe blood transfusion is provision of well-matched blood which are cross matched and screened at least for five who suggested transfusion transmitted infections, human immunodeficiency virus (hiv), hepatitis c (hcv), hepatitis b (hbv), syphilis and malarial parasite (dipta & rahman, 2009). objectives of the study were to guesstimate occurrence of hepatitis b and c in blood donors of local area and recommend measures for safe blood transfusion. in this study, we intended to estimate the prevalence of hiv, hbv, hcv and syphilis among apparently healthy blood donors. it would also reflect on the blood safety dimensions and can be carefully extended to provide estimation about the disease burden in the community. hiv: hiv is the contraction used for the human immunodeficiency virus. hiv attacks the body's immune system. normally, the immune system produces white blood cells and antibodies that attack viruses and bacteria. the infection combating cells are called t-cell lymphocytes. months to years after a person is infected with hiv, the virus destroys all the t-cell lymphocytes. this disables the immune system to shield the body against diseases and tumors. various infections will be able to develop; the opportunistic infections take benefit of the body's destabilized immune system. these infection which normally won't cause severe or lethal health problems will eventually cause the death of the hiv patient (rombauts, 1997). untreated hiv disease is a continual progressive process that begins with infection, is often followed by a "primary hiv syndrome," and progresses in adults over a median period of more than 10 years to the late stage: aids. from the time of infection, the virus continuously and swiftly replicates, mutates, and as a result diversifies and evolves in response to selective pressure. immune system damage also begins upon infection. the viral load and the volume of this process occur in lymphoid tissue, and the immune system struggles to seize the process in check. slowly, but persistently, the process destroys vital components of the host immune system. progression is often accelerated in infants with prenatal hiv infection. eventually the host becomes increasingly liable to and eventually dies as a result of complications of opportunistic infections and malignancies resulting from immune system dysfunction (cohen, 1998). epidemiology of hiv: the viruses that are the source of acquired immune deficiency syndrome (aids), human immunodeficiency virus types 1 and 2 (hiv1 and hiv-2), are lentiviruses (hahn, 2000). the global prevalence of hiv-1 has stabilized at 0.8%, with 33 million people breathing with hiv/aids, 2.7 million new infections, and 2.0 million aids deaths in 2007 (the official site of the national aids program of pakistan nacp)during the last three decades, the hiv pandemic has entered our realization as an inexplicable disaster. hiv and aids has already taken a dreadful human toll, lying assert to millions of lives, inflicting pain and grief, causing dread and doubt and threatening economic devastation. while sub-saharan africa remains the nastiest affected region in the world, there is mounting concern about the emergence of hiv and aids in asia, which is home to more people than any other constituency of the world. though the epidemic in asia is less severe than in other parts of the world and rates of infectivity in the general population remain comparatively low, it is clear that hiv has ongoing spreading swiftly through the region and enormous asian population is gradually more at risk. (usaid, 2004) pakistan is the second largest country in south asia that stands only a few steps at the back of india and nepal in terms of hiv epidemic. in spite of many efforts, the hiv infection rate has enlarged considerably over the past few years and infect, the country has moved from a low occurrence to intense epidemic with hiv prevalence of more than 5% among injecting drug users (idus) in at least eight major cities (nacp-hasp, 2007).other high-risk groups, such as men who have sex with men (msm), hijra sex workers (hsws) and female sex workers 57 vol 4 (2), july 2013; 56-63 58 vol 4 (2), july 2013; 56-63 (fsws), also look set to reach this threshold level. many bridging populations, totaling almost five million persons, are in direct sexual contact with these groups and are exposed to hiv infection through unprotected sexual activity. the heterogeneity and interlinking of high risk injecting and sexual behavior, combined with low levels of hiv knowledge and prevention, and high levels of other sexually transmitted infections (stis), indicates that hiv could spread rapidly to marriage partners or sex clients and result in generalized epidemic.hiv is not currently a dominant epidemic in pakistan. however, the number of cases is growing (the official site of the national aids program of pakistan nacp) recent variation in the epidemiologic data regarding hiv prevalence in pakistan suggests alarm. unpublished data indicate a recent increase in hiv prevalence from surveys of iv drug users (idus) all around the nation. according to sindh aids control program hiv seroprevalence rate of idus in karachi is about 9%. in 2002-2003 family health international sponsored cross-sectional studies suggest 23% seroprevalence among idus in karachi. hiv/aids surveillance project (hasp) of canadian international development agency results in 2004 suggested 26% seroprevalence among idus in karachi. now hiv is reported routinely among idus in other areas of county,including rawalpindi, lahore and sargodha (the official site of the national aids program of pakistan nacp)there is no doubt that pakistan has moved from a nascent epidemic into one with a intense epidemic, with high rate of prevalence in at least one major high risk population. hepatitis b and c: the hepatitis b and c virus infections are known to occur in the general population and because of blood and blood products transfusion, it has made the stipulation of safe blood difficult, that’s why screening of blood completely necessary (olokoba, et al, 2008). these viruses infect mankind from centuries but in 1970s hepatitis b virus was discovered and hepatitis c in 1990s. both viruses can cause acute illness where patients have acute jaundice and very high serum alanine transaminase (a part of liver function tests). in legal period 90% of adults, incisive infectious disease b resolves within ii-tierce calendar month with entire improvement. the assumption is different in new born and up to quintet geezerhood of age where full recovery is seen only in 10% while xc% develop chronic liver disease. in acute liver disease c full recovery appear in only 20-thirty% of cases while70 eighty% require treatment for clearance of disease as they may go on to develop chronic liver disease. if the mother is hbsag empiricist philosophy and is also positive for hbeag (a marker for infectivity) than there is a large integer% encounters that she will transmit the disease to her newborn. if the mother is hbsag positive and not hbeag positive, than the probability goes down to 20-30%. it is therefore recipient to equal pregnant parent for these 2 viruses. for the former scenario the newborn has to receive 2 immunizations within large integer hours of biological process, the hyper immune globulin at one internet site and the infectious disease b vaccine at another site. for the latter forgather only hepatitis b vaccine is recommended. epidemiology of hbv & hcv: hbv linguistic process come alongs all over the world. the who has estimated that there are more than two 1000000000 hbv infected folk and about 37digit million chronic vectors worldwide. there are approximately 620 000 hbv related killing each year. in addition, approximately 4-5 million new hbv infections occur worldwide each year, of which a quarter progresses to liver unwellness. in high endemic region, like key asian republics, sou'-east asia, sub-saharan africa and the parrot sink, the hbv carrier rate is over 8%. in low endemic body part, like the united states, northern europe, land and surroundings of south america, hbsag ratio is less than 2%. the middle east, some eastern dweller countries and the mediterranean basin are considered areas of liaise endemicity with a carrier rate between 2% and 8% (who. department of communicable diseases surveillance and response). in many countries, after the introduction of mass immunization operation, the generality of hbv notably changed, resulting in a reduction of the hbsag carrier place 59 vol 4 (2), july 2013; 56-63 and hcc incidence (zanetti, 2008). it was estimated that somebody cancer transpose approximately 4% of all new cancer cases diagnosed worldwide and that more than 50% of viscus person were attributable to hbv. the highest epoch-adjusted incidence rate (> 20 per c 000) was reported from southeast asian and sub-nilo-saharan language individual countries that are endemic for hbv linguistic process. up to 90% of child septic during the first year of life and 30%-50% of children infected between one to four years of age develop chronic linguistic process and about 25% of someone who become chronically infected during childhood die from hbv-related liver cancer or cirrhosis (who-expanded programme on immunization hepatitis b vaccine). pakistan is endemically high in hbv (noorali et al., 2008) with nine million people infected with hbv (hepatitis prevention & control program sindh 2009) and its infection rate is on a steady rise (hakim et al., 2008). the reason may be the deficiency of proper upbeat facilities, poor system status and less public consciousness about the transmission of major communicable diseases including hbv, hcv and hiv (alam et al., 2008). globally, an estimated 130-170 million persons (2%3% of the populace\'s population) are beingness with hepatitis c virus (hcv) infection (who, 2004). this corruptness, particularly in its chronic form, is associated with sizable morbidity and mortality. more than 350 000 destruction are attributed to hcv infection each year, most of which are caused by inhabitant cirrhosis and hepatocellular malignant neoplastic disease. (perz et al., 2006). an estimated 27% of liver unwellness and xxv% of hcc can be attributed to hepatitis c planetary, and disease revenue enhancement can be even more square in countries with a high burden of communication. for example, in japan, up to 90% of all reported cases of hepatocellular carcinoma caused by hcv infection (perz et al., 2006). in development countries where resources and facilities may be significantly limited, the ratio of hcv is higher as compared to the developed homo. roughly 10 million make full have been septic with hcv in asian nation. the majority of patients have acquired their infection through unsafe injections, apply of syringes and needles and community barber patronise used for face up and armpit shaving. more than two-thirds of hcv patients were 40 to fifty years old. development of a vaccine against hcv is more problematic fixed cost to the genetic nonuniformity of the representation. however, with large integer% of hcc in processing countries attributable to hcv (approximately 93,000 cases per class) a vaccine would make a major contribution to cancer hindrance. (wild and hall, 2000) syphilis: spirochetes are spiral-shaped, motile bacteria that are divided into the families s p i r o c h a e t a c e a e , b r a c h y s p i r a c e a e , a n d leptospiraceae (paster and dewhirst. 2006). spirochaete taxon, which are members of the family spirochaetaceae, are fastidious anaerobiotic or microaerophilic host associated spirochetes. while the majority of spirochete taxonomic group are found in the ?ora of humans and animals, a few species are infective for humans. treponema globus pallidus subspp. pallidum, endemicum, and pertenue, the functionary of reproductive organ syphilis, endemic syphilis, and framboesia, respectively, together with treponema carateum, the agent of pinta, are primary pathogens of humans that have eluded in vitro cultivation (stamm, 2001). spirochete denticola and certain other oral treponema species that are associated with man periodontal illness are arable, opportunistic pathogens (holt and ebersole, 2006). syphilis causes symptoms in three different stages (primary, secondary, and tertiary), separated by periods when no symptoms go on (latent stages). syphilis is extremely transmittable during the primary election and secondary traveling. infection is usually beddispersed through intimate contact. a single sexual encounter with a material assemblage who has early-represent sexually transmitted disease statement in unhealthiness about one third of the time. the bacterium enters the body through mucous animal tissue, such as those in the channel or mouth, or through the life. within hours, the bacteria reach nearby liquid body substance nodes, and then spread throughout the body through the bloodstream. 60 vol 4 (2), july 2013; 56-63 syphilis can also be spread in other construction. it can infect a fetus during pregnancy, causing birth defects and other trouble. it can also be spread through contact with skin. however, the bacteria cannot go retentive outside the homo body. people with syphillus often have other pathological process, including other sexually transmitted diseases (stds).each stage of indication (primary, secondary, and tertiary) is progressively worse. if not tempered, syphilis can persist without grounds for many years and may price the heart or brainiac, maybe leading to death. if detected and treated former, syphilis can be cured, and there is no permanent alteration. epidemiology of syphilis: syphilis is a multistage disease that is usually genetic through contact with active lesions of a intimate soul or from an infected meaning woman to her craniate (paster and dewhirst, 2006). efforts to eliminate syphilis have met with only mild success (hook and peeling, 2004). the world health organization (who) estimated that there were 12 large integer new caseful of venereal disease in 1999, with more than xc% of the cases occurring in developing countries congenital syphilis is a leadership cause of stillbirth and prenatal mortality in many of these countries (schmid et al., 2007; tichonova et al., 1997). despite the accessibility of new diagnostic tests and antibiotic medical care, venereal disease has reemerged in several developed countries. while the widespread epidemics of syphilis that occurred in soviet union in the 1990s and more new in nationalist china mostly mired heterosexuals, smaller occurrent in the united suggest, canada, and european country predominately involved men who have sex with men (schmid et al., 2007). however, late amount in syphilis rates for u.s. women and infants suggest that heterosexually transmitted syphilis may be an emergent problem in the cooperative states (martin et al., 2009.). a major concern associated with increased rates of venus's curse is that someone, early venereal infection (i.e., primary and secondary initiate) improve transmission of human immunode?ciency virus (virus infection) by 2to 5-fold, thus promoting the spread of hiv (douglas, 2009). by comparing international studies with our study on seroprevalence of syphilis in idus, our resolution are quite heights (25.89%). when compared with the study results of 2005 in idus of karachi (18.2%) (government of pakistan. 2005. however a significant (p<0.05) decrease in syphilis was observed in karachi. when the same study results (3.9%) in lahore. syphilis in idus were compared with our study results (25.89%), a decrease in seroprevalence of syphilis was noted, which was highly significant (p<0.001). materials and methods total 13170 donors who were recruited in this study and all are males. we collected the blood from voluntary and replacement blood donors. replacement donors are donors who donate blood on behalf of their patient and family members. voluntary blood through blood donation camps organized by man organizations and student bodies including the pupil of medical colleges, commercial enterprise organization in metropolis. name, age (eighteen-60 years), sex, edible fruit of bear, address and contact confine were recorded for each donor, while giving them a unique identification number. donors with history of any febrile illness in the late past, free weight loss, uncontrolled looseness of the bowels, recent distort, liver disease, cardiovascular disease, pulmonary disease, evilness, epilepsy, malaria, strange or overweening bleeding, recent donation of rakehell, receipt of descent, and winning contraindicated medicine were excluded. detailed history of immunization was taken. weight, pulse, blood somaesthesia and somesthesia were recorded for each donor. screening for fern genus was done clinically along with copper sulfate specific gravitation method. scrutiny was made for any marks of drug abuse or any skin lesions/ infections at the vein puncture information processing system. a written informed consent was taken from each donor before the blood donation. proper sterilization and other care were taken during the blood collection and blood units were stored by appropriate method acting. after collection all samples are screened for human immunodeficiency virus (hiv) by hiv 61 vol 4 (2), july 2013; 56-63 ag/ab combo kit on abbott’s (i-2000 and i 2000 sr) chemiluminescent microparticle immuno assay technology (cmia).hepatitis b by hbsag qualitative and hbsag qualitative ii kit on abbott’s (i-2000 and i 2000 sr) chemiluminescent m i c r o p a r t i c l e i m m u n o a s s a y t e c h n o l o g y (cmia).hcv by anti hcv kit on abbott’s (i2 0 0 0 a n d i 2 0 0 0 s r ) c h e m i l u m i n e s c e n t microparticle immuno assay technology (cmia). treponema pallidum: detection of treponemal antibodies (reagin) by rapid plasma reagin test (rpr) with spinreact and teco kits. calibrators and controls are run before samples all controls are satisfactory. results and discussion most of the bestower, who were recruited in this study, came from public sentience. it was to be noted that maximum public presentation of donors came from get on group eighteen-60 years. it may reflect proper awareness among the young population about blood donation. blood donors are of various type voluntary blood donors, replacement or family blood donors, plasma and platelet donor and commercial donors. form the above donors the voluntary donor are the safest donor because they donate blood regularly and know their medical status. replacement donor and family donors are at risk because they donate blood when they need blood, they are the donors which can transmit the infectious agent which they have in family. they don’t know anything about their medical status. commercial donors are highly at risk because they sell their blood to fulfill their needs they mostly are iv drug user because of hygiene and close contacts with each other. in this study we work on two types of donor replacement and voluntary donors. this is the comparative study of infective rate between these two types of donors. according to our data which we collected from the husaini blood bank karachi. we screened 13170 donors from which 6585 are replacement donors and 6585 are voluntary donors. these donors are screened for hepatitis c, b hiv and syphilis. we observe that in voluntary blood donors 260 donors are reactive and in replacement blood donor is 368 donors. this observation shows that the replacement donor show high reactive rate as compare to voluntary as shown in table i. highest reactive rate among voluntary blood donors was observed of hepatitis b while highest reactive rate among replacement blood donors was observed of hcv. all donors were screened for hiv replacement donor show more hiv positive then voluntary donors infective. voluntary donors are 0.02% infected among them and replacement donors are 0.09% infected. (table ii). in replacement donation 127 donors and 115 voluntary donors are hbsag reactive. in our data the ratio of reactivity in both types of donor is very similar because in our region hepatitis b is the major disease which is growing very fast and lack of awareness about this disease is also a reason of less difference in a reactive numbers. anti hcv only 99 persons are found reactive of hepatitis c in voluntary rather 159 individual are reactive in replacement blood donor. in syphilis 76 donors are r.p.r positive in replacement donor but 45 donors are reactive. in pakistan hepatitis c is the major disease which is spread by transfusion of blood and its products table i. reactive rate of voluntary blood donors and replacement blood donors. vol.reactive rate 115 99 1 45 260 rep.reactive rate 127 159 6 76 368total reactive test hepatitis b hepatitis c hiv syphilis vol.donor 6585 6585 6585 6585 rep.donor 6585 6585 6585 6585 table ii. reactive rate of hepatitis b, c.hiv and syphilis. % 0.09 0.02 hbsag +ve 127 115 donors replacement voluntary total screened 6,585 6,585 hiv +ve 6 1 % 1.93 1.75 hcv +ve 159 99 % 2.41 1.50 rpr +ve 76 45 % 1.15 0.68 62 vol 4 (2), july 2013; 56-63 and needle stick injuries. we also perform hepatitis c pcr for these donor and we found one (1) donor is rna positive among 6585 donors in replacement but voluntary donors are all are rna negative. rpr done for syphilis but this test have shown false positive result when in some conditions such as iv drug use, lyme disease, certain types of pneumonia, malaria, pregnancy, systemic lupus erythematosus (sle) and some other autoimmune disorders, tuberculosis (tb) but these donors are also screened for malaria as well because it is also a mandatory test for screening as per who recommendation. that’s why the chance of false positive result is less in these donors. conclusion in pakistan the ration oaf replacement donor is 7 % and voluntary donor is 2.7 % .but according to our data it very from the donor survey in our country because we collect the specific data of specific range but the survey is done on yearly basis. the reactive rate is increased in voluntary donor because of less awareness about the blood donation and bias also occurs in verbal screening or donor recruitment because some people tell lie when we ask these question. we have to promote the voluntary blood donation and also give awareness to our young youth because some have a doubt in blood donation that if they donate blood they get infection or weakness. voluntary blood donation is the safest donor for the recipient. in our country we have less blood donation but the need of blood is high. references alam mm, zaidi sz, malik sa, naeem a, shaukat s, sharif s, angez m, khan a, butt ja.2007.serology based disease status of pakistani population infected with hepatitis b virus. bmc infect dis, 7:64. bhattacharya p, chandra pk, datta s, banerjee a, chakrabortys, rajendran k et al. 2007. significant increase in 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e s s f u l l l e n g t h a r t i c l e therapeutic attributes of stevia rebaudiana leaves in diabetic animal model fatma hussain*, javaria hafeez department of biochemistry, faculty of sciences, university of agriculture, faisalabad, pakistan. a b s t r a c t background: medicinal plants contain organic chemicals with different properties. as synthetic medications can create deleterious effects, therefore, use of safe natural medicinal adjuncts like stevia rebaudiana is endorsed. objectives: the purpose of this study was to assess the antidiabetic, antioxidant, antihyperlipidemic, hepatoprotective and renoprotective attributes of stevia rebaudiana leaves in the diabetic rat model. methodology: single dose of alloxan monohydrate was given to induce diabetes mellitus in the rats. plant extract treatment along with synthetic drug glibenclamide was given to rats for about 28 days to check their efficacies (antidiabetic, antioxidant, antihyperlipidemic, hepatoprotective and reno-protective) by using commercially available kits. results: treatment showed a significant decrease in blood glucose, glycated hemoglobin hba1c, and a rise in insulin, although it could not normalize these biomarkers after 28 days of treatment. catalase (cat) activity was restored yet it was not significantly improved in the case of superoxide dismutase (sod) and reduced glutathione (gsh). changes in lipid peroxidation products were trivial. ingestion of stevia rebaudiana significantly reduced alanine transaminase (alt) and aspartate transaminase (ast) levels, however, changes in gamma-glutamyl transpeptidase (ggt), and total protein (tp) were not significant. similarly, treatment with stevia rebaudiana reduced serum urea, creatinine and urinary albumin in diabetic animals. conclusion: it is established that stevia rebaudiana leaves have multiple benefits and can be an exceptional nutraceutical. keywords antidiabetic, antioxidant, hepatoprotective, renoprotective, stevia rebaudiana. *address of correspondence fatmauaf@yahoo.com article info. received: october 28, 2020 accepted: april 02, 2021 cite this article hussain f, hafeez j. therapeutic attributes of stevia rebaudiana leaves in diabetic animal model. rads j biol res appl sci. 2021; 12(1):1-7. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n stevia rebaudiana bertoni (asteraceae family) is taken as a sugar substitute or artificial sweetener. different types of glycosides present in stevia leaves are stevioside, rebaudioside (a to e), steviolbioside, and isosteviols1-4. stevioside with about 300 times more sweetness than sucrose has commercial value worldwide as an alternative sugar. numerous studies have suggested that steviosides possess anti-infertility, hypotensive, antiseptic, diuretic, anti-fertility, cardiotonic, antimicrobial, anticancer, antidiabetic and antioxidant potentials5-7. phytoconstituents contribute prominent insulinotropic, glucagonostatic, and antiplatelet effects8. stevia rebaudiana leaves contain various antioxidant compounds such as ascorbic acid and phenolic compounds including flavonoids and tannins9. it is found that stevia powder enhances the reduced level of glutathione10. one of the major therapeutic interventions to cure diseases is the use of medicinal plants, an option recently focused by researchers and the pharmaceutical sector. as synthetic medications can create deleterious effects, therefore, use o r i g n a l a r t i c l e therapeutic attributes of stevia rebaudiana leaves vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 2 of safe natural medicinal adjuncts like stevia rebaudiana is endorsed. henceforth, current project was planned to assess the selected therapeutic effects of stevia rebaudiana leaves in diabetic animals. m a t e r i a l a n d m e t h o d s plant sample collection and extract preparation s. rebaudiana leaves powder was extracted in methanol at ambient temperature. after solvent removal, it was dried on rotary evaporator. same procedure was performed thrice after 3 days and final plant sample was reconstructed in dimethyl sulfoxide11. experimental design healthy albino male rats (weight 200-250g) were kept in stainless steel cages at adequate environmental conditions (25 ± 2°c temperature, 60 ± 5% humidity, 12hours lightdark cycle). ethical approval for trial was granted by institutional biosafety committee. induction of diabetes mellitus was done by alloxan monohydrate (60mg/kg body weight) freshly prepared in normal saline as 5% solution. rats with blood glucose levels more than 200mg/dl at fasting were assumed as diabetics. animals were divided into four groups of seven animals each. first (dc) and second (nc) groups were diabetic control and normal control (non-diabetic), respectively. these groups were given standard diet and water ad libitum. diabetic rats that received s. rebaudiana extract (12-15mg) and synthetic drug glibenclamide were included in third (dse) and fourth (dgd) groups, respectively. s. rebaudiana extract dose of 500ppm/kg body weight/day was given orally for 28 days. all the rats were slaughtered, blood sample and tissue homogenates were collected12. biochemical analysis antidiabetic (fasting glucose, glycated hemoglobin hba1c, insulin), antioxidant (superoxide dismutase, lipid peroxidation product, glutathione, catalase), antihyperlipidemic (total cholesterol, hdl-c, ldl-c, triglycerides), hepatoprotective (ast, alt, ggt), and reno-protective (serum urea, creatinine, urinary albumin) profiles were assessed by commercially available kits. statistical analysis the data were presented as mean ± sem. a comparison among variables was assessed by analysis of variance technique with p-value < 0.05 as significant. spss software was used for data analysis. r e s u l t s antidiabetic potential a momentous decrease in blood glucose, glycated hemoglobin hba1c, and a rise in insulin were estimated after treatment with stevia (table. 1). the diabetic control group had higher blood glucose levels as compared to normal control. in diabetic group treated with stevia extract (dse), significant decline (151.8 ± 29.49mg/dl) in elevated glucose concentrations was observed as compared to dc group (265.6 ± 30.26mg/dl). however, synthetic drugs exhibited the highest hypoglycemic effect (120.1 ± 39.47mg/dl). diabetic animals (dc) had reduced insulin concentration (4 ± 0.3u/ml) and treatment with stevia extract improved these levels in dse group (7.5 ± 1.8u/ml). whereas, diabetic glibenclamide drug group (dgd) showed highly significant effect as drug treatment increased insulin levels (9.3 ± 1.7u/ml). regarding glycated hemoglobin levels, significant decline was noted in the dse group (6.9 ± 1.6%) as compared to dc group (10.6 ± 0.7%). glibenclamide treatment showed highly significant reduction (4.9 ± 0.7%) as compared to dc group. although treatment with stevia extract exhibited significant changes in blood glucose, hba1c and insulin levels, it could not normalize these biomarkers after 28 days of treatment. antioxidant potential alloxan administration reduced hepatic antioxidant enzyme activities significantly by 59% for sod, 31.7% for gsh, and 50.57% for cat in diabetic animals (table. 1). decreased concentrations of antioxidant enzymes in diabetes are associated with increased generation of reactive oxygen species (ros). when rats were fed with stevia leaves extract, although cat activity was restored yet it was not significantly improved in the case of sod and gsh. cat activity was enhanced by up to 37.7%. changes in lipid peroxidation products were trivial in the present study. therapeutic attributes of stevia rebaudiana leaves vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 3 antihyperlipidemic activity prominent antihyperlipidemic activity of plant extract was observed as the administration of s. rebaudiana extract reversed hyperglycemic condition by significantly reducing almost all lipid parameters, except high density lipoprotein-cholesterol (hdl-c) that remained almost the same after treatment (table. 1). table 1. antidiabetic, antioxidant and antihyperlipidemic effects of stevia rebaudiana. parameters / potentials study groups dc nc dse dgd antidiabetic profile serum glucose (mg/dl) 265.6 ± 30.26 99.5 ± 13.12 151.8 ± 29.49* 120.1 ± 39.47** serum insulin (u/ml) 4 ± 0.3 12 ± 1.2 7.5 ± 1.8* 9.3 ± 1.7** hba1c (%) 10.6 ± 0.7 5.5 ± 0.6 6.9 ± 1.6* 4.9 ± 0.7** antioxidant profile superoxide dismutase (units/mg protein) 3.69 ± 0.18 9.20 ± 0.42 5.07 ± 0.31ns 6.43 ± 0.42** glutathione peroxidase (red) (units/mg protein) 6.44 ± 0.29 9.43 ± 0.43 7.10 ± 0.30ns 8.52 ± 0.43** catalase (units/mg protein) 41.20 ± 3.76 83.33 ± 2.93 66.22 ± 3.16* 71.30 ± 3.60** lipid peroxidation (mm/100g of tissue) 1.77 ± 0.08 0.85 ± 0.03 1.51 ± 0.44ns 0.96 ± 0.07** antihyperlipidemic potential triglycerides (mg/dl) 166.81 ± 4.3 89.15 ± 6.37 136.33 ± 11.4* 78.32 ± 5.7** total cholesterol (mg/dl) 210.2 ± 4.77 54.67 ± 7.64 179.17 ± 3.49* 123.75 ± 2.80** hdl-c (mg/dl) 20.86 ± 3.65 27.99 ± 7.21 23.892 ± 2.5 27.54 ± 1.67 ldl-c (mg/dl) 132.98 ± 0.9 8.85 ± 5.61 109.19 ± 1.81* 56.55 ± 2.97** data expressed as mean or percentage ± sem of triplicate measurements for groups of seven animals each. dc: diabetic control group, nc: normal non-diabetic control group, dse: diabetic stevia extract group, dgd: diabetic glibenclamide drug group. * significant p < 0.05 as compared to diabetic control. ** highly significant p < 0.05 as compared to diabetic control. ns: non-significant. figure 1. hepatoprotective effect of stevia rebaudiana. therapeutic attributes of stevia rebaudiana leaves vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 4 figure 2. renoprotective effect of stevia rebaudiana. data expressed as mean or percentage ± sem of triplicate measurements for groups of seven animals each. dc: diabetic control group, nc: normal non-diabetic control group, dse: diabetic stevia extract group, dgd: diabetic glibenclamide drug group. hepatoprotective activity ingestion of stevia rebaudiana significantly reduced ast and alt levels, however, changes in ggt and total protein were not significant (fig. 1). the diabetic group (dgd) that received synthetic drug showed maximum reduction in hyperlipidemia, as drug ameliorated alloxan-induced hepatic damage normalizing the lipid profile. renoprotective activity stevia rebaudiana had a protecting role as it declined renal function parameters (fig. 2). treatment with stevia rebaudiana reduced serum urea, creatinine and urinary albumin in diabetic treated group. data expressed as mean or percentage ± sem of triplicate measurements for groups of seven animals each. dc: diabetic control group, nc: normal non-diabetic control group, dse: diabetic stevia extract group, dgd: diabetic glibenclamide drug group. d i s c u s s i o n bioactive compounds like steviosides exert antihyperglycemic potential by affecting the pancreas to enhance insulin secretion3-4, 13. in current study, although dse group exhibited decline in blood glucose, hba1c levels, and increase in insulin concentration, but it was unable to normalize these parameters. current results are in accordance with previous studies13-16. insulin, pparγ and mrna expression levels are increased by treatment with stevia. steviosides and other bioactive ameliorate alloxan-induced hepatic damage, leading to an increase in insulin concentrations and hypoglycemic effect13, 14. earlier, shivanna et al.17 stated that stevia rebaudiana leaf extracts significantly reduced glucose and hba1c. steviosides may sensitize insulin receptors and stimulate pancreatic beta cells to release insulin in diabetic animals. the overall effect is improved carbohydrate metabolism15. glycated hemoglobin levels were reduced by stevia treatment (dse group) as compared to diabetic control group (dc) but stevia treatment was not effective in normalizing this biomarker. phytoconstituents present in s. rebaudiana leaves may initiate glycogenesis15 thereby, reducing hba1c levels in dse group and exhibiting an antidiabetic attribute. regarding antioxidant effects of stevia in present study, current results are not consistent with the previous reports that recognized the achievement of either normal or increased levels of antioxidant enzymes along with reduced lipid peroxidation after administration of stevia extracts10, 17. it has been reported that a new polyphenol family known as chlorogenic acid along with therapeutic attributes of stevia rebaudiana leaves vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 5 hydroxycinnamic acid and quinic acid have exceptional antioxidant potential along with stevioside18, 19. our results can be justified by the fact that stevia has the potential to reduce free radicals directly instead of moderating mitochondrial antioxidant enzymes system20. reduction in triglycerides (tg), total cholestrol (tc) and low density lipoprotein-cholestrol (ldl-c) might be due to activation of lipase activity or receptor up-regulation by saponin glycosides of stevia rebaudiana leaves21. however, hdl-c level was increased by 14%. it is noteworthy that none of the lipid profile parameters was restored to the normal level either by plant extract or by the synthetic drug. the results of the present research are confirmed by earlier findings of assaei et al.,13; singh et al.,14; and ritu and nadini16 that stevia has the potential to ameliorate hyperlipidemia. assaei et al.,13 and najafi et al.,22 stated that stevia caused a decline in aminotransferases activities in treated rats and prevented hepatic damage. previously, ibrahim et al.,9 reported that in diabetic control rats, serum alt and ast levels were significantly higher than normal rats. in another study, kuntal and kathirity23 observed that the levels of liver enzymes (ast, alt, ggt) were significantly decreased with the administration of stevia extract. probably stevia rebaudiana exhibits hepatoprotective potential due to its stimulation of nuclear factor-e2-related factor 2 nrf2 expression, repression of nuclear factor kappa-b and obstruction of various profibrogenic signaling paths24. renoprotective role of stevia evaluated in the current study is supported by previous research works17, 25-28. it is suggested that stevia attenuate diabetes-induced renal impairment by opposing oxidation, inflammation, and apoptosis through bio-signaling pathways26. c o n c l u s i o n it is concluded stevia leaves have strong therapeutic potentials especially with reference to diabetic manifestations and it can be incorporated in daily diet as a value-added healthy component. further studies on underlying mechanisms by which s. rebaudiana defends against different ailments should be planned. e t h i c a l a p p r o v a l institutional biosafety/ bioethics committee gave ethical approval (d. no. 5497/ oric; dated: 11-10-2018) for in vivo animal trial under the guidelines of national biosafety committee (nbc) and punjab biosafety rules 2014. c o n f l i c t o f i n t e r e s t none. f u n d i n g s o u r c e none. a c k n o w l e d g m e n t s the authors appreciate the encouragement and support of prof. dr. amer jamil, department of biochemistry, faculty of sciences, university of agriculture, faisalabad, pakistan. l i s t o f a b b r e v i a t i o n s ast aspartate transaminase alt alanine transaminase cat catalase dc diabetic control nn normal non-diabetic control dse diabetic stevia extract dgd diabetic glibenclamide drug ggt gamma-glutamyl transpeptidase gsh glutathione (reduced) hdl-c high density lipoprotein cholesterol ldl-c low density lipoprotein cholesterol nn normal non-diabetic control pparγ peroxisome proliferator-activated receptor-γ ros reactive oxygen species sem standard error mean sod superoxide dismutase spss statistical package for social sciences tc total cholesterol tg triglyceride therapeutic attributes of stevia rebaudiana leaves vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 6 r e f e r e n c e s 1. momtazi-borojeni aa, esmaeili sa, abdollahi e, sahebkar a. a review on the pharmacology and toxicology of steviol glycosides extracted from stevia rebaudiana. curr pharm des. 2017; 23(11):1616‐22. 2. ruiz-ruiz jc, moguel-ordoñez yb, segura-campos mr. biological activity of stevia rebaudiana bertoni and their relationship to health. crit rev food sci nutr. 2017; 57(12):2680‐90. 3. rojas e, bermúdez v, motlaghzadeh y, mathew j, fidilio e, faria j, et al. stevia rebaudiana bertoni and its effects in human disease: emphasizing its role in inflammation, atherosclerosis and metabolic syndrome. curr nutr repor. 2018; 7(3):161-70. 4. samuel p, ayoob kt, magnuson ba, wölwer-rieck u, jeppesen pb, rogers pj, et al. stevia leaf to stevia sweetener: exploring its science, benefits, and future potential. j nutr. 2018; 148(7):1186s-1205s. 5. hossain s, alam mb, asadujjaman m, islam mm, rahman ma, islam ma, et al. antihyperglycemic and antihyperlipidemic effects of different fractions of stevia rebaudiana leaves in alloxan induced diabetic rats. int j pharm sci res. 2012; 2(7):1722-9. 6. moradi mt, asadi-samani m, bahmani m, shahrani m. medicinal plants used for liver disorders based on the ethnobotanical documents of iran: a review. int j pharm tech res. 2016; 9(5):407-15. 7. carrera-lanestosa a, moguel-ordóñez y, seguracampos m. stevia rebaudiana bertoni: a natural alternative for treating diseases associated with metabolic syndrome. j med food. 2017; 20(10):933‐ 43. 8. salehi b, lópez md, martínez‐lópez s, victoriano m, sharifi‐rad j, martorell m, et al. stevia rebaudiana bertoni bioactive effects: from in vivo to clinical trials towards future therapeutic approaches. phytother res. 2019; 33(11):2904-17. 9. ibrahim na, el-gengaihi s, reyad s, el-rigal ns, hamed ma. hypoglycemic, hypolipidemic and antioxidant potency of the aqueous extract of stevia rebaudiana (bert.) leaves. inter j pharm pharma res. 2015; 3(3):15-32. 10. sharma r, yadav r, manivannan e. study of effect of stevia rebaudiana bertoni on oxidative stress in type-2 diabetic rat models. biomed aging pathol. 2012; 2(3):126-31. 11. mehmood n, zubair m, rizwan k, rasool n, shahid m, ahmad vu. 2012. antioxidant, antimicrobial and phytochemical analysis of cichorium intybus seeds extracts and various organic fractions. iranian j pharm res. 2012; 11(4):1145-51. 12. rekha n, balaji r, deecarman m. effect of aqueous extract of syzgium cumini pulp on antioxidant defense system in streptozotocin induced diabetic rats. iranian j pharmacol ther. 2008; 7(2):137-45. 13. assaei r, mokarram p, dastghaib s, darbandi s, darbandi m, zalf, et al. hypoglycemic effect of aquatic extract of stevia in pancreas of diabetic rats: pparγdependent regulation or antioxidant potential. avicenna j med biotechnol. 2016; 8(2):6572. 14. singh s, garg v, yadav d. antihyperglycemic and antioxidative ability of stevia rebaudiana (bertoni) leaves in diabetes induced mice. int j pharm pharma sci. 2013; 5(2):297-302. 15. ahmad u, ahmad rs. antidiabetic property of aqueous extract of stevia rebaudiana bertoni leaves in streptozotocin-induced diabetes in albino rats. bmc complement altern med. 2018; 18(1):179-85. 16. ritu m, nandini j. nutritional composition of stevia rebaudiana, a sweet herb, and its hypoglycaemic and hypolipidaemic effect on patients with non-insulin dependent diabetes mellitus. j sci food agric. 2016; 96(12):4231‐4. 17. shivanna n, naika m, khanum f, kaul vk. antioxidant, anti-diabetic and renal protective properties of stevia rebaudiana. j diabetes complic. 2013; 27(2):103-13. 18. myint kz, wu k, xia y, fan y, shen j, zhang p, et al. polyphenols from stevia rebaudiana (bertoni) leaves and their functional properties. j food sci. 2020; 85(2):240-8. 19. gawel-beben k, bujak t, niziol-lukaszewska z, antosiewicz b, jakubczyk a, karas m, et al. stevia rebaudiana bert. leaf extracts as a multifunctional source of natural antioxidants. mol. 2015; 20(4):546886. 20. vaško l, vašková j, fejerčáková a, mojžišová g, poráčová j. comparison of some antioxidant properties of plant extracts from origanum vulgare, salvia officinalis, eleutherococcus senticosus and stevia rebaudiana. in vitro cell dev biol anim. 2014; 50(7):614-22. 21. ahmad u, ahmad rs, arshad ms, mushtaq z, hussain sm, hameed a. antihyperlipidemic efficacy of aqueous extract of stevia rebaudiana bertoni in albino rats. lipids health dis. 2018; 17(1):175-9. therapeutic attributes of stevia rebaudiana leaves vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 7 22. najafi f, goodarzi n, zangeneh mm, zangeneh a, hagh-nazari l. antidiabetic and hepatoprotective effects of bitter fraction of stevia rebaudiana alcoholic extract on streptozotocin-induced diabetic male mice. j rafsanjan uni med sci. 2017; 16(6):493-504. 23. kuntal das, kathiriy ak. hepatoprotective activity of stevia rebaudiana bert. leaves against thioacetamide induced toxicity. turk j pharm. 2012; 9(3):343-52. 24. ramos-tovar e, flores-beltrán re, galindo-gómez s, camacho j, tsutsumi v, muriel p. an aqueous extract of stevia rebaudiana variety morita ii prevents liver damage in a rat model of cirrhosis that mimics the human disease. ann hepatol. 2019; 18(3):472‐9. 25. kujur rs, singh v, ram m, yadava hn, singh kk, kumari s et al. antidiabetic activity and phytochemical screening of crude extract of stevia rebaudiana in alloxan-induced diabetic rats. pharma res. 2010; 2(4):258-63. 26. potočnjak i, broznić d, kindl m, kropek m, vladimirknežević s, domitrović r. stevia and stevioside protect against cisplatin nephrotoxicity through inhibition of erk1/2, stat3, and nf-κb activation. food chem toxicol. 2017; 107:215-25. 27. el-mesallamy a, hussein s, hussein aam, mahmoud sa, el-azab km. reno-protective effect of methanolic extract of stevia rebaudiana bertoni and bioactive phenolic compounds in type-1-diabetes. egyptian j chem. 2018; 61(4):609-15. 28. rizwan f, yesmine s, banu sg, chowdhury ia, hasan r, chatterjee tk. renoprotective effects of stevia (stevia rebaudiana bertoni), amlodipine, valsartan and losartan in gentamycin-induced nephrotoxicity in the rat model: biochemical, hematological and histological approaches. toxicol rep. 2019; 6:683-91. introduction aloe vera has a long history of use as a therapeutic agent with many reported medicinal properties. amongst its therapeutic properties, it has been shown to have anti-inflammatory activity (azfal, et al. 1991; malterud, et al. 1993) immunostimulatory activity (ramamoorthy and tizard, 1998), and cell growth stimulatory activity (tizard, et al. 1994; rodriguezbigas, 1988). furthermore, activity against a variety of infectious agents has been attributed to aloe vera; for instance, antibacterial (ferro, et al. 2003), antiviral (kahlon, et al. 1991) and anti fungal (kawai, et al. 1998). aloe has a history of traditional use by native americans for stomach disorders and intestinal disorders including constipation, hemorrhoids, and colitis and colon problems. it is said to be a natural cleaner, powerful in penetrating tissues, relieving pain associated with joints and muscles, bactericidal, a strong antibiotic, virucidal when in direct contact with long periods, fungicidal, anti inflammatory, instrumental in increasing circulation to the area, breaking and digesting dead tissue and moisturizing tissues. the skin absorbs aloe vera up to four times faster than water, it appears to help pores of the skin open and receive moisture and nutrients of the plants. additionally, numerous constituents within aloe vera have demonstrated enhancement of immune system functioning within the body. aloe vera also has the ability to stimulate macrophages (davis, et al. 1997). the efficacy of aloe vera liquid as an antibacterial agent is shown to have a wide range of activity against gram positive and gram negative bacteria. the antimicrobial agents of aloe vera gel was reported to effectively kill or greatly reduce or eliminate the growth of staphylococcus aureus, klebsiella pneumoniae, streptococcus pyogenes, pseudomonas aeruginosa, escherichia coli, propionibacterium acne, helicobacter pylori and salmonella typhi (lawless, et al. 2000; pugh, et al. 2001; reynolds, et al. 1999; reynolds, et al. 1999). antimicrobial activity of aloe vera against pathogenic bacteria shumaila malik1*, sayyada ghufrana nadeem1 and shazia tabassum hakim2 1medical mycology research and reference laboratory, department of microbiology, jinnah university for women, karachi-74600, pakistan. 2virology and tissue culture laboratory, department of microbiology, jinnah university for women, karachi-74600, pakistan. abstract present study was conducted to determine the antimicrobial activity of aloe vera with ethanol against gram positive (staphylococcus aureus) and gram negative (escherichia coli, klebsiella pneumoniae and pseudomonas aeruginosa). the agar well diffusion method was used to test the antimicrobial activity. the result shows that maximum inhibition observed against staphylococcus aureus and klebsiella pneumoniae (1mm---4mm), minimum inhibition was observed against pseudomonas aeruginosa while negligible activity observed against escherichia coli. this is important to used aloe vera for cosmetic and food purpose. key words: antimicrobial agent, pathogen, aloe vera, medicinal plant. *corresponding author: sho.malik-14@hotmail.com 24 vol 3 (1), january 2012; 24-26 material and methods sample collection: the aloe vera plant was collected from bahria nursery, karachi. collection of plant material: the collect plant gel was freeze dried and then grinds to get crude extract. the crud extract is filtered through whatmann filter paper. the plant extract were prepared according to the method describe by (ahmed, et al. 1998) with minor modification. briefly 1 gm gel extract was mix in 5ml of ethanol and mixed well and kept it under shaker for overnight (lin, et al. 1999) after overnight incubation the mixture was filtered through whatmann no.1 paper and it was evaporated at room temperature. after evaporation pellet was resuspended with 0.5ml of dimethyl sulfoxide (dmso) using micro syringe and recollect it for further use. selection of solvent: polar solvent such as ethanol were used for this study. bacterial strain: bacterial strains were obtained from the department of microbiology, jinnah university for women, karachi. the bacterial strains such as escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae and staphylococcus aureus were used for antimicrobial assay. all the strains were grown in nutrient agar at ph 7.2 and incubated at 370c for 24 hour. testing of antimicrobial activity: take 0.5 ml each bacterial culture inoculated into each mark nutrient broth incubate at 370c for 24 hour. make a lawn by spreading 0.1 ml of each culture on their respective nutrient agar plates. take sterile borer and heat on flame then make well on agar plate. inoculate 0.1ml aloe vera extract on respective well and incubate at 370c for 24 hour. result and discussion antimicrobial activity of aloe vera with ethanol against staphylococcus aureus, klebsiella pneumoniae, pseudomonas aeruginosa and escherichia coli was detected. the zone of inhibition observed was range between 1mm to 4mm as shown in table i. table i. zone of inhibition against test organisms. aloe vera as medicinal important plant gives best result against all pathogen that used in study but not show inhibitory effect on escherichia coli. this result could be responsible for the popular use of aloe vera gel and leaf to relieve many types of gastrointestinal irritations (foster, 1999; grindlay and reynolds, 1986) since staphylococcus aureus form part of the normal microbial flora of the skin, upper respiratory tract and intestinal tract (cheesbrough,1984). references azfal m., ali r.a., hassan, h., sweedan, n., dhami, m.s. 1991. identification of some prostanoids in aloe vera extracts.êplanta medica,ê57: 38-40. cheesbrough, m. 1984. medical laboratory manual for tropical countries. great britain: cambridge university press. pp. 372-391. davis, h.r. 1997. aloe vera: a scientific approach. new york: vantage press. ferro, v.a., bradbury, f., cameron, p., shakir, e., rahman, s.r., stimson, w.h. 2003. in vitro susceptibilities of shigella flexneri and vol 3 (1), january 2012; 24-26 test organism staphylococcus aureus klebsiella pneumoniae pseudomonas aeruginosa escherichia coli zone of inhibition 4 mm 3.1 mm 1.7 mm 0 mm 25 vol 3 (1), january 2012; 24-26 26 streptococcus pyogenes to inner gel of aloe barbadensis miller.antimicrobial agents and chemotherapy,47(3):1137-1139. foster, s. 1999. aloe vera: the succulent with skin soothing cell protecting properties. herbs for health magazine. health world online. available at:http://www.healthy.net/library/articles /hfh/aloe.htm> [accessed 14 june 2012]. kahlon, j., kemp, m.c., yawei, n., carpenter, r.h., mcanalley, h.r., shannon, w.m., mcdaniel, b.h. 1991. in evaluation of the synergistic antiviral effects of acemannan in combination with azidothymidine and acyclovir. molecular biotherapy, 3: 214-223. kawai, k., beppu, h., shimpo, k., chihara, t., yamamoto, n., aggatsu, t., ueda, h., yamada, y. 1998. in vivo effects of aloe arborescens miller var natalensis berger (kidachi aloe) on experimental tinea pedis in guinea pig feet. p h y t o t h e r a p y r e s e a r c h , 1 2 : 1 7 8 1 8 2 . lawless, j., allan, j. 2000. the clinical composition of aloe vera,in: aloe vera natural wonder cure. london: thorsons, publishing ltd. pp 161-171. pugh, n., ross, s.a., elsohly, m.a., pasco, d.s. 2001. characterization of aloeride, a new high molecular weight polysaccharide from aloe vera with potent immunostimulatory activity. j. agri. food. chem., 49(2): 1030-1034. ramamoorthy, l. and tizard, i.r. 1998. induction of apoptosis in a macrophage cell line raw 264.7. molecular pharmacology, 53: 415-421. reynolds, t., dweck, a.c. 1999. aloe vera leaf gel: a review update. j. ethnopharmacol. 68: 3-37. urch, d. 1999. aloe vera the plant. in: aloe vera nature’s gift. bristol: blackdown publication. pp 8-17. antimicrobial activity of fresh and old honey aliya hayat1* and gul jahan1 1department of microbiology, jinnah university for women karachi pakistan abstract honey is a thick sugary natural substance that is produced by honeybee from the nectar of flowers of different plants. the objective of this study was to determine the antimicrobial activity against different clinical isolates and to compare the activity of fresh honey with old honey. unbranded honey samples were taken from a local market of karachi. one sample was fresh and other was four year old. this activity was assessed by agar well diffusion method. honey samples were diluted as 100%, 80% and 50% with saline as control. undiluted and diluted old honey was inhibitory to gram positive and gram negative species. fresh and old honey showed good antibacterial activity against e. coli, acinetobacter, s. paratyphi a, s. dysenteriae, k. pneumoniae, citrobacter, s. aureus, s. saprophyticus, b. subtilis, with the strongest activity seen against p. fluorescens. s. typhi was resistant to both old and fresh honey. s. pneumonia was inhibitory to fresh honey only. both honey samples also inhibit candida a l b i c a n s ( y e a s t ) . a n t i b a c t e r i a l a c t i v i t y o f o l d h o n e y i s m o re t h a n f re s h h o n e y. keywords: honey, antibacterial activity, agar well diffusion method, bacteria. introduction honey is a sweet food made by bees using nectar from flowers. the variety produced by honey bees (the genus apis) is the one most commonly referred to and is the type of honey collected by beekeepers and consumed by humans. honey bees transform nectar into honey by a process of regurgitation, and store it as a primary food source in wax honeycombs inside the beehive. honey gets its sweetness from the monosaccharides fructose and glucose, and has approximately the same relative sweetness as that of granulated sugar. it has attractive chemical properties for baking, and a distinctive flavor that leads some people to prefer it over sugar and other sweeteners. most microorganisms do not grow in honey because of its low water activity of 0.6. however, honey sometimes contains dormant endospores of the bacterium clostridium botulinum, which can be dangerous to infants, as the endospores can transform into toxin-producing bacteria in the infant's immature intestinal tract, leading to illness and even death (anonymous, 2011). honey is an ancient remedy for the treatment of infected wounds, gastroenteritis, gastric ulcers, diabetes etc. it can be effective against multi-drugresistant strains of bacteria. the antibacterial properties of honey include the release of low levels of hydrogen per oxide while some honeys have an additional phytochemical antibacterial component (molan, 2001). in current study, the antimicrobial activity of local pakistani ber/sidder honey was examined against 20 different bacterial strains. material and methods honey sample: two samples of honey (1 fresh and one 3 years old) were used for antibacterial potential against different micro-organism. clinical isolates: twenty different isolates belonging to 11 genera of microorganisms viz., e. coli (5) pseudomonas *corresponding author: aliyaap@yahoo.com 17 vol 3 (1), january 2012; 17-19 fluorescens (2), klebsiella pneumonia (1), citrobacter (1), salmonella typhi (1), salmonella para typhi a (1), acinetobacter (1), shigella dysenteriae (1), s.aureus (3), streptococcus pyogenes (1), b. subtilis (1) and candida albicans (2) were obtained from essas’s laboratory, abbasi shaheed hospital and al-khidmat welfare hospital karachi were included in study. dilution of honey sample: 100% (pure honey), 80%, 50% and 0% (control) dilutions of old and fresh honey samples were prepared as following protocol. preparation of mcfarland nephlometer standard: mcfarland nephlometer standard tube no 5 was prepared by mixing 0.05 ml 1.175 % , barium chloride and 9.95 ml 1% sulfuric acid, to get bacterial density of 1.5 x 108 cfu / ml . prepration of inoculum: nutrient broth (5ml) was inoculated with given culture and incubated for 24 hours at 37 o c. after incubation, tubes were matched with 0.5 mcfarland tube to standardize the inoculum. agar well diffusion method: a lawn was prepared by using a sterile cotton swab dipped in standardized inoculum on nutrient agar plates. wells were cut by sterile borer having 5mm diameter. diluted samples were poured in each well. plates were incubated at 37º c for 24 hours. measure the zone of inhibition of respected wells. results discussion in this study two dark coloured berry honey samples were used. they were analysed by agar well diffusion method against different gram negative and positive bacteria. ziziphus jojoba is another economically important plant in pakistan. it is commonly known as ‘sidder’ or ‘ber’ and is indigenous plant species to pakistan it is a bush like tree found in karak, kohat and bannu districts of n.w.f.p, attock, chakwal and êmianwali districts of punjab, karachi, hyderabad and nawabshah districts of sindh province (qamer et al., 2007). most bacteria showed similar growth inhibition patterns for both honey tested, but some variations were detected. fresh and old honey showed good antibacterial activity against p. fluorescens .the zone s. pneumoniae b. substilis gram negative salmonella paratyphi a shigella dysenteriae e .coli k.pneumoniae p. florescense s. typhi citrobacter acinetobacter yeast candida albicans 01 01 01 01 05 01 02 01 01 01 02 13 23 23 20 26 27 15 20 28 12 20 22 20 20 25 16 20 28 19 19 21 17 40 11 18 honey sample 10 ml 8ml 5ml sterile distilled water 2ml 5ml 10ml dilution (%) 100% 80% 50% control vol 3 (1), january 2012; 17-19 table 2. antimicrobial activity of old honey organism gram positive s. aureus s. saprophyticus s. pneumoniae b. substilis gram negative s. paratyphi a shigella dysenteriae e. coli k. pneumoniae p. florescense salmonella typhi citrobacter acinetobacter yeast candida albicans no of isolates 02 01 01 01 01 01 05 01 02 01 01 01 02 control (0%) 100 % 20 30 32 24 30 30 34 25 28 35 25 25 80% 15 30 21 25 20 27 20 35 20 20 18 50% 22 20 20 20 15 27 26 36 30 15 zone of inhibition (mm) organism gram positive s. aureus s. saprophyticus no of isolates 02 01 100 % 30 41 80% 20 32 50% 15 30 zone of inhibition (mm) table 1. antimicrobial activity of fresh honey control (0%) 19 vol 3 (1), january 2012; 17-19 of inhibition obtained in this study indicated the 50% (minimum) concentration of honey needed to kill 50 % of bacteria. all honey solutions were freshly prepared before each assay. the above information shows that in microbiological and clinical tests, honey offers advantages in controlling bacterial growth and treatment of certain health problems. even in modern day society, the medical use of honey still has a place. both of the honey samples (old and fresh) inhibited s. aureus at concentrations of 100 %, 80 % and 50 % dilutions of honey. e. coli was sensitive against both honey samples at concentrations of 100%, 80 % and 50 % dilutions. in the present study, the activities against s. aureus and e. coli were confirmed; however some omani and african honey samples also showed activity against p. aeruginosa (zulma et al ., 1989). although honey has been reported to have antifungal activity against c. albicans, our sample shows the zone of inhibition at concentrations of 100 % and 80 %. different formulations of honey has significantly inhibited growth of pathogenic microorganisms, s. aureus , e. coli, c. albicans and a. nigar when compared to control group, which is an evidence that honey is a therapeutic agent being used since ancient time throughout the world (gulfraz et al ., 2010). the antimicrobial activity against s. saprophyticus was almost equal in fresh and old honey samples. strept. pneumoniae was resistant to both honey samples at concentration 50% . both of the honey samples (old and fresh) inhibited b. subtilis at 80%, 50% and 100% dilutions of honey. the highest zone of inhibition for old and fresh honey was p. fiuorescens. s.typhi was resistant in both old and fresh honey. both of the old and fresh honey inhibited s. paratyphi a at concentrations of 100%, 80% and 50%. sh. dysenteriae was inhibited at concentrations 100%, 80% and 50% in both old and fresh honey samples. k. pneumoniae was inhibited in old honey while resistant in fresh honey. references anonymous.2005.honey.htt.//en.wikipedia.org/ wiki/honey. gulfraz, m., iftikhar, f., raja, s., asif, s., mehmood, s., anwar, z., and kaukob, g. 2010. quality assessment and antimicrobial activity of various honey types of pakistan. afr. j. biotech., 9(41): 6902-6906. molan, p.c. 2001. the antibacterial activity of honey.1.the nature of the antibacterial activity. bee world, 73 (1): 5-28. qamer, s., ehsan, m., nadeem, s. and shakoor, a.r. 2007. free amino acids content of pakistani unifloral honey produced by apis mellifera. pak. j. zool., 39(2)., 99-102. zulma, a. and lulat, a. 1989. honeya remedy re discovers. j. roy. soe. med., 82:384-5 nutrition & therapeutics speciality of opuntia-ficus-indica vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 144 r a d s j . b i o l . r e s . a p p l . s c i . 144 open access f u l l l e n g t h a r t i c l e opuntia ficus-indica as nutritious food ingredient; prevalence for therapeutic speciality saniya ramzan1,*, maryam saeed2 1national institute of food science and technology, university of agriculture, faisalabad, pakistan. 2institute of agricultural sciences, university of the punjab, lahore, pakistan. a b s t r a c t background: it is predicted that the world’s population, specifically in arid and semi-arid regions, usually with complications such as water scarcity, climate change, soil erosion, and wind erosion, is facing food insecurity issue. moreover, the outbreak of several diseases is further a burden on the people living in such areas. the growth of improved crop varieties which is compatible with agroclimatic conditions of the site such as opuntia ficus-indica is the ray of hope. multipurpose native crops like opuntia ficus-indica is cultivated owing to it is drought tolerant ability and growth in different ranges of the environment. furthermore, it has natural components that can not only be served as food but also the therapeutic ability for numerous diseases. objectives: the purpose of this review is concerning global issues such as food insecurity, malnutrition and their solution through natural products i.e. nutraceuticals. the target is to find one of the numerous approaches that can be adapted to focus on dealing with such problems. methodology: opuntia ficus-indica has various nutraceutical uses that had been utilized in past but can further be accommodated for commercial utilization and public benefit. in this review, the therapeutic aspects of opuntia ficus-indica have been elaborated with the utilization of its components to fight different diseases. different studies from various scientists published in the form of research/review articles have been overviewed proving its physico-chemical elements to be beneficial in this aspect. results: opuntia ficus-indica is not only a source of food but also has exponential components that serve as nutraceuticals including phytochemicals such as phenolics, vitamin c, flavonoids, betalains, and carotenoids. these components impart diverse beneficial effects on health and act as anti-ulcerogenic, anti-carcinogenic, antioxidant, hepatoprotective, anti-atherogenic, and immunomodulator. conclusion: opuntia ficus-indica is a miracle of nature that has been bestowed upon us to reduce the risk of many calamities. it can not only be served as food but also imparts beneficial effects on health that not only reduces the chances of getting sick but also helps in alleviating the drastic effects of disease exposure. keywords antioxidant, apoptosis, hepatotoxicity, fortified, extract. *address of correspondence saniyaramzan8@gmail.com article info. received: august 14, 2020 accepted: november 24, 2020 cite this article: ramzan s, saeed m. opuntia ficus-indica as nutritious food ingredient; prevalence for therapeutic speciality. rads j biol res appl sci. 2020; 11(2):144-153. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n cacti (singular cactus) have unique and unusual properties that are adaptive to extremely hot and arid/semi-arid environments. they show a variety of functional and structural characteristics which make them more interesting like water conservation and drought tolerance capabilities. as for drought tolerance ability, it comes from the phenomenon of co2 fixation capacity (cam). moreover, its leaves have the shape of spines, while the stem is transformed into the succulent structure containing chlorophyll1. commonly, it is native to north r e v i e w a r t i c l e nutrition & therapeutics speciality of opuntia-ficus-indica vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 145 r a d s j . b i o l . r e s . a p p l . s c i . 145 america, venezuela, bolivia, south africa, argentina, jordan, israel, and west indies, but now it is commonly growing in india, italy, spain, mexico, northern africa, and the usa where they are now a significant part of people’s dietary needs. it can be found in range of the environment from the dessert area below sea level to high altitude areas such as mountains of peru2. the genus opuntia has approximately 300 different species which produces edible fruit varieties but the most important of these are opuntia ficus indca, opuntia robusta, opuntia streptocantha, o. amyclaea, opuntia. megacantha, o.hiptiacantha, and opuntia ficus indica (l) mill. in different countries, a cactus pear is cultivated for different purposes e.g. to ensure food security (control the wind, water, and soil erosion, utilize as forage and as fence), use in the food processing industry (natural colorant, juice, jam, vinegar, flour, and food supplement), as a pharmaceutical product (essential oils, herbal extracts, in medicine), and in cosmetic industry (seed oil, soap, and shampoo)3. opuntia ficus-indica (l.) mill., common name: prickly pear/nopal cactus, family: cactaceae (dicotyledonous angiosperm) is an important cactus species in agriculture of non-irrigated lands due to its utility in food, fodder, dye, source of energy, ecosystem remediation, and prevention of soil erosion4. it is also recognized as “fruit for the poor”, “treasure under spines”, “future plant”, “scared plant” and “monster tree” and these names depict its significance in human life5. the largest producer of opuntia ficus-indica species in mexico with 72000 hectare (ha) area under fruit cultivation and 10,500 for nopalitos. in brazil, north african countries (morocco, egypt, tunisia, algeria, and libya) italy, and chile, the area under cultivation is 40000ha, 16000ha, 2500ha and 1100ha respectively6,7. without any technical involvements, the crop is produced in two seasons per year i.e. february-april and julyseptember with the covered area of 934.4ha in chile. among african countries i.e. algeria, ethiopia, morocco, south africa, tunisia where optunia species are grown, ethiopia is the largest producer of cactus pear with the covered area of 360,000ha. the major purposes of its cultivation are fencing, delicious fruit, essential oil, pharmaceutical products, food products, cosmetics, livestock feed, and forage8. it is a cheap source of food i.e., nopal/cladodes and prickly pear, fodder, and ornamental use. cladodes are used as salads. prickly pear is an elongated and fleshy berry having different sizes, color, and shape which can be consumed in the fresh, dried, and preserved form such as syrups, jams and candy-like products9. in most of the west asian countries, the cactus is established for food and feed production, also utilized as hedges around the houses for enhancing aesthetic value. the modern cactus pear production in australia is very small around 200ha and most established in the home gardens. among the european countries, italy is famous for its appreciation of opuntia ficus-indica and it was recognized as “bread for poor” and also being used as an emergency fodder. portugal has also promoted the production of cactus pear and planned to plant on more than 500ha which was before 200ha and also made the plan to engage the unemployed persons2. opuntia ficus-indica contains 92% moisture in addition to 4-6% total fiber and 1-2% protein10. it is of great nutritional profile having high sugars i.e., glucose, fructose, and sucrose, other macronutrients like protein, lipid, ash, fiber, and minerals. moreover, there is an increased amount of free amino acids including glutamine, proline, serine, methionine, arginine, aminobutyric acid, and histidine. there is considerable amount of bioactive compounds available in opuntia ficus-indica for example ascorbic acid, carotenoids, and taurine11,12. it is known to have pigments and molecules which are usable in nutritive and medicinal aspects that make it among the list of functional foods13. these pigments can be classified according to the chromophore structure such as chromophores with conjugated systems i.e. carotenoids, caramel, betalains, lakes, and synthetic pigments, and metal coordinated porphyrins such as chlorophyll and myoglobin. the amount of bioactive compounds in the pulp of cactus pear varies with the variety and place of growth14. t h e r a p e u t i c p r o p e r t i e s anti-oxidant activity the oxidation process in the body is the stimulant of various diseases. polyphenols present in opuntia ficusindica possesses anti-oxidant activity which is divided into flavonoids, stilbenes, phenolic acids, and lignin & suberin. through the interaction of flavonoids with carbohydrates, lipids, and proteins, the oxidation process nutrition & therapeutics speciality of opuntia-ficus-indica vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 146 r a d s j . b i o l . r e s . a p p l . s c i . 146 is inhibited15. according to slimen14, the mechanisms behind the antioxidant activities of polyphenols are given in fig 1. fig 1. polyphenols of opuntia ficus-indica: flavonoids, quercetin and silibin working as antioxidants. (slimen et al., 2016). moreover, betalains also plays a vital role in antioxidant properties owing to their hydroxyl, imino, and tetrahydropyridine groups. betalains are pigments present in the vacuole in the form of betalamic acid which condenses and convert into betacyanins (violet color) and betaxanthins (yellow color). in opuntia ficus-indica these are present in pulp and peel. in the pulp fruit, betanin, betanidin, neobetanin, isobetanin, and indicaxanthin are detected16. while in the peel portion, betanin and indicaxanthin are present. the two pigments betanin and indicaxanthin have the ability to donate their electrons that help in the inhibition of lipid peroxidation and heme disintegration. betanin is more effective in the radical scavenging process rather than indicaxanthin17. betanidin is another potent antioxidant that acts against nitric acid and peroxyl radical and has additive interaction with αtocopherol18,19. yahia & mondragon-jacobo7 have concluded during their study that tocopherols and carotenoids are not responsible for the antioxidant activity but phenolic compounds, betalains, and vitamin c are the ones to carry out the antioxidant property. during experimental studies, it has been observed that opuntia ficus-indica is effective for a decrease in lipid oxidation and an increased amount of antioxidant i.e. reduction in glutathione. this ultimately results in improving the erythrocyte’s redox potential. hence, 500g of prickly pear supplement on daily basis is effective for lowering the oxidative stress in a healthy person within two weeks20. similarly, siriwardhana21 also showed that opuntia ficusindica (whole fruit) extract has the potential effect as an antioxidant which helped in 60% decrease in impaired lymphocytes dna. hence, the utilization of prickly pear and its products are effective in free radical’s reduction that leads to alleviation in the development of chronic ailments. anti-inflammatory and anti-thrombogenic properties the flavonoids present in opuntia ficus-indica helps in the prevention of inflammation. the mechanism behind this is that they inhibit arachidonic acid metabolism which results in anti-thrombogenic and anti-inflammatory characteristics14. moreover, isorhamnetin glycosides flavonoids namely isorhamnetin-3-o-glucosyl-rhamnoside (igr) and isorhamnetin3-o-glucosyl-rhamnosylrhamnoside (igrr) regulates anti-inflammatory activities by inhibition of the nfк-b activation and the cox-2 (cyclooxignase-2) gene expression with suppressing the tnf-ɑ production as well. antunes-ricardo22 has done the study using alkaline extraction and purification through semi-preparative chromatography of opuntia ficus-indica and the results suggest that it significantly affects cox-2 and decreased secretion of cytokines and nitric oxide thus exhibiting the anti-inflammatory properties (fig 2). fig 2. flavonoids exhibiting anti-inflammation characteristics in the human body. (antunes-ricardo et al., 2015). tesoriere23 observed that indicaxanthin from cactus pear (opuntia ficus-indica l. mill) fruit prevents eryptosis (programmed cell death of erythrocytes) induced by oxysterols. eryptotic erythrocytes may contribute to thrombotic complications; as if phosphatidylserine (ps) is exposed to the surface of eryptotic cells may activate nutrition & therapeutics speciality of opuntia-ficus-indica vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 147 r a d s j . b i o l . r e s . a p p l . s c i . 147 coagulant enzymes which can cause thrombosis and thrombo-occlusive disease. further, it will also attach to the endothelial cells and can cause vascular damage. this problem can be prevented by cactus pear (opuntia ficus-indica) fruit because polyphenol such as betalamic acid and indicaxanthin obtained from it have the reducing potential and amphipathic in nature, interacts with membranes, enters various cells, including erythrocytes and counteracts oxidative damage induced by various events such as the presence of reactive oxygen species (ros), the release of prostaglandin (pge2), the opening of prostaglandin dependent calcium channels and exposure of phosphatidylserine (ps). the indicaxanthin of prickly pear fruit in the concentration range of (1.0-5.0mm) can eliminate the ps externalization and cell shrinkage. anti-carcinogenic and anti-ulcerogenic characteristics opuntia ficus-indica also possesses chemo-preventive activity. it was found effective against the inhibition of cancerous cells. zou24 has studied the effect of prickly pear extract on tumor cell growth24. he made a comparison of the effects of prickly pear extract and retinoid n-(4-hydroxipernil) retinamide (4-hpr) on cancer cells of cervical, bladder, and ovary. the results showed that there is an obvious increase in cellular apoptosis and inhibition in cancerous cells development (fig 3). fig 3. prickly pear’s extract working in tumor growth prevention. (zou et al., 2005). furthermore, betanin extract from opuntia ficus-indica is also beneficial in terms of cellular apoptosis induction. in myeloid leukemia, betalains impose inhibitory action on the cellular growth25. gastric mucous is a protective layer of gastric cells but if any damage occurs to it, it results in the impairment of cells and many problems such as ulcers, gastritis, and cancer take place. nonetheless, antioxidants present in the opuntia ficus-indica have the ability to prevent such problems to arise. galati26 has studied anti-ulcerogenic effect of opuntia ficus-indica during a trial on wistar mice. moreover, the mechanism behind such activity is demonstrated by jiménez-aguilar27 that is shown in fig 4. fig 4. flavonoids bioactivity in gastric ulcer reduction. (aguilar et al., 2014). another study based on the lyophilized cactus pear fruit juice reported that it has certain constituents that work against the stress-induced gastric lesions particularly betanins and opuntia ficus-indica var. saboten fruit juice and maltodextrin (ofsm). it was proven that it is effective against the prevention of gastric lesions and gastric mucosal tumor (tnf-a)28 (fig 5). fig 5. betanin bioactivity to prevent tumor growth. (kim et al., 2012). nutrition & therapeutics speciality of opuntia-ficus-indica vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 148 r a d s j . b i o l . r e s . a p p l . s c i . 148 immunomodulatory activity human body defence system has the ability to fight the disease causing factors and prevent the body, but there are some causalities like an autoimmune disease where the immune system starts to act against the body organs and tissues for example psoriasis, atopic dermatitis, type i diabetes and rheumatoid arthritis. aires29 has conducted the study to find the solution to such a problem and results showed that opuntia ficus-indica has immunemodulatory effects which are attained by minimizing jukart t cell multiplication (fig 6). fig 6. pathway of polyphenolic compounds of opuntia ficus-indica acting as immunity boosters. (aires et al., 2004). hepatoprotection liver is a fundamental human body organ as its major role is to detoxify, bile excretion, protein and fat metabolism, and storage of carbohydrates and vitamins. opuntia ficusindica has proven to be effective for the protection and cure of liver functionality. galati30 has worked on the opuntia ficus-indica fruit juice for its evaluation of protective working against carbon tetrachloride (ccl4) induced hepatotoxicity and found that injury caused ccl4 was cured in 72hrs. similarly, djerrou31 find out that ccl4 induced hepatotoxicity, can be cured by using aqueous extract of opuntia ficus-indica. in the results, it was prominent that there was a raised value of aspartate ammonia transferase and alanine ammonia transferase in the groups intoxicated with ccl4 dose which indicate opuntia ficus-indica aqueous extract’s hepatoprotective capability. cellular membrane protection cladodes of opuntia ficus-indica are identified for having three carotenoids including β-carotene, lutein, and αcryptoxanthin. the highest content of carotenoids was detected in orange color cultivars. while in the case of young cladodes the values fall in 0.047 to 0.077mg/100g range and for fruits, the concentration varies between 1.77mg/100g and 2.65mg/100g. the peeling part contains 2.97mg/100g of carotenoids32. carotenes and xanthophylls like zeaxanthin, cryptoxanthin, s-carotene, lycopene, and α-carotene are potential singlet oxygen (1o2) and peroxyl radical quenchers. they scavenge the peroxyl radical and make them stabilized in the form of carbon-centered radical. moreover, they possess lipophilic properties. both of these characteristics make them vital for the protection from oxidative impairment of cellular membranes and lipoproteins14. cactus pear was used in traditional medicine for their cicatrisant activity. the cladodes of opuntia ficus-indica have carbohydrate polymers such as a mixture of mucilage and pectin. the results showed that its cladodes, particularly pectic polysaccharide has the crypto-protection phenomenon by breaking the epithelial cell, increased mucus production, and enhance the number of secretory cells. ulcer healing is either through regeneration or migration of cells which results in thickening the mucus membrane33. gastrointestinal tract recently, cactus cladodes are considered as fiber source for human consumption as its powder contains 43% fiber including 28.5% of insoluble fiber. soluble fibers can impose potential health benefits as they stabilize the intestinal food transit. moreover, insoluble fibers such as hemicellulose, cellulose, and lignin are beneficial for microbiota, water retention, bile acid absorption, and ionic exchange14. sánchez-tapia34 has checked the prebiotic effect of nopal (opuntia ficus-indica) and the results of the study suggest that more consumption of high fat and high sugar food, enhance the chances to get affected with the conditions such as obesity, gut dysbiosis, inflammation, and gut barrier disruption. the reason is the disturbance in the balance of gut microbiota. nopal is a rich source of dietary fibers, vitamin c, and polyphenols which are effective in the reduction of obesity by nutrition & therapeutics speciality of opuntia-ficus-indica vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 149 r a d s j . b i o l . r e s . a p p l . s c i . 149 regulating the gut microbiome and prevent metabolic endotoxemia. blood glucose and cholesterol opuntia ficus-indica cladodes have soluble fibers including gums, pectin, mucilage, and hemicellulose that help in the lowering of cholesterol level and glycemic index in humans14. polysaccharides, alkaloids, neobetanin, indicaxanthin, and flavonoids extracted from cladodes are capable with special effects of antidiabetic and antiglycation35. the redox imbalance is one of the main factors leading to the induction of a genetic basis for diabetes36. berraaouan37 suggested that opuntia ficusindica seed oil have a high amount of polyunsaturated components and antioxidant compounds which are useful against the diabetes mellitus (dm) as it increased the survival rate by 78% against the diabetes mellitus. according to milán-noris38 prickly pear is the fruit which is enriched with many phytosterols, betalains, phenolic acids, and flavonoid. especially, its peel has many bioactive compounds which can be extracted and used as a functional ingredient. his observation proved that the prickly pear peel is enriched with total phytosterols (65.65+1.5mg/g) such as, sitosterol (76.6%), campesterol (19.5%), and stigmasterol (3.9%) which show the strong hypocholesterolemic effect. also, it contains the highest phenolic content which will produce a synergistic effect also. benign prostatic hypertrophy the prostate is a part of the mammalian male reproductive organ which contributes to secretions. it surrounds the bladder and proximal portion of the urethra. usually, these glandular muscles enlarge and press the urethra which causes hindrance in urination. opuntia ficus-indica is thought to cure such complications especially the decoction made from the flowers of cactus pear. in north africa, the flowers of opuntia combined with barley seeds and corn-silk is used to treat urine obstruction. during a study on clinical trials of its 250mg capsules by palevitch39, results have shown that patients got relief from the symptoms of obstruction during urination and there was no change in urino-dynamics and kidney functions of the patients. moreover, flowers of opuntia ficus-indica are also effective in the cure of benign prostatic hypertrophy40. wound healing traditionally, opuntia ficus-indica cladodes were used to promote wound healing through the formation of a scar. an ointment prepared by using 15% lyophilized cladodes has shown that it produces more organized tissue reconstruction, fibroblast and fibers will arrange in derma properly and piliferous bulbs will recover in 5 days41. for wound healing, it is mandatory to keep the area well hydrated. so, opuntia ficus-indica oil stimulates the scarring process by the development of epithelium over the granulation muscle and prevents the wound from dehydration as well42. u s e s a s f o o d opuntia ficus-indica has found its place in a variety of products to utilize in daily routine. this was possible due to its contents that are beneficial for health and also its production is easy and adaptive to the environment. the young tender stem is called “nopalitos” which are used in diverse dishes including salads, soups, snacks, pickled and desserts43. indigenous people utilize the nopal cactus seed extract in tea, juice, jam, or consume as dry or fresh fruits. moreover, peeled and sliced pads are dried and called as leather britches that help add texture and fibrous material in soups and stews35. the cactus pear pulp with effective natural colors is utilized for the preparation of additive-free i.e. artificial color/flavor toppings44. it is used in the fortification purpose to make high quality products with high contents of phenolics and antioxidants such as stirred yogurt45. the fruit wine is also obtained by the blend of opuntia ficus-indica and lantana camara46. furthermore, cactus pear peel extraction can be successfully used in the substitution of wheat flour biscuits47. its pulp is also utilized in the production of fortified rice milk product48. in the southwestern united states, cactus pear is commonly added to the omelets which are very famous over there. n u t r a c e u t i c a l u s e s neopuntia prepared by bio serae laboratories is an innovative functional food product that is made of dehydrated leaves of opuntia ficus-indica containing soluble and insoluble fibers. this product is effective against lipid metabolism disorder and has hypolipidemic characteristics1. furthermore, cactus preparation is also nutrition & therapeutics speciality of opuntia-ficus-indica vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 150 r a d s j . b i o l . r e s . a p p l . s c i . 150 proved to be useful for the prevention of alcohol addiction49. traditionally, phytochemical rich products were prepared from opuntia ficus-indica extracts for herbal medicines35. pharmacopeia, traditional medicine of sub-sahara is prepared from cactus. its flowers are effective anti-hemorrhoid medicine while fruits and flowers are antidiarrheal and anti-ulcerogenic mediators. its cladode sap is the remedy for whooping cough. it involves heating the pads and places it on the chest of the patient which helps in relieving the congestion50. alcoholic extracts of opuntia ficus-indica is used as an antiviral, hypoglycemic, and anti-inflammatory. similarly, it has been a part of traditional medicine and curative for various ailments like catarrhal gastritis, hyperlipidemia, edema, burns, wounds, and obesity in different countries51. opuntia ficus-indica cladodes were used by sicilian folk as cicatrizant (promote the healing through the formation of the scar)41. dehydrated nopal of cactus pear has also served in the nutraceutical market as dietary supplements for having high nutritional value52. not only the extract of cactus pear is helpful in the remedy of high blood sugar level, but also the cooked form plays a significant role in lowering the blood sugar level. it may be due to the presence of high fiber content as pectin absorbs sugar and supports the body in slow release of sugar throughout the day. similarly, the stem portion of this plant is found to be useful in use for traditional medicine for edema and indigestion53. tea made from the pad has been utilized for the cure of lung diseases54. moreover, it is a traditional cure for various ailments like diuretic action, gastritis, antiulcer activity, arteriosclerosis, antioxidant property, diabetes, cartilage alteration protection, and hyperglycemia55. a process known as chinese medicine is the “moxibustion”. in this process, plant material is burned on the skin to cure the infection and irritation. likewise, the lakota tribe prepares tea from prickly pear that is found to be helpful in childbirth13. opuntia ficusindica has been utilized as a treatment for the ailments such as bronchial asthma, diabetes, abdominal ache, indigestion and burns in south korea56. o t h e r u s e s extract of opuntia ficus-indica cladodes is found to be useful in the reduction of salt stress in the agriculture sector57,58. it is also utilized as cattle fodder which imparts quality characteristics in milk. in addition, to enhance the flavor and color of the milk, it also brings nominal changes in the medium and longchain fatty acids in milk profile59. oil extracted from prickly pear seeds are utilized in cosmetics as well35. biogas is an essential energy source in the rural and agricultural areas. cactus pear cladodes are served for the production of biogas as it has a high potential of biomass production2. the cactus pear (both spiny and spineless) was utilized as ruminant feed in zimbabwe which was imported from south africa60. the juice prepared by nopal is used as a hair massage which results in soft and shiny hair. opuntia ficus-indica is utilized in the formation of moisturizers for skin55. for many centuries in mexico, it was utilized in the formation of waterproof paints that are used to paint houses, churches, and marking of property lines. moreover, it is also useful as a protective barrier against predators. in central africa, its juice is proved to be nominal mosquito repellent. it has its ornamental use as well due to its attractive colors and desert landscape. the gum obtained from cactus is usually used for stiffening the clothes. it is also a good source of dye. additionally, essential oils obtained from its flowers and seeds are consumed as perfumes and oils13. c o n c l u s i o n in this modern era, industrialization has made everything possible and we get many alternates of natural products. there are some natural elements have also proven to be effective as well. global food insecurity, natural resources shortcomings and increase in population are the issues that have already targeted our globe but will become drastic in the future. to avoid such a situation, we need to find the solutions within nature. opuntia ficus-indica is a natural product that can grow without many natural resources consumption. moreover, it is proven that it has therapeutic effects for various diseases. hence, it can become one of the major nutraceutical product in near future owing to its marvelous physical and chemical characteristics. c o n f l i c t s o f i n t e r e s t none. nutrition & therapeutics speciality of opuntia-ficus-indica vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 151 r a d s j . b i o l . r e s . a p p l . s c i . 151 f u n d i n g s o u r c e none. a c k n o w l e d g e m e n t s none. l i s t o f a b b r e v i a t i o n s cam crassulacean acid metabolism ccl4 carbon tetrachloride cox-2 cyclooxignase-2 dm diabetes mellitus ha hectare hrs hours igr isorhamnetin-3-o-glucosyl-rhamnoside igrr isorhamnetin-3-o-glucosyl-rhamnosylrhamnoside pge2 prostaglandin ps phosphatidylserine ros reactive oxygen species r e f e r e n c e s 1. shetty aa, rana mk, preetham sp. cactus: a medicinal food. j food sci tech. 2012; 49:530-36. 2. inglese p, mondragon c, nefzaoui a, saenz c. crop ecology, cultivation and uses of cactus pear. 2017. food and agriculture organization of the united nations (fao). 3. feugang jm, konarski p, zou d, stintzing fc, zou c. nutritional and medicinal use of cactus pear (opuntia spp.) cladodes and fruits. frontiers biosci. 2006; 11:2574-89. 4. small e, catling pm. blossoming treasures of biodiversity 11. cactus pear (opuntia ficus-indica) miracle of water conservation. biodiv. 2004; 5:27-31. 5. jiménez ea. (2013b). preámbulo: importancia de la tuna [cactus pear importance]. proceedings of the second meeting for the integral use of cactus pear and other cacti and first south american meeting of the fao-icarda cactusnet, santiago del estero, argentina. 175. 6. yahia em, mondragon-jacobo c. nutritional components and antioxidant capacity of ten cultivars and lines of cactus pear fruit (opuntia spp.). food res int. 2011; 44:11-8. 7. yahia em, sáenz c. cactus pear (opuntia species). in postharvest biology and technology of tropical and subtropical fruits. woodhead publishing; 2011; 290331e. 8. targa mg, leguizamon g, coronel de renolfi m, ochoa mj. economic feasibility of scozzolatura in traditional and improved orchards of cactus pear in santiago del estero, argentina. acta horticulturae. 2013; 995:189-200. 9. piga a. cactus pear: a fruit of nutraceutical and functional importance. j professional assoc cactus dev. 2004; 9-22. 10. brinker fnd. prickly pear as food and medicine. j diet suppl. 2009; 6:362-76. 11. kuti j. antioxidant compounds from four opuntia cactus pear fruit varieties. food chem. 2004; 85:52733. 12. stintzing f, herbach k, moβhammer m, carle r, yi w, sellappan s, et al. color, betalain pattern, and antioxidant properties of cactus pear (opuntia sp.) clones. j agric food chem. 2005; 53:442-51. 13. saleem m, kim hj, han ck, jin c, lee ys. secondary metabolites from opuntia ficus-indica var. saboten. phytochemistry. 2006; 67:1390-4. 14. slimen ib, najar t, abderrabba m. opuntia ficusindica as a source of bioactive and nutritional phytochemicals. j food nutr sci. 2016; 4:162-69. 15. jakobek l. interactions of polyphenols with carbohydrates, lipids and proteins. food chem. 2015; 175:556-67. 16. yeddes n, cherif jk, guyot s, sotin h, ayadi mt. comparative study of antioxidant power, polyphenols, flavonoids and betacyanins of the peel and pulp of three tunisian opuntia forms. antioxidants. 2013; 2:37-51. 17. gandia-herrero f, escribano j, garcia-carmona f. purification and antiradical properties of the structural unit of betalains. j nat prod. 2012; 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13:445-58. 22. antunes-ricardo m, gutiérrez-uribe ja, lópezpacheco f, alvarez mm, serna-saldívar so. in vivo anti-inflammatory effects of isorhamnetin glycosides isolated from opuntia ficus-indica (l.) mill cladodes. indus crops prod. 2015; 76:803-8. 23. tesoriere l, attanzio a, allegra m, livrea ma. dietary indicaxanthin from cactus pear (opuntia ficus-indica l. mill) fruit prevents eryptosis induced by oxysterols in a hypercholesterolaemia-relevant proportion and adhesion of human erythrocytes to endothelial cell layers. br j nutr. 2015; 114:368-75. 24. zou dm, brewer m, garcia f. cactus pear: a natural product in cancer chemoprevention. nutr j. 2005; 4:25-37. 25. sreekanth d, arunasree mk, roy kr, chandramohan t, reddy gv, reddanna p. betanin: a betacyanin pigment purified from fruits of opuntia ficus-indica induces apoptosis in human chronic myeloid leukemia cell line-k562. phytomedicine. 2007; 14:739-46. 26. galati em, mondello mr, giuffrida d. chemical characterization and biological effects of sicilian opuntia ficus indica (l.) mill. fruit juice: antioxidant and antiulcerogenic activity. j agric food chem. 2003; 51:4903-8. 27. jiménez-aguilar dm, mújica-paz h, welti-chanes j. phytochemical characterization of prickly pear (opuntia spp.) and of its nutritional and functional properties: a review. curr nutr food sci. 2014; 10:5769. 28. kim sh, jeon bj, kim dh, kim ti, lee hk, han ds. prickly pear cactus (opuntia ficus indica var. saboten) protects against stress-induced acute gastric lesions in rats. j med food. 2012; 15:968-73. 29. aires v, adote s, hichami a, moutairou k, boustani ee, khan na. modulation of intracellular calcium concentrations and t cell activation by prickly pear polyphenols. mol cell biochem. 2004; 260:103-10. 30. galati em, mondello mr, lauriano er, taviano mf, galluzzo m, miceli n. opuntia ficus-indica (l.) mill. fruit juice protects liver from carbon tetrachloride induced injury. phytother res. 2005; 19:796-800. 31. djerrou z, maameri z, halmi s, djaalab h, riachi f, benmaiza l, et al. hepatoprotective effect of opuntia ficus-indica aqueous extract against carbon tetrachloride-induced toxicity in rats. j biol sci. 2015; 15:36-41. 32. chougui n, louaileche h, mohedeb s, mouloudj y, hammoui y, tamendjari a. physico-chemical characterization and antioxidant activity of some opuntia ficus-indica varieties grown in north algeria. afr j biotechnol. 2013; 12:299-307. 33. galati em, pergolizzi s, miceli n, monforte mt, tripodo mm. study on the increment of the production of gastric mucus in rats treated with opuntia ficus indica (l.) mill. cladodes. j ethnopharmacol. 2002; 83:229-33. 34. sánchez-tapia m, aguilar-lópez m, pérez-cruz c, pichardo-ontiveros e, wang m, donovan sm. nopal (opuntia ficus indica) protects from metabolic endotoxemia by modifying gut microbiota in obese rats fed high fat/sucrose diet. sci rep. 2017; 7:1-16. 35. el-mostafa k, el-kharrassi y, badreddine a, andreoletti p, vamecq j, el-kebbaj mhs et al. nopal cactus (opuntia ficus-indica) as a source of bioactive compounds for nutrition, health and disease. mol. 2014; 19: 14879-901. 36. droge w. free radicals in the physiological control of cell function. physiol rev. 2002; 82:47-95. 37. berraaouan a, abderrahim z, hassane m, abdelkhaleq l, mohammed a, mohamed, b. evaluation of protective effect of cactus pear seed oil opuntia ficus-indica (l.) mill. against alloxan-induced diabetes in mice. asian pac j trop med. 2015; 8:5327. 38. milán-noris ka, chavez-santoscoy a, olmosnakamura r, gutiérrez-uribe aa, serna-saldívar os. an extract from prickly pear peel (opuntia ficusindica) affects cholesterol excretion and hepatic cholesterol levels in hamsters fed hyperlipidemic diets. curr bioact compd. 2016; 12:10-16. 39. palevitch d, earon g, levin i. treatment of benign prostatic hypertrophy with opuntia ficus-indica (l.) mil. j herbs spices med plants. 1993; 2:45-9. nutrition & therapeutics speciality of opuntia-ficus-indica vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 153 r a d s j . b i o l . r e s . a p p l . s c i . 153 40. british herbal phannacopeia (bhp). the british herbal medicine association, west york, england. 255 p. 1983. 41. galati em, mondello mr, monforte mt, galluzzo m, miceli n, tripodo mm. effect of opuntia ficus-indica (l.) mill. cladodes in the wound-healing process. j prof asso cactus develop. 2003; 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(eds.). phytochemicals of nutraceutical importance. 2014; cabi. 53. park eh, chun mj. wound healing activity of opuntia ficus-indica. fitoterapia. 2000; 72:165-7. 54. foster s, duke ja. field guide to medicinal plants and herbs of eastern and central north america. 2nd ed, boston: houghton mifflin company, 2000. 55. damasceno gab, silva rmac, fernandes jm, ostrosky ea, langassner smz, ferrari m. use of opuntia ficus-indica (l.) mill extracts from brazilian caatinga as an alternative of natural moisturizer in cosmetic formulations. braz j pharm sci. 2016; 52:450-70. 56. kim jh, park sm, ha hj. opuntia ficusindica attenuates neuronal injury in in vitro and in vivo models of cerebral ischemia. j ethnopharmacol. 2006; 104:257-62. 57. sáenz c, berger h, félix ar, galletti l, garcía jc, sepúlveda e, et al. agro-industrial utilization of cactus pear. food and agriculture organization of the united nations. 2013. 58. rai a, cherif a, cruz c, nabti e. extracts from seaweeds and opuntia ficus-indica cladodes enhance diazotrophic-pgpr halotolerance, their enzymatic potential, and their impact on wheat germination under salt stress. pedosphere. 2017. 59. oliveira vsd, ferreira mda, guim a, modesto ec, arnaud bl, silva fmd. effects of replacing corn and tifton hay with forage cactus on milk production and composition of lactating dairy cows. revista brasileira de zootecnia. 2007; 36:928-35. 60. makumbe mt. a review of the distribution, use and potential of cactus pear (opuntia ficus-indica (l.) mill. as ruminant feed in zimbabwe. improved utilization of cactus pear for food, feed, soil and water conservation and other products in africa. 2010; 3643. https://www.hindawi.com/journals/ecam/ analysis of helminth associated infections vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 96 op e n ac c e s s f u l l l e n g t h a r t i c l e helminth associated infections among residents of karachi aly khan1,*, nasira khatoon2, syed shahid shaukat3, samina waheed2, adnan khan4, rozmeen nil2 1crop diseases research institute, pakistan agricultural research council, university of karachi, karachi-75270, pakistan. 2department of zoology, university of karachi, karachi-75270, pakistan. 3institute of environmental studies, university of karachi, karachi-75270, pakistan. 4department of microbiology, university of karachi, karachi-75270, pakistan. a b s t r a c t background: helminth parasite affects approximately 2 million individuals yearly throughout the world. these infections are extremely important while often receive inadequate consideration. helminths are among the gastrointestinal parasites mainly involved in an increased ratio of inflammatory bowel disease, especially in underdeveloped and developed countries. objectives: to study the prevalence of helminth eggs in stool samples collected from five hospitals of gulistan-e-johar area of karachi, pakistan, among different age groups of male and female patients with the targeted age groups < 1-14, 15-40 and > 40 yrs. methodology: all the samples included in the study were collected according to ethical review board policy of the participating hospital. stool samples were obtained from a total of 1042 patients, of which 198 samples were found to be positive for helminth eggs. the helminths included were ascaris lumbricoides, ancylostoma duodenales and hymenolepis nana. results: the association between helminth infection (a. lumbricoides, a. duodenale and h. nana) and age group was found to be significant. results are based on the chi-square test. samples from males showed a noticeable prevalence of helminths than females samples. single, double and triple infections were also recorded. conclusion: present study could be very beneficial for developing programs not only for awareness in the urban and rural populations, but also for the control of infections due to helminths in pakistan. keywords helminth infection, a. lumbricoides, a. duodenale, h. nana. *address of correspondence aly.khan@hotmail.com article info. received: september 25, 2020 accepted: december 23, 2020 cite this article khan a, khatoon n, shaukat ss, waheed s, khan a, nil r. helminth associated infections among residents of karachi. rads j biol res appl sci. 2020; 11(2):96-101. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n the helminth parasites affect more or less two million people worldwide every year1. so far, 342 species of helminths are associated with humans, amongst those 197 are the inhabitant of the human gastrointestinal tract2. a high rate of infection with helminth parasites may lead to severe anemia, malnutrition and loss of productivity in the workplace. it was reported that almost 819 million people are infected with ascaris, 464 million with trichuris and 438 million with hookworms (ancylostoma and necator) amongst the rural population of the world especially, where sanitation facilities are improper3. in random samples of 1313 children aged 1 month to 12 years in villages, in nine rural districts of southern laos peoples democratic republic was enrolled and examined for helminths prevalence using duplicate kato-katz thick smears. they recorded considerable morbidity among the surveyed o r i g n a l a r t i c l e analysis of helminth associated infections vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 97 children which included hepatomegaly (13.7%), abdominal pain (13.2%), and anemia (60.4%) with significant signs of low body mass index and stunting. the most common worms were hookworms and opisthorchis viverrini4. this study summarized the clinical aspects, biology, diagnosis, treatment and epidemiology for the common and rarer human intestinal cestodes5. the immunology of infections with helminths was also reported6. increased prevalence of intestinal helminths infection during pregnancy in the sub-saharan african community was reported in this survey 105 pregnant women were recruited and found positive as compared to the same number of non-pregnant7. in another study, intestinal helminth parasites with respect to nutritional status of children aged 6-14 yrs attending school in waskiso district, central uganda was noted and found that the prevalence of stunting was 22.5%, underweight 5.3% and moderate acute malnutrition 18.5%, respectively. the prevalence of parasites namely trichuris trichiura was 10.9%, schistosoma mansoni was 1.9% and ascaris lumbricoides was 0.2%8. it was reported that in studies conducted in pakistan, the focus was mostly on school children, low-income group and rural populations9. in southern malawi, the urban/rural difference along with risk factors for intestinal helminth infection was studied. surprisingly, the overall prevalence of helminth infection was higher in urban as compared to rural subjects10. relations of helminths with human allergy was studied and suggested that they are of significant importance to epidemiologist as well as immunologists11. the relationship between the frequency distribution of a. lumbricoides and intensity of infection in human communities was reported. the factors responsible for this relationship in the human population required further study but may involve some combinations of (i) density-dependent reduction in worm number within individuals, (ii) density-dependent parasiteinduced host motility or (iii) self-treatment by heavily infected host12. the prevalence of taenia saginata and hymenolepis nana infection amongst farmers, education concerned people, and shepherds of swat were studied and found shepherds to be more prone to infection as compared to other groups13. the incidence of intestinal parasitic infections for inand outpatient visiting king fahd medical city, riyadh, s. arabia for a period of five years was reported. t. trichiura and h. nana were prevalent and further suggested that updating the epidemiological survey of parasites at regular intervals using statistical methods14. work on treatment and diagnosis of intestinal helminths including hymenolepis, taenia, diphyllobothrium latum and dipylidium caninum was carried out. niclosamide and praziquantel are the drugs for choice for tapeworm infections15. studies on the intestinal helminths risk factors for developing active tuberculosis in patients were made16. the most prevalent helminths in humans were a. lumbricoides, t. trichiura and hookworms, and there are now approximately one billion infections with each of these worldwide17. since, the economic burden of helminths in humans along with their co-endemicity with acquired immunodeficiency syndrome (aids) and malaria18, is also very obvious in many of the study groups, therefore, it is important to launch awareness for controlling human helminth infection in karachi. the drugs currently being used as anti-helminth are albendazole, praziquantel, oxamniquine and ivermectin along with the drugs developed in the early twentieth century i.e. diethylcarbamazine and mebendazole, which are the only compound medicines being used around the world. moreover, scarcity of available anti-helminth drugs reflects the deprecating commercial markets for human helminth infections and shows how little we know about the metabolism and mechanisms of helminths by which these parasites avoid human defense against them. as far as the soil-transmitted helminths are concerned, who has suggested the use of both prevalence and intensity of infection to explain communities into transmission categories19. the present study was carried out in age groups <1-14 yrs, 15-40 yrs and > 40 yrs, to study the prevalence in male and female patients of different age groups, and whether there was single, double or triple helminth ova in various age groups of ascaris lumbricoides20, ancylostoma duodenale21 and hymenolepis nana22,23 among individuals visiting five different hospitals in gulistan-e-johar area of karachi, pakistan. analysis of helminth associated infections vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 98 m a t e r i a l a n d m e t h o d s from oct 2018 to sep 2019, 198 samples were found to be positive from 1042 examined with helminths namely ascaris lumbricoides, ancylostoma duodenale and hymenolepis nana eggs in stool samples collected from alshifa medical hospital, family care and children health hospital, darul-sehat hospital, batool general hospital and darul shifa hospital, gulistan-e-johar, karachi. the subject belonged to all age groups visiting these hospitals. the age groups were grouped < 1-14, 15-40 and > 40 yrs. the samples were collected in a sterile stool collection container carrying an identification number and a disposable wooden spatula. all samples were transported to the parasitology laboratory, department of zoology, university of karachi where samples were immediately examined by direct smear technique for helminth eggs24. eggs were photographed using (optiphot-2) photomicrographic camera. for quality control, all the slides were double read by an exchange between the parasitologists. for each data set chi-square (χ²) contingency table was analyzed to test the association of age groups and helminth infection. r e s u l t s the three types of helminth eggs recorded in the present study were a. lumbricoides, a. duodenale and h. nana (fig.1a-c). fig. 1a-c. eggs of a, ascaris lumbricoides; b, ancylostoma duodenale and c, hymenolepis nana isolated from stool samples. the chi-square between age groups and infection with three types of helminths eggs was found to be significant (χ² = 28.038, with df = 4, p < 0.001) (table. 1). a. lumbricoides and a. duodenale were most frequent in age group < 1-14 yrs, whereas h. nana prevalence was equally found in age groups < 1-14 yrs and 15-40 yrs. the chi-square between age groups in male/female individuals with helminth infection was found highly significant (χ² = 81.332, df = 10, p < 0.001) (table. 2). male patients had more prevalence of helminths eggs in stool as compared to females. the association tested by chi-square between age groups having single, double and triple infection with helminths was found to be significant (χ² = 58.205, df = 4, p < 0.001) (table. 3). in the age group, 0-14 yrs single infection was most common, while in the age group 15-40 yrs and > 40 yrs triple infection was most prevalent. table 1. infection of three different helminths in various age groups. (total tested samples; n=1042) age (years) ascaris lumbricoides helminth species ancylostoma duodenale hymenolepis nana < 1-14 30 10 0 15-40 46 6 12 > 40 24 24 4 (p < 0.001) chi-sq = χ² = 28.038 df= 4 analysis of helminth associated infections vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 99 table 2. infection in male and female individuals showing positive helminth infection. (total tested samples; n=1042) age positive male female positive male female positive male female < 1-14 32 16 24 0 0 0 15-40 36 10 4 2 10 2 > 40 24 24 16 32 8 8 (p < 0.001) chi-sq = χ² = 81.332 df= 10 table 3. number of individuals with single, double and triple helminth in various age groups. (total tested samples; n=1042) age total individuals positive single infection double infection triple infection < 1-14 64 32 16 16 15-40 64 4 6 54 > 40 60 20 0 40 (p < 0.001) chi-sq = χ² = 58.205 df= 4 d i s c u s s i o n soil-transmitted human helminth infections occur in large parts of the world, including pakistan. in the urban areas due to inappropriate sanitation, bad water quality and frequent use of open defecation locations in slum areas, there is no proper community education to prevent and control helminth infection9,25-29. actual prevalence must have been higher than what was obtained in this study, because the majority of the urban population takes medicines prior to stool test simply because of diarrhea. at the same time, since only a single stool sample was collected, the actual number of parasites or their eggs/ova may vary from time-to-time and day-today. it was reported that collecting a single sample may not show the actual population of helminths30,31, while three consequent samples are always considered more appropriate. it was stated that approximately one billion people are currently infected with the directly transmitted nematodes; a. lumbricoides and a. duodenale32. h. nana infection have been found to be more common as compared to a. lumbricoides in konkor, gadap district east, karachi33. h. nana was recorded 1.3% in medical students of multan31, whereas approximately 0.95% h. nana and 15.4% a. lumbricoides were reported in a study conducted in sargodha34. h. nana was found to be more common compared to a. lumbricoides, but did record a. duodenale in islamabad and rawalpindi areas35. in another study, a. lumbricoides (11.5%), a. duodenale (4.95%) and h. nana (4.18%) were found in samples from food handlers30. in muzaffarabad district, a. lumbricoides (3.8%) and h. nana (1.7%) were found in tested samples, but a. duodenale was not reported9. a vast majority of infections are asymptomatic pathological manifestations and depend on metabolism, size, the activity of the worm. the most obvious forms of direct damage is blockage of internal organs or from the effects of pressure caused by growing parasites, thus, the mucosa of intestine reflects chemical and physical damage of tissue36. it was found that h. nana and enterobius remained relatively common parasites of humans in many parts of iran. it was reported that hymenolepis nana was found (19%) in children aged 2 to 15 years who were referred to the main pediatric health center of lorestan province, iran37. the changes occurring in climate plays an important role in determining the future viability of human helminth parasites38. similarly, the time distribution pattern of disease may alter as a result of changing lifestyle of the analysis of helminth associated infections vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 100 people of a particular region39. the most common drugs being prescribed by medical practitioners in these five medical centers were mebendazole and albendazole. determination of occurrence and intensity of human helminth infection is an important tool / method for selective use of preventive cytotoxic drugs and to develop comprehensive programs both in rural as well as urban population along with mass awareness programs and must be launched for the helminth control. c o n c l u s i o n s the direct damage of helminths is from the blockage of internal organs or the pressure caused by growing parasites. contaminated food must be avoided, hands must be thoroughly washed before eating, after using the bathroom and in a direct contact with animals, faeces and pre-school aged children. avoid eating improper washed salads and usage of water that may be contaminated with sewage, faeces or wastewater runoff near storm drains. c o n f l i c t s o f i n t e r e s t none. f u n d i n g s o u r c e none. a c k n o w l e d g m e n t s none. l i s t o f a b b r e v i a t i o n s a. lumbricoides ascaris lumbricoides a. duodenale ancylostoma duodenale h. nana hymenolepis nana yrs years r e f e r e n c e s 1. brindley pj, mitreva m, ghedin e, lustigman s. helminth genomics: the implications of human health. plos negl trop dis. 2009; 3:e538. doi: 10.1371/journal.pntd.0000538. 2. horton jms. human gastrointestinal helminth infection: are they now neglected diseases? trends parasitol. 2003; 19:527-31. 3. hedley l, wani rls. helminth infections: diagnosis and treatment. pharm j. 2015; 295:1-13. 4. sayasone s, utzinger j, akkhavong k, odermatt p. multiparasitism and intensity of helminth infections in relation to symptoms and nutritional status among children: a cross-sectional study in southern lao peoples democratic republic. acta tropica. 2015; 141:322-31. 5. craig pa, ito a. intestinal cestodes. curr opin infect dis. 2007; 20:524-32. 6. allen je, maizels rm. immunology of human helminth infection. int arch allergy immunol. 1996; 109:3-10. 7. adegnika aa, agnandji st, chai sk, ramharter m, breitling l, kendjo e, et al. increased prevalence of intestinal helminth infection during pregnancy in a subsaharan african community. wien klin wochenschr. 2007; 119:712-6. 8. lwanga f, barbara ek, christopher go. intestinal helminth infection and nutritional status of children attending primary schools in waskiso district, central uganda. int j environ res public health. 2012; 9:2910-21. 9. chaudhry zh, afzal m, malik ma. epidemiological factors affecting prevalence of intestinal parasites in children of muzaffarabad district. pakistan j zool. 2004; 36:267-71. 10. phiri k, whitty cj, graham sm, sembatya-lule g. urban/rural differences in prevalence and risk factors for intestinal helminth infection in southern malawi. ann trop med parasitol. 2000; 94:381-7. 11. cooper pj. intestinal worm and human allergy. parasite immunol. 2004; 26:455-67. 12. guyatt hl, bundy da, medley gf, grenfell bt. the relationship between the frequency distribution of ascaris lumbricoides and the prevalence and intensity of infection in human communities. parasitol. 1990; 101:139-43. 13. khan w, noor-un-nisa, nawaz ma. incidence of tapeworm infection in human population of swat, pakistan: an occupation based study. pak j zool. 2018; 50:639-45. 14. amer oso, al-malki es, waly mi, alageel a, lubbad my. prevalence of intestinal parasitic infections among patients of king fahd medical city of riyadh region, saudi arabia: a 5-year retrospective study. j parasitol res. 2018; 8076274. doi: 10.1155/2018/8076274. 15. tanowitz hb, weiss lm, witter m. diagnosis and treatment of intestinal helminths. i. common intestinal cestodes. gastroenterol. 1993; 1:265-73. analysis of helminth associated infections vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 101 16. elias d, mengistu g, akuffo h, britton s. are intestinal helminths risk factors for developing active tuberculosis? trop med int health. 2006; 11:551-8. 17. chan ms. the global burden of intestinal nematode infections – fifty years on. parasitol today.1997; 13:438-43. 18. hotez pj, brindley pj, bethony jm, king ch, pearce ej, jacobson j. helminth infections: the great neglected tropical diseases. j clin invest. 2008; 118:1311-21. 19. montresor a, crompton dwt, hall a, bundy dap, savioli l. guidelines for the evaluation of soiltransmitted helminthiasis and schistosomiasis at community level. who, geneva. 1998. who/ctc/sip/98.1. 20. linnaeus c. systema naturae per regna tria naturae, secundum classes, ordines, genera, species, cum characteribus, differentiis, synonymis, locis. editio decimal, reformata. holmiae. (salvius). tomus. 1758; i:1-824. 21. dubini a. nuovo verme intestinale umano ancylostoma duodenale, constitutente un sesto genere dei nematoidei proprii dell’uomo. ann univ med milano.1843; 106:5-13. 22. bilharz t. ein beitrag zur helminthographia humana, au s brieflichen mittheilungen des dr. bilharz in cairo, nebst bemerkungen von prof. c. th. von siebold in breslau. zeitschrift für wissenschaftliche zoologie. 1851; 4:53-76. 23. ransom wh. an account of the tapeworm of the genus hymenolepis parasitic in man, bull. no. 18, hyg. lab. u. s. public health and marine-hospital service, washington. 1901. 24. cheesbrough m. medical laboratory manual for tropical countries. vol 1, 2nd ed. cambridge university press, cambridge. 1992. 25. bilqees fm, khan a, ahmad a. a survey of intestinal protozoan and helminth parasites in karachi. pak j med res.1982; 21:54-7. 26. khan w, noor-un-nisa, khan a. an investigation on incidence of intestinal parasites under and above 15 years age in farmers of swat, pakistan. proc parasitol. 2011; 52:43-53. 27. khan w, noor-un-nisa, khan a. diversity of intestinal parasites in male and female students and workers of education department. pak j zool. 2015; 47:65-8. 28. khan w, noor-un-nisa, khan a. soil transmitted helminthiasis in different occupational groups in swat, khyber pakhtunkhwa, pakistan. pak j pharm sci. 2017; 30:1345-50. 29. khan w, noor-un-nisa, khan a. prevalence and risk factors associated with intestinal parasitic infections among food handlers of swat, khyber pakhtunkhwa, pakistan. j food nutr res. 2017a; 5:331-6. 30. rajper gm. significant role of parasitic infestation in people concerned with food handling. med channel. 1999; 5:17-25. 31. farooki ma. intestinal parasites burden of 224 medical students. j pak med assoc.1965; 15:27-66. 32. anderson rm. the population dynamics and control of hookworm and roundworm infections. in: anderson, r.m. editors. population dynamics on infectious diseases: theory and applications. london: chapman and hall. 1982; 67-109. 33. siddiqui mi, bilqees fm, lliyas m, perveen s. prevalence of parasitic infections in a rural area of karachi, pakistan. j pak med assoc. 2002; 52:315-20. 34. ghauri sa, alam m. the pattern of intestinal parasitic infestations in sargodha area: a comparative study. pak pathol. 1992; 3:99-101. 35. qureshi ah, karamat ka, qamer rh, malik ia. intestinal parasitic infestation in rawalpindi / islamabad area: a study of 12640 stool samples. pak j pathol. 1992; 3:31-9. 36. wakelin d. helminths: pathogenesis and defenses. in: baron, s. editor. medical microbiology. 4th edition. galveston (tx): university of texas medical branch at galveston (chaptor 87). 1996. 37. mahmoudvand h, badparva e, khalaf ak, niazi m, khatami m, nazer mr. prevalence and associated risk factors of intestinal helminthic infections in children from lorestan province, western iran. parasite epidemiol control. 2020; 9:e00136. doi:10.1016/j.parepi.2020.e00136. 38. blum aj, hotez pj. global “worming”: climate change and its projected general impact on human helminth infections. plos negl trop dis. 2018; 12:e0006370. doi: 10.1371/journal.pntd.0006370. 39. saki j, khademvatan s, foroutan-rad m, gharibzadeh m. prevalence of intestinal parasitic infections in haftkel county, southwest of iran .int j infect. 2017; 4:e15593. doi: 10.5812/iji.15593. histopathological impacts of bisphenol-a on mice liver vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 90 op e n ac c e s s f u l l l e n g t h a r t i c l e the assessment of histopathological impacts of bisphenol-a on the liver in mice model faheem nawaz1,*, asmat ullah1, chaman ara1, madeeha mehboob1, muhammad idnan2 1department of zoology, university of the punjab, main quaid-azam campus lahore, pakistan. 2department of zoology, faculty of life sciences, university of central punjab lahore, pakistan. a b s t r a c t background: bisphenol-a (bpa) is one of the synthetic materials which is the chief part of polycarbonate plastics and it is the cheap alternate for metals available at time. it is considered as an ancient ecological contaminant which has harmful and severe effects on living beings all over the world. a worrisome effect of bpa is that there is incorporation in the living beings through the use of domestic appliances. objectives: the current study was carried out to investigate the lethal effects of bpa on the liver in a mice model. methodology: following complete random design-model, forty (40) mice (mus musculus) (24g ± 5g) were categorized into 4 groups (n=10) and administered with oral bpa as “low dose” (300 mg/kg/bw) and “high dose” (600mg/kg/bw), respectively. the doses were planned by keeping in view the ld50 value of the drug. this experimental treatment was conducted for 28 days, consecutively. numerical data were analyzed statistically through anova by using software spss (statistical program for social science version 20) followed by the tukey’s test to observe the differences among the groups. results: a substantial difference was observed between the treated and control groups. there was a significant elevation in the biochemical analysis of serum. microscopic and micrometric examination indicated that bpa has reduced the body and liver weight in treated groups as compared to control group. histopathological (h & e stained sections) studies revealed that there were deleterious impacts found in hepatic cells which were symptoms of hepatotoxicity. necrosis due to bpa disintegrates the normal composition of the liver, causing depression of body and liver weight when compared with control group. conclusion: the findings indicated bpa as a toxicant that is capable of acting on hepatocytic cells, resulting in histopathological alterations. bpa also show negative effects on liver function tests (lfts). keywords bisphenol a, hepatotoxicity, ld50, mus musculus, liver function tests, necrosis. *address of correspondence faheem263@gmail.com article info. received: december 14, 2020 accepted: september 22, 2021 cite this article nawaz f, ullah a, ara c, mehboob m, idnan m. the assessment of histopathological impacts of bisphenol-a on the liver in mice model. 2021; 12(2):90-97. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n back in 1891, alexander pavlovich dianin, a russian chemist was the one who first depicted the artificial capability of bpa polymers1. for the first time in 1950, it was used as a component in the production of polycarbonate. it is a lightweight, transparent, heat resistant and almost indestructible chemical. bpa was considered as one of the highest capacities of the chemical manufacturers with a worldwide consumption. in the last 80 years, bpa has grown widespread, and it is now on the list of pollutants linked to estrogenic disruption. the impending disastrous impacts of bpa on human health hazards were documented2. this artificial compound has its gigantic pervasiveness in our environment as an anthropogenic pollutant. regardless of this, it is continuously used in chief o r i g i n a l a r t i c l e histopathological impacts of bisphenol-a on mice liver vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 91 industries including food and brewing industry, medicals and safety appliances, water and milk bottles, and toys3. the underprivileged quality of plastic proliferates its leaking capacities. the toddlers having less body weight are more prone to its harmful effects4. a worrisome effect of bpa is that there is incorporation in the living beings through the use of domestic appliances5 (schematic 1). there is also a reported strong association between upraise of bpa and diabetes. many republics banned the use of bpa in edibles and their allied equipment. unfortunately, asian countries realized the hazardous impact of bpa in recent decades on living beings and injuries of organs6. hines et al., 2018 documented that employees of the bpa industries have more chances to become the victim of it7. a unique observation from the piles of the experiment revealed the bio-magnifications of the bpa in the organism8. the most recent observation evaluated long-term exposure casing organ catastrophe and having a casual role in making behavior anomalies9. nowadays, hormones disruptor mechanism of bpa is the leading cause to declare bpa as carcinogenic. lots of investigations were done to study the intrusions of bpa toxicity, signposted by a preliminary survey of the literature10. the political and economic interference has crafted a misconception regarding bpa fatal effects in layman’s mind. although, there is a wide-range use of bpa products however, the statistics about liver toxicity via bpa is still inconclusive, and unluckily in pakistan there is little awareness about bpa. therefore, this study was carried out to investigate the lethal effects of bpa on the liver. schematic 1. representation of bisphenol-a (bpa) toxicity on liver. histopathological impacts of bisphenol-a on mice liver vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 92 m a t e r i a l s a n d m e t h o d s animals: in this investigation, 40 male albino mice (swiss strain) were used. the mice were hosted in an animal house at the department of zoology, university of the punjab, quaid-azam campus, lahore, pakistan. the animals were familiarized with the native circumstances of the animal house a week before. bioethical protocol permitted by the committee of the university of punjab, lahore was applied in this research. husbandry: to diminish the peripheral interference, stainless steel cages were used under control conditions. up to 12hrs of light and dark cycle were upheld during investigational work. chick feed no. 14 (national) was fed to the animals along with water ad libitum. chemicals: all chemicals used in the study were 99.9% pure, by duskan usa. dosing: two dosages were used in this research. these doses were decided based on the ld50 value. bisphenol-a was administered orally via gavages (introduction of material into the stomach by a tube). initial body weight was noted before dosing. the dose was administered daily for twentyeight days. soya bean oil was used as vehicle control. investigational scheme: this investigational scheme consists of four groups, having 10 mice in each group (n=10). group (c): this group act as a negative control, (was not given any dose except water and feed for 28 days). group (ld): this group was treated with a low dose of bpa 300mg/kg body weight for 28 days. group (hd): this group was treated with a high dose of bpa 600mg/kg body weight for 28 days. group (vc): this group acts as a positive control and is given oil via gavages for 28 days. blood samples: the mice were kept fastened overnight, weighed, and anesthetized. blood samples were collected 24hrs after the last treatment before dissection, by cardiac puncture, for biochemical analysis. after this, mice were dissected and liver tissues were removed carefully. the liver was instantaneously preserved in preservatives after trimming extra fats. histology: liver samples were tranquilized in 10% formalin for histopathological analysis. to observe the liver morphology, several laboratory phases were performed for slide preparation. liver samples were dehydrated by series of ethanol, cleared with xylene, embedded in paraffin wax, and sliced into 5µm sections by a rotatory microtome. tissues were processed and embedded in paraffin wax. slides were examined using microscope model no.m4000d swift, japan supported by portable camera “ease-iimageur universal” with h & e staining. statistical analysis: all the data were expressed as mean ± / standard error of the mean. the data were statistically analyzed by spss (statistical program for social science version 20) software using one-way analysis of variances (anova), followed by tukey test for multi-comparison at significance level p < 0.05. r e s u l t s on the initial day of the treatment, the body weight (g) of mice have been recorded. the average final body weight (g) before dissection of mice recorded in distinct groups has been summarized in table 1. table 1. the table shows the effects of bpa on the body weight and liver weight of mice. groups body weight (initial) (g) body weight (final) (g) liver weight (mg) control (c) 20.075 ± 1.33 23.785 ± 1.44 1506.8 ± 41.87 high dose (hd) 23.815 ± 1.91 23.125 ± 1.54 1256.1 ± 29.11 low dose (ld) 24.080 ± 1.70 23.397 ± 1.73 1322.2 ± 61.87 vehicle control (vc) 23.400 ± 1.86 23.732 ± 1.64 1545.6 ± 5.98 histopathological impacts of bisphenol-a on mice liver vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 93 bpa treatment caused a significant reduction in the body as well as the liver weight of both treated groups (hd & ld) as compared to the control group (c). however, the intensity of weight reduction is associated with exposure to bpa dose and vice versa. besides, no discrepancy differences (p < 0.05) were noted between control and vehicle control. the damage due to bpa that occurred in (ld) group was less unlike in the (hd) group. the occurrence of alterations in the fundamental structure of the liver are revealed in fig 1. histological sections were observed at three different magnifications i.e., (10×04) (10×10) (10×40). sections of liver tissues were evident in massive destructions and dysfunctions in the bpa exposed groups. there was the finding of cytoplasmic vacuolization and blocking (congestion) of the blood vessel within the liver cells, and inflammatory cellular and infiltration occurred in the bile duct. the destructive symptoms of necrosis were prominent in treated groups. the whole architecture of the liver cells was deformed. centrally arranged leucocytes were scattered from their original position. clumps of leucocytes with the impact of microsteatosis as well as macrosteatosis were prominent in the high dose (table 2; fig 1). figure 1. hematoxylin and eosin (h & e) staining. histopathological analysis of the hepatic cells. c and vc are the images from liver slides of the control group illustrating normal architecture. hd and ld sections show effected liver sections by bpa doses. both shows distended and chocked main vital veins and hepatic sinusoids. liver sections of the high dose of bpa experimental group show hepatocytes with marked vacuolar degeneration, h & e stain shows constant changes. 10x04 (1st column), 10x10 (2nd column). serum analysis shows that there was a significant decrease in alp and ast, however, alt was normal and does not show any change in the treated groups (fig. 2a-i). histopathological impacts of bisphenol-a on mice liver vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 94 table 2. histopathological impacts of bpa on mice liver serum assay. s. no. liver lesions control (c) high dose (hd) 600mg/kg/b.wt low dose (ld) 300mg/kg/b.wt vehicle control (vc) 1 hepatic necrosis and periportal congestion no yes yes but less than hd negligible 2 cellular infiltration of lymphocytes in the portal area negligible yes yes no 3 disarrangement of hepatic plates with swollen no yes yes no 4 pyknotic nucleus no yes yes but less than hd no 5 proliferative bile ducts negligible yes yes no 6 cytoplasmic vacuolization no yes yes no fig 2 (a-i). fluctuations in different parameters of serum analysis in bpa exposure. histopathological impacts of bisphenol-a on mice liver vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 95 d i s c u s s i o n a significant reduction in body and liver weight was found in this study, similar to the previous results by verstraete et al., (2018) where they found that bpa exposure caused significant decrease in the body as well as liver weight. they also concluded dysfunctions and abnormal variation in the level of serum. alp and ast decreased significantly whereas alt remained undisturbed via bpa exposure11. the preliminary study of elswefy (2016) on bpa’s toxic effect on the histology of the liver clearly shows symptoms of necrosis. this likelihood of bpa includes chronic damage and fibrosis creation within the liver. the indication of conclusive necrosis correlated with this study outputs12. there are dysfunctions and necrosis in the physiological and structural properties of the liver respectively. bpa disjunctions the regular gap between the liver cells radiating from the central vein of blood vessels. it was revealed that the potency of dose determines the extent of the deformation in the internal anatomy of the liver13. the finding of kamal et al., (2018) supported the current outputs as similar consequences are also part of his studied results. our results also suggest the noticeable physical change in normal liver architecture with dilated sinusoids in treated groups. bpa was definite, intense and concrete in dose-related dysfunctions and injurious to the liver. the hostile consequence of bpa was studied by kamal et al., (2018)14. the current results coincided with the findings of wang et al., (2019) which revealed a massive degree of apoptosis is the common mean of damage in the liver. they also found an association between hepatotoxicity and the destruction15. the output correlated with the reports of faheem et al., (2016) as they concluded that bpa has a toxic impact on the liver. its prolonged exposure caused significant serious degenerative changes in the liver16, 17. the researchers revealed that hepatocyte cells have a unique sensitivity. the bpa toxicity is not only destructive and eccentric in hepatocytes but prominent. such type of damage to the liver and its parameters was observed frequently in both treated groups of this study. manna et al., (2018) classified bpa as an endocrine-disrupting chemical, after finding changes in hepatocytes of oreochromis mossambicus in his study. they validated the results as they matched concerning serum variations in mice18. serum concentration transformations after oral disclosure can be directly endorsed to the fluctuations in hepatic and gastric glucuronidation rates among the correspondents. significant changes were found in serum analysis in treated groups when compared with control groups. a sheer elevation of alt and ast were noted. however, the total bilirubin and its various components were also elevated in the treated groups. it was also revealed that variation in serum analysis in the exposed organism was more likely to be affected by molecular disturbance, leads to metabolic sickness19. they also verified that bpa is well associated with developmental mechanisms, as in these research outputs. these results correlated with our studies. tassinari et al., (2020) ascertained via an investigation that the liver, besides the thyroid gland, is the most sensitive organ to bpa exposure during the early stage of rodents20. bpa is liable for inducing the basic mechanism disorders significantly involved in lipid metabolism creating irregularity in it; they also found that this abnormality is associated with the intensity of disorder in metabolism21. elwakeel et al., (2018) revealed and proved bpa a toxicant for the liver. he observed histopathological alternation in mice liver exposed to bpa22. bpa-induced genes that played a contributory role in liver steatosis. results are supported by the work of lin et al., (2017)23. histological studies revealed bpas’ negative impacts include hepatocellular degeneration, edema formation, and necrosis of liver cells. these were finding of khan et al., (2016) which matched with the present study results. they also proposed during their study that with bpa hepatotoxicity, there was an elevation in the serum level of aminotransferase aspartate aminotransferase, alkaline phosphate, and dehydrogenase, which are the symptoms of bpa deleterious24. serum analysis found and proved bpa responsible for necrosis via his studies. the present results also have similar findings25. mahmoudi et al., (2018) demonstrated that daily oral administration of bpa caused a significant abnormal increase of lipids, which lead to its dysfunctions26. eid et al., (2015) and faheem et al., (2019) worked separately and noted significant degeneration in the liver due to bpa exposure27, 28. histopathological impacts of bisphenol-a on mice liver vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 96 c o n c l u s i o n the findings of this study confirm that ingesting bpa has major deadly consequences, including serious malfunction as well as degenerations in the liver's normal architecture (hepatic toxicity). it also harms the liver cells' compatible activities. the bpa is dangerous to use, especially for the liver and it was discovered that it was linked to blood congestion. severe haemorrhages and congestion in bpaaffected rats result in hormonal changes. e t h i c a l a p p r o v a l ethical approval for the study was obtained from the ethical board of the university of punjab, lahore, pakistan. c o n f l i c t s o f i n t e r e s t the authors declare no conflict of interest. f u n d i n g s o u r c e university of the punjab, lahore, pakistan provided us with all necessary facilities and funds. a c k n o w l e d g m e n t s i owe my sacred and humble acknowledgment to my supervisor “prof. dr. asmatullah”. moreover, i am thankful to all the contributors. l i s t o f a b b r e v i a t i o n s anova analysis of variance alp alkaline phosphatase ast aspartate aminotransferase alt alanine transaminase bpa bisphenol a h & e hematoxylin and eosin ld50 lethal dose 50 spss statistical package for the social sciences r e f e r e n c e s 1. mir sh, baddar a. association between urinary bisphenol a concentration and obesity prevalence in children and adolescents: bisphenol a and its effects on humans. in handbook of research on environmental and human health impacts of plastic pollution 1st ed. india, igi global; 2020; 214-45. 2. kumar p. role of plastics on human health. ind j pediatr. 2018; 85(5):384-9. 3. jensen tk, mustieles v, bleses d, frederiksen h, trecca f, schoeters g, et al. prenatal bisphenol a exposure is associated with language development but not with adhd-related behavior in toddlers from the odense child cohort. environ res. 2019; (3)170:398405. 4. zhang yf, shan c, wang y, qian ll, jia dd, zhang yf, et al. cardiovascular toxicity and mechanism of bisphenol a and emerging risk of bisphenol s. sci total environ. 2020; (3):18:13. 5. burakov ae, burakova iv, galunin ev, mkrtchyan es, melezhik av. highly efficient graphene‐based nanocomposites for environmental application. the elsi handbook of nanotechnology: risk, safety, elsi and commercialization. usa, scrivener publishing. 2020; (3)4:25-50. 6. huang q, chen y, lin l, liu y, chi y, lin y, et al. different effects of bisphenol a and its halogenated derivatives on the reproduction and development of oryzias melastigma under environmentally relevant doses. sci total environ. 2017; (10)1595:752-8. 7. hines cj, christianson al, jackson mv, ye x, pretty jr, arnold je, et al. an evaluation of the relationship among urine, air, and hand measures of exposure to bisphenol a (bpa) in us manufacturing workers. ann work expo health. 2018; (7):840-51. 8. suyamud b, thiravetyan p, gadd gm, panyapinyopol b, inthorn d. bisphenol a removal from a plastic industry wastewater by dracaena sanderiana endophytic bacteria and bacillus cereus ni. int j phytoremed. 2020; 22(2):167-75. 9. minatoya m, araki a, nakajima s, sasaki s, miyashita c, yamazaki k, et al. cord blood bpa level and child neurodevelopment and behavioral problems: the hokkaido study on environment and children's health. sci total environ. 2017; (12)31607:351-6. 10. xiong y, wen x, liu h, zhang m, zhang y. bisphenol a affects endometrial stromal cells decidualization, involvement of epigenetic regulation. j steroid biochem mol. 2020; 1(200):105640-9. 11. verstraete sg, wojcicki jm, perito er, rosenthal p. bisphenol a increases risk for presumed non-alcoholic fatty liver disease in hispanic adolescents in nhanes 2003-2010. environ health. 2018; 17(1):12-9. histopathological impacts of bisphenol-a on mice liver vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 97 12. elswefy se, abdallah fr, atteia hh, wahba as, hasan ra. inflammation, oxidative stress and apoptosis cascade implications in bisphenol a‐induced liver fibrosis in male rats. int j exp pathol. 2016; 97(5):369-79. 13. khawar mb, mehmood r, abbasi mh, sheikh n. assessing the hepatotoxicity of industrial leachate; histopathology and heavy metal contents in liver of wistar rats. rads j biol res appl sci. 2018; 9(2):82-7. 14. kamel ah, foaud ma, moussa hm. the adverse effects of bisphenol a on male albino rats. j basic appl zool. 2018; 79(1):1-9. 15. wang w, zhang x, qin j, wei p, jia y, wang j, et al. long-term bisphenol s exposure induces fat accumulation in liver of adult male zebrafish (danio rerio) and slows yolk lipid consumption in f1 offspring. chemosphere. 2019; (4):500-10. 16. faheem m, jahan n, lone kp. histopathological effects of bisphenol-a on liver, kidneys and gills of indian major carp, catla catla (hamilton, 1822). j anim plant sci. 2016; (2):514-22. 17. meng z, tian s, yan j, jia m, yan s, li r, et al. effects of perinatal exposure to bpa, bpf and bpaf on liver function in male mouse offspring involving in oxidative damage and metabolic disorder. environm pollut. 2019; (4):935-43. 18. manna a, amutha c. early maturation and liver necrosis in the fingerling stage of oreochromis mossambicus due to bpa can cause an ecological imbalance. rsc adv. 2018; 8(23):12894-9. 19. azevedo lf, carneiro mf, dechandt cr, cassoli js, alberici lc, barbosa jr f. global liver proteomic analysis of wistar rats chronically exposed to lowlevels of bisphenol a and s. environ res. 2020; (3):109080-6. 20. tassinari r, narciso l, tait s, busani l, martinelli a, di virgilio a, et al. life persuaded project group valeri mauro mancini francesca romana cianfarani stefano germani daniela gastaldelli amalia barsotti graziano ciociaro demetrio latta veronica della distante graziella gaggini melania landi patrizia toffol giacomo. juvenile toxicity rodent model to study toxicological effects of bisphenol a (bpa) at dose levels derived from italian children biomonitoring study. toxicol sci. 2020; (2):387-401. 21. gao p, wang l, yang n, wen j, zhao m, su g, et al. peroxisome proliferator-activated receptor gamma (pparγ) activation and metabolism disturbance induced by bisphenol a and its replacement analog bisphenol s using in vitro macrophages and in vivo mouse models. environ int. 2020; (1):105328-35. 22. elwakeel sh, abd el-monem dd. ameliorative effect of melatonin and quercetin against bisphenol a induced reproductive toxicity in male albino mice. cienc tec. 2018; (11):31-64. 23. lin y, ding d, huang q, liu q, lu h, lu y, et al. downregulation of mir-192 causes hepatic steatosis and lipid accumulation by inducing srebf1: novel mechanism for bisphenol a-triggered non-alcoholic fatty liver disease. bba-mol cell biol lipids. 2017; (9):869-82. 24. khan s, beigh s, chaudhari bp, sharma s, aliul hasan abdi s, ahmad s, et al. mitochondrial dysfunction induced by bisphenol a is a factor of its hepatotoxicity in rats. environ toxicol. 2016; 31(12):1922-34. 25. zaulet m, kevorkian se, dinescu s, cotoraci c, suciu m, herman h, et al. protective effects of silymarin against bisphenol a-induced hepatotoxicity in mouse liver. exp ther med. 2017; (3):821-8. 26. mahmoudi a, hadrich f, feki i, ghorbel h, bouallagui z, marrekchi r, et al. oleuropein and hydroxytyrosol rich extracts from olive leaves attenuate liver injury and lipid metabolism disturbance in bisphenol a treated rats. food funct. 2018; 9(6):3220-34. 27. eid ji, eissa sm, el-ghor aa. bisphenol a induces oxidative stress and dna damage in hepatic tissue of female rat offspring. j basic appl zool. 2015; (8)71:109. 28. faheem m, jahan n, khaliq s, lone kp. modulation of brain kisspeptin expression after bisphenol-a exposure in a teleost fish, catla catla. fish physiol biochem. 2019;(2) 15;45(1):33-42. assessment of pt, aptt & pc in t2dm vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 108 op e n ac c e s s f u l l l e n g t h a r t i c l e assessment of variations in pt, aptt and platelet count and their correlation with glycated hemoglobin in type 2 diabetes mellitus sajjad ahmad1,*, haji muhammad rashid2, wali ur rahman3, muhammad mujahid4, hafiz muhammad waleed5, whied ul hussan shahzad6 1department of pathology, quaid-e-azam medical college, bahawalpur, pakistan. 2department of chemical pathology, university of health sciences, lahore, pakistan. 3department of medical lab technology, university of haripur, pakistan. 4pathology department, bsl-3 lab, dhq hospital sargodha, pakistan. 5pathology department, thq hospital, mianchannu, pakistan. 6institute of industrial biotechnology, government college university, lahore, pakistan. a b s t r a c t background: diabetic patients are at higher risk of cardiovascular disease than non-diabetics. elevated glucose level in type 2 diabetes mellitus (t2dm) can induce variations in blood composition and blood vesicles, which lead towards coagulation abnormalities and cardiovascular disease. prothrombin time (pt) activated partial thromboplastin time (aptt) and platelet count are commonly used tests to assess the coagulation abnormalities of blood. objectives: to evaluate the variations in pt, aptt and platelet count in t2dm and their correlation with glycated hemoglobin. methodology: this cross sectional study was performed at asia diagnostic center, islamabad. pre-diagnosed 52 adults with type 2 diabetes mellitus, from 35 to 65 years of age, and 52 sex and age matched healthy subjects were considered as control in this study. glycosylated hemoglobin (hba1c), aptt, pt and platelet counts were measured in both groups. results: pt and aptt were significantly lower in t2dm patients (p value < 0.0001) and platelet count was slightly higher in t2dm (p value = 0.13) than the control group. pt (r2= -0.23) and aptt (r2= -0.16) were negatively correlated with hba1c, while platelet count was positively correlated with hba1c (r2=0.23) in t2dm group. conclusion: from our study, it was concluded that low pt and aptt with relatively high platelet count in t2dm than control group may induce coagulopathies that can lead toward thrombosis in t2dm patients. keywords activated partial prothrombin time, diabetes, hba1c, platelet count, prothrombin time, t2dm. *address of correspondence sajjad.ahmadbwp@gmail.com article info. received: june 01, 2021 accepted: june 29, 2021 cite this article ahmad s, rashid hm, rahman wu, mujahid m, waleed hm, shahzad wuh. assessment of variations in pt, aptt and platelet count and their correlation with glycated hemoglobin in type 2 diabeties mellitus. 2021; 12(2):108-112. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n diabetes mellitus is the most common disease, which is associated with hyperglycemia. it is mainly divided in to two types. first is type 1 diabetes that is called as insulin dependent diabetes mellitus (iddm) and second is type 2 diabetes that is non-insulin dependent diabetes mellitus (niddm)1. in these two types, different genetical and o r i g i n a l a r t i c l e assessment of pt, aptt & pc in t2dm vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 109 environmental factors are involved that can cause the destruction of β-cells of pancreas or they suppress its function2. diabetic patients are prone to develop a number of serious life threatening health complications, resulting in increased mortality rate3. persistent high level of blood glucose in t2dm causes generalized endovascular injury, which affects the heart, eyes, kidneys, muscles and nerves4. worldwide prevalence of diabetes is 8.5%, amongst which 7.3% men and 9.6% women are the affectees5. international diabetic federation (idf) states that pakistan is at number seven among the countries with highest prevalence of diabetes. about 6.9 million were diabetic in 2007 and it is assumed that there will be 11.5 million diabetic people by the end of 20256. diabetes mellitus is frequently associated with hypercoagulability resulting in 2 to 4 time higher risk of thrombosis7. the main mechanisms of increased risk of thrombosis in diabetes are diverse, and involve multiple pathways8. diabetic patients having endovascular dysfunction result in premature atherosclerosis, which enhance the chances of a thrombus. these diabetic patients have a high tendency of thrombosis because of increased activation of thrombocytes, excessive activation of prethrombotic coagulation factors, and decreased fibrinolysis9. prethrombosis status can be determined by elevated plasma levels of fibrinogen, elevated plasminogen activator inhibitor (pai)-1 and abnormal platelet function10. different studies reported different types of variations in coagulation profiles among diabetic patients9. this study was aimed to determine the variation in pt, aptt and platelet count in t2dm patients along with the analysis of correlation of these coagulation profile parameters with glycemic control (hba1c) in t2dm. m a t e r i a l s a n d m e t h o d s this cross sectional study was performed at asia diagnostic center, islamabad from 1st january, 2019 to 31st december, 2019. already diagnosed type 2 diabetic patients (both male and females) from 35 to 65 years were diagnosed according to the 1998 world health organization (who) guidelines11 were taken as cases. males and females having same age group were taken as control. patients who had a previous history of susceptibility to hypercoagulation, such as patients with a history of venous thromboembolism, hereditary coagulopathy, malignancy, gestation, recent surgery, hyperthyroidism, type 1 diabetes, liver and kidney disease were excluded from this study12. coagulopathies were excluded by medical history, physical examination for petechiae, and bruises and lab investigations for thrombocytosis. eventually, the study included 52 pre-diagnosed type 2 diabetic patients and 52 non-diabetic healthy individuals as controls. t2dm was confirmed by medical history of adult onset of hyperglycemia, fasting glucose level >126mg/dl and negative urinary ketones11. venous blood samples were collected in sodium citrate tubes for prothrombin time (pt) and activated partial thromboplastin time (aptt) tests. ethylene diamine tetra acetic acid (edta) tubes were used for hba1c and platelet count. pt and aptt tests were measured by a coagulation analyzer sysmax 50. platelet count were measured by an automated blood analyzer sysmex xp and hba1c was measured on a point-of-care testing device i-chroma. ichroma was an instrument of choice to measure hba1c because it is being used for determination of hba1c in different studies13, due to user friendly operation. this also covers the unavailability of high performance liquid chromatography (hplc); a reference method for hba1c measurement. r e s u l t s microsoft excel was used for analysis and presentation of data in tabular form. frequency and %age distribution of males and females in this study is given in table 1. table 1. frequency and percentage distribution of males and females. patients t2dm % control group male 32 (61%) 28 (54%) females 20 (38%) 24 (46%) total 52 52 assessment of pt, aptt & pc in t2dm vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 110 table 2. comparison of pt, aptt and platelet count mean by two sample t-test. tests control group t2dm p-value pt 12.3 ± 1.3 9.4 ± 1.2 <0.0001 aptt 32.5 ± 2.1 27.6 ± 3.1 <0.0001 platelet count 266.6 ± 42.8 280.8 ± 52.7 0.1366 table 3. pearson correlation coefficient of pt, aptt and platelet count with hba1c (9.2 ± 1.2) in t2dm. test parameter pearson correlation of co-efficient with hba1c pt -0.23 aptt -0.16 platelet count 0.223 the mean levels of pt, aptt, and platelet count between t2dm and the control group were compared with a twosample t-test, as shown in table 2. p-value < 0.05 was considered significant. mean pt (9.4 ± 1.2) was significantly lower in diabetic group when it was compared in control group (12.3 ± 1.3) (p < 0.0001). the mean aptt (27.6 ± 3.1) in the t2dm group was also lower than that in the control group (32.5 ± 2.1). the average platelet count of t2dm patients (280.8 ± 52.7) was slightly higher than that of the control group (266.6 ± 42.8), but the p value of 0.1366 was not significantly different. correlation analysis of pt, aptt and platelet count with blood glucose control (hba1c) was performed with pearson correlation. table 3 shows the pearson correlation coefficients of coagulation parameters with hba1c. we observed a weak negative correlation of pt, aptt and a weak positive correlation of platelet count with hba1c in t2dm. d i s c u s s i o n our results indicates the tendency of coagulation profile towards hypercoagulability in t2dm. we observed decrease in pt, aptt and increased platelet in t2dm group as compared with control group. t2dm patients are considered to be more susceptible than non-diabetic patients to macrovascular complications and thrombotic events14. type 1 or type 2 diabetic patients with increased activation of the coagulation system has been previously reported. this coagulation activation is an important factor in the occurrence of vascular complications of diabetes15. many researches have shown that coagulation activation markers such as antithrombin-thrombin complex and prothrombin activating fragments are elevated in diabetes. the plasma levels of coagulation factors, such as fibrinogen level, factor vii, viii, xi, xii (kallikrein) and von villi brond factor (vwf) are increased in diabetes16. t2dm is correlated with decreased aptt and elevated fibrinogen levels12. similar results were reported for pt and aptt in a study conducted by ephraim et al in 20179. here, we also performed correlation of pt, appt and platelet count with hba1c in t2dm and the results were also supporting the tendency of hypercoagulability with poor glycemic control. the uk prospective diabetes study (ukpds) has shown that the risk of complications in patients with t2dm is closely related to previous hyperglycemia. decrease in hba1c may reduce the risk of complications, hba1c within the normal range (<6.0%) is good indicator of lowest risk of atherosclerosis17. in a study conducted by kaur et al. in 2018, they also observed that diabetic patients develop complications according to the glycemic control and risk of microvascular complication and thrombus formation is decreased with good glycemic control14. some studies indicate that strength of clot formation is correlated with blood sugar level in diabetes18. in diabetic patients with non-insulin dependence, there is structural damage and functional impairment of the endothelium, which has proven to be essential for stimulating different coagulation factors. it is believed that high levels of von villi brand factor (vwf) in dm indicate excessive activation of coagulation factors in diabetes, assessment of pt, aptt & pc in t2dm vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 111 which may result in shorter pt and aptt in diabetic patients than the healthy control, as shown in table 2 of our study19. diabetic patients are at high risk of developing hypercoagulability and good glycemic control can reduce the risk of thrombosis20. c o n c l u s i o n this study concluded that mean pt and aptt are shorter and mean platelet count is higher in t2dm patients than controls, and that is the indication of hypercoagulability and higher tendency of thrombus formation in t2dm patients. decrease in pt, aptt and increase in platelet count are also associated with glycemic control status of the t2dm patients. therefore, it can be said that t2dm induce hypercoagulability and tendency of thrombus formation which is associated with glycemic status of the patient. e t h i c a l a p r o v a l ethical approval for the study was obtained from ethical board of medical laboratory technology, university of haripur, pakistan. c o n f l i c t s o f i n t e r e s t the authors declare that there is no conflict of interest regarding the publication of this article. f u n d i n g s o u r c e the project was not funded by any external funding body. the required funds were managed by self-contributions of authors. a c k n o w l e d g m e n t s we are thankful to ceo of asia diagnostic center, blue area islamabad, for technical support. l i s t o f a b b r e v i a t i o n s aptt activated partial prothrombin time dm diabetes mellitus edta ethylene diamine tetra acetic acid hba1c glycosylated hemoglobin iddm insulin dependent diabetes mellitus idf international diabetic federation niddm non-insulin dependent diabetes mellitus pai-1 plasminogen activator inhibitor-1 pt prothrombin time t2dm type 2 diabetes mellitus ukpds uk prospective diabetes study vwf von willebrand factor r e f e r e n c e s 1. bishop ml, fody ep, schoeff le. clinical chemistry: techniques, principles, correlations/ [edited by] michael l. bishop, edward p. fody, larry e. schoeff.; 2010. 2. zaccardi f, webb dr, yates t, davies mj. pathophysiology of type 1 and type 2 diabetes mellitus: a 90-year perspective. postgra med j. 2016; 92(1084):63-9. 3. cho n, shaw j, karuranga s, huang y, da rocha fernandes j, ohlrogge a, et al. idf diabetes atlas: global estimates of diabetes prevalence for 2017 and projections for 2045. diabetes research and clinical practice. 2018; 138:271-81. 4. chawla a, chawla r, jaggi s. microvasular and macrovascular complications in diabetes mellitus: distinct or continuum? indian j endocrinol metab. 2016; 20(4):546. 5. bouguerra r, alberti h, salem lb, rayana cb, atti j, gaigi s, et al. the global diabetes pandemic: the tunisian experience. eur j clin nutr. 2007; 61(2):1605. 6. meo sa, zia i, bukhari ia, arain sa. type 2 diabetes mellitus in pakistan: current prevalence and future forecast. j pak med assoc. 2016; 66(12):1637-42. 7. park hs, gu j-y, yoo hj, han se, park ch, kim yi, et al. thrombin generation assay detects moderateintensity statin-induced reduction of hypercoagulability in diabetes. clin app thrombosis / hemostasis. 2018; 24(7):1095-101. 8. tabit ce, chung wb, hamburg nm, vita ja. endothelial dysfunction in diabetes mellitus: molecular mechanisms and clinical implications. rev endo metab dis. 2010; 11(1):61-74. 9. ephraim rk, awuku ya, adu p, ampomah lt, adoba p, panford s, et al. high risk of coagulopathy among type-2 diabetes mellitus clients at a municipal hospital in ghana. ghana med j. 2017; 51(3):101-7. assessment of pt, aptt & pc in t2dm vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 112 10. imperatore g, riccardi g, iovine c, rivellese aa, vaccaro o. plasma fibrinogen: a new factor of the metabolic syndrome: a population-based study. diab care. 1998; 21(4):649-54. 11. alberti kgmm, zimmet pz. definition, diagnosis and classification of diabetes mellitus and its complications. part 1: diagnosis and classification of diabetes mellitus. provisional report of a who consultation. diab med. 1998; 15(7):539-53. 12. zhao y, zhang j, zhang j, wu j. diabetes mellitus is associated with shortened activated partial thromboplastin time and increased fibrinogen values. plos one. 2011; 6(1):17-28. 13. omer ma, ali ae. relationship between hba1c levels and inflammatory biomarkers (c-reactive protein, il6 and tnf-alpha) among type 2 diabetes mellituskhartoum-sudan. int j med biomed 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185:55-62. 19. junbin h, wenning w, guizhi d, li y, zhongpin l. variations and clinical significance of coagulation and fibrinolysis parameters in patients with diabetes mellitus. j tongji univ nat sci. 1998; 18(4):233-5. 20. agarwal c, bansal k, pujani m, singh k, chauhan v, rana d, et al. association of coagulation profile with microvascular complications and glycemic control in type 2 diabetes mellitus-a study at a tertiary care center in delhi. hematol transf cell therapy. 2019; 41(1):31-6. gut microbiota hypolipidemic modulating role with parkia biglobosa (fabaceae) seeds vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 102 r a d s j . b i o l . r e s . a p p l . s c i . 102 op e n ac c e s s f u l l l e n g t h a r t i c l e gut microbiota hypolipidemic modulating role in diabetic rats fed with fermented parkia biglobosa (fabaceae) seeds olayinka anthony awoyinka1,*, tola racheal omodara2, funmilola comfort oladele1, margret olutayo alese3, elijah olalekan odesanmi4, david daisi ajayi5, gbenga sunday adeleye6, precious bisola sedowo2 1department of medical biochemistry, college of medicine, ekiti state university ado ekiti nigeria. 2department of microbiology, faculty of science, ekiti state university, ado ekiti, nigeria. 3department of anatomy, college of medicine, ekiti state university, nigeria. 4department of biochemistry, faculty of science, ekiti state university, ado ekiti, nigeria. 5department of chemical pathology, ekiti state university teaching hospital, ado ekiti, nigeria. 6department of physiology, college of medicine, ekiti state university, ado ekiti, nigeria. a b s t r a c t background: modulation and balancing of host gut microbiota by probiotics has been documented by several literature. prebiotic diets such as locust beans have been known to encourage the occurrence of these beneficial microorganisms in the host gut. objectives: to study the modulating role of gut microbiota in the hypolipidemic effect of fermented locust beans on diabetic albino wister rats as animal models. methodology: albino rats (wistar strain), averagely weighing 125g were successfully induced with alloxan. there after this induction, anti-diabetic treatment was carried out on various groups of rats by feeding them ad libitum with a diet of milled fermented and unfermented parkia biglobosa seeds, respectively. results: after three weeks of treatment, it was observed that fermented locust beans caused a significant reduction (p ≤ 0.05) in glucose, total triglycerides, total cholesterol and ldl, while the hdl levels were significantly elevated (p ≤ 0.05). results of fecal analysis showed that the fermented locust beans modulated the gut microbiota through the occurrence of probiotic bacteria, bacillus subtilis in the gut and faeces of the rats. conclusion: this study support that fermented locust beans is a prebiotic diet that encourages the growth of bacillus subtilis in the gut of animals and is associated with hypolipidemic activities which alleviate diabetes as portrayed in these rat models. keywords gut, microbiota, probiotics, hypolipidemic, bacillus subtilis, diabetes. *address of correspondence olayinka.awoyinka@eksu.edu.ng article info. received: september 10, 2020 accepted: december 22, 2020 cite this article: awoyinka o a, omodara t r, funmilola comfort oladele f c, alese m o, odesanmi e o, ajayi d d, adeleye g s, sedowo p b. gut microbiota hypolipidemic modulating role in diabetic rats fed with fermented parkia biglobosa (fabaceae) seeds. rads j biol res appl sci. 2020; 11(2):102-111. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n in recent times, the future of disease and infection management has been directed towards the development and use of natural products, combined with medical methods as well as diet and nutrition, genetic engineering, phytopharmacy etc. diabetes mellitus is one of the common metabolic diseases that have plagued man over o r i g i n a l a r t i c l e gut microbiota hypolipidemic modulating role with parkia biglobosa (fabaceae) seeds vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 103 r a d s j . b i o l . r e s . a p p l . s c i . 103 centuries and has become more prevalent across the world’s population. it is sometimes described as “hyperglycemia”, a condition of elevated blood sugar beyond normal1-5. diabetes is a major threat to global public health with increasing prevalence and mortality. studies have shown that worldwide, diabetes affects over 170 million people and an exponential increase in the prevalence over the next few decades have been predicted2,3,6. hyperglycemia is plaguing the world and causing individuals, communities, governments, health organization and the world at large to invest millions in its treatment and management7-8. the management of this disease is dependent on four fundamental factors: patient education about the disease, physical exercise, diet, and the use of hypoglycemic agents9-10. the most popular hypoglycemic agent used clinically to control diabetes is insulin. although there has been successful advancement with the management and treatment of diabetes, the increase in prevalence of the disease and its associated complications remains unchanged11. asides the associated side effects of most orthodox antidiabetic agents, this mode of treatment cannot reach the rural populace and poor urban dwellers because of the cost of the drug and other factors like easy availability. hence, the needs for alternative therapies such as medicinal plants, without adverse reactions, have been experienced with the use of synthetic antidiabetic drugs12-13. there have been many studies on medicinal plants with antidiabetic properties14-16. in most low and middle income countries such as nigeria, many people still rely on medicinal plants in their natural environment for the treatment of diabetes11,14. parkia biglobosa belongs to the family fabaceae17. they are widely cultivated in various habitats in africa, and the parts of the tree are routinely used as food additives and for medicinal purposes18-19. it is generally grown throughout the west african savannah where it is commonly referred to as african locust bean20. the seeds are well known for their use in the production of local condiment due to its outstanding protein quality21. although frequently consumed by the locals, parkia biglobosa is overlooked as a gem in disease management22-24. however, its use in folkloric medicine for the treatment of diabetes mellitus has been reported in senegal and south western part of nigeria25-26. therefore, providing information on a more affordable and effective treatment methods such as diet on fermented african locust bean seeds will go a long way in combating hyperglycemia and diabetes. recently, many researches have been focused on improving health through cheaper and more effective alternatives such as diet and natural resources. researchers like mduduzi et al.60 and aderiye b et al.61 in their review stated that, diverse african traditional fermented foods and beverages produced using different types of fermentation have been used since antiquity because of their numerous nutritional values. in their reports, fermented product such as iru, from parkia biglobosa containing bacillus and staphylococcuss spp. as the major microorganism implicated as probiotics. the answer to a multitude of health problems lies in the ability to utilize and fully exploit environmental resources. these include the modulation of gut microbiota by probiotics (from a prebiotic-diet) to treat metabolic disorders such as diabetes. african locust beans seeds (parkia biglobosa) have been associated with the treatment of diabetes by various studies62-63. however, there is dearth of information on the relationship between fermentation of the seeds, the gut microbiota and the lipid profile of the host as a trialogue of potential importance in the treatment of diabetes. thus, this project would be a contribution to such innovations. hence, we analyzed the hypoglycemic ability of fermented parkia biglobosa seeds on albino wistar rats in relation to its function as prebiotic. m a t e r i a l a n d m e t h o d s collection and preparation of materials unfermented seeds of parkia biglobosa were procured at a local community market in ado ekiti. these were identified and authenticated by the chief botanist of plant science at our institution and deposited in the university herbarium (uhae 2020063). the method of aderibigbe et al.27 was adopted for the fermentation process. briefly, the dried seeds were hand-picked to remove dirt and boiled under pressure for 3hrs; thereafter, the testa was removed by dehulling and thorough washing. following re-boiling of the cotyledons for about 1hr, 300g of the boiled substrate each was weighed separately into twenty sterile baking pans. then 2ml of suitably dialyzed starter gut microbiota hypolipidemic modulating role with parkia biglobosa (fabaceae) seeds vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 104 r a d s j . b i o l . r e s . a p p l . s c i . 104 cultures were used to inoculate each of the baking pans containing the substrate. the inoculated substrate were mixed using flamed spatula and incubated at 35°c for 36hrs. preparation of starter culture for this, 6.25g of nutrient broth was weighed and dissolved in 250l of distilled water in a sterilized 500l conical flask. it was allowed to homogenize completely for 40-45min in a water bath; a clear yellow solution was formed. the homogenized clear solution was sterilized in an autoclave for 15min at 121°c to eliminate all the microorganisms present. a pure previously isolated bacillus subtilis was obtained in an inactive slant form and was fed in the homogenized solution. this was then put in an incubator for 24hrs at 37°c, and turned turbid confirming activation. subsequently, 10ml of each sample was centrifuged at 3000g. the cells were then rinsed off the broth and later homogenized with 5ml of water and set aside for fermentation. fermentation process twenty (20) fermenting cans with suitable covers were purchased from market dealers. the cans were sterilized by swabbing with cotton wool soaked in ethanol; 500g of the prepared locust beans were weighed and kept in the fermenting cans. then, 2ml of the bacillus cells were sprinkled with the aid of a syringe on the locust beans and mixed with a spatula. the cans were covered and kept in an incubator at 37ºc for 2 days. at the end of fermentation, a whitish substance was formed on the parkia biglobosa indicating a successful fermentation. the fermenting cans were put in a freezer to stop fermentation before microbiota analysis. microbiota analysis this was investigated using dilution streak plate method as described by satish28. 1000mg of the fecal sample from each group was measured and kept in sterile test tubes. this was followed by the addition of 10ml of sterile water and the feces allowed to dissolute. thereafter, 1ml of the suspension was pipetted into clean test tubes with 9ml of distilled water and shaken. this was repeated until a dilution of 10¹ obtained. aseptically, already prepared nutrient agar was decanted in duplicates in petri dishes and labeled correctly. a loopful from each of dilution (10-3) was streaked on the already prepared nutrient agar and then incubated at a temperature of 37ºc for 24hrs. the morphological characteristics and number of the colonies was observed and then sub-cultured on new plates containing nutrient agar for pure isolation of microorganisms. the pure colonies were transferred to a slant for further identification. various biochemical tests were conducted on the fecal samples to detect and identify microorganisms. the tests include: gram staining, motility test, catalase, indole, coagulase, citrase, oxidase, urease and test for various sugars. animals and alloxan administration following ethical approval from the experimental animal research ethics committee, ekiti state university (ord/ethics/ad/043); 20 male albino wistar rats averagely weighing 125g were used. they were kept under standard environmental conditions and fed rat pellets and water ad libitum. after one week acclimatization, they were divided into four groups. diabetes mellitus was induced in all the rats by administration of freshly prepared alloxan (120 mg/kg) (british drug house, london, uk) solution intraperitoneally using 0.9% w/v nacl as the vehicle. diabetes was confirmed with fasting blood glucose above 80mg/dl. group 1 was the diabetic control group (no treatment). groups 2 and 3 were treated with a diet of milled fermented and unfermented parkia biglobosa seeds (60g mixed with 40g per cage of grower’s mash) respectively, while group 4 was treated with oral administration of a reference anti-diabetic drug glibenclamide (0.01mg/150g body weight). all the treatments lasted for 3weeks. determination of fasting blood sugar as described by airaodion et al.29, following a 12hr overnight fast, the blood was collected from the tails of the rats and sugar was estimated with a digital glucometer (sinocare, china). animal sacrifice and collection of serum on day 22, rats in each study group were sacrificed under ketamine anesthesia following an overnight fast as described by alese et al.21. thereafter, venous blood was collected from each rat and transferred into sterile bottles gut microbiota hypolipidemic modulating role with parkia biglobosa (fabaceae) seeds vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 105 r a d s j . b i o l . r e s . a p p l . s c i . 105 before centrifuging at 3000rpm for 5min. the supernatants were decanted and stored at 4ºc until use. lipid profile analysis the aliquot samples were used for determination of lipid profile according to manufacturer’s instruction (randox, usa). the hdl-c was determined using the enzymatic spectrophotometric method; the samples were precipitated by the addition of phosphotungstic acid and magnesium chloride. after centrifugation at 3000g for 10min at 25ºc, the clear supernatant contained the hdl fraction, which was assayed for cholesterol concentration using a randox kit while ldl calculated using the formula of friedwald et al.30. statistical analysis all the data were subjected to t-test and one-way analysis of variance with the use of statistical graph pad-prism software31. statistical significance was set at p ≤ 0.05. fig 1. fasting blood sugar level through the period of the experiment. table 1. identification of isolated organisms from fecal sample of rats. s. no. group no. of colonies edge color shape size surface organism detected 1 fermented locust beans 35 ± 0.01 serrated cream round/ irregular small smooth/rough escherichia coli bacillus subtilis 2 unfermented locust beans 7 ± 0.1 serrated cream/ yellow round small flat/ elevated proteus vulgaris citrobacter freundii 3 glibenclamide 91 ± 0.1 smooth cream/ pink round small flat citrobacter freundii proteus vulgaris enterobacter aerogenes 4 control group 20 ± 1.5 smooth pink/ cream round small flat staphylococcus aureus bacillus subtilis enterobacter aerogenes gut microbiota hypolipidemic modulating role with parkia biglobosa (fabaceae) seeds vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 106 r a d s j . b i o l . r e s . a p p l . s c i . 106 fig 2. effect of locust beans on total cholesterol of diabetic rats. fig 3. effect of locust beans on total triglycerides of diabetic rats. fig 4. effect of locust beans on high density lipoprotein of diabetic rats’ serum. fig 5. effect of locust beans on low density lipoprotein of diabetic rats. r e s u l t s a n d d i s c u s s i o n diabetes mellitus is a metabolic condition characterized by hyperglycemia over a prolonged period with myriads of complications with associated morbidity and mortality32-33. untreated or poorly treated diabetes can result in many complications including, neuropathy, retinopathy, nephropathy, cardiovascular complications, anemia, diabetic ketoacidosis, infection and inflammation, hyperosmolar hyperglycemic state and death33. previous studies have demonstrated the hypoglycemic and insulinstimulatory effects of various medicinal plants8. in nigeria, parkia biglobosa seeds are commonly used as ingredients in local dishes; besides this, it is used as alternative medicine for the management of diabetes. hence, the need to verify its efficacy in mitigating diabetic complications and its effects on the patient34-36. in this study, it was observed that fermented parkia biglobosa reduced the level of blood glucose and lipid profiles in the experimental rats. this reduction was reflected as progression or regression of the concentrations of these metabolites (glucose and lipids). following alloxan treatment, there was a significant gain (p ≤ 0.05), in blood glucose levels of the rats (fig. 1). however, there was a significant lowering of blood sugar level following treatment with both unfermented (p ≤ 0.05) and fermented (p ≤ 0.05) locust beans when examined with the untreated group. also, the observed reduction in the blood glucose level of the animals fed with fermented locust beans (mean value, 59mg/dl) tallied favorably with the hypoglycemic effect of glibenclamide (mean value, gut microbiota hypolipidemic modulating role with parkia biglobosa (fabaceae) seeds vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 107 r a d s j . b i o l . r e s . a p p l . s c i . 107 62mg/dl). likewise, fermented locust beans proved more effective than the unfermented seeds in lowering the glucose levels of the blood as there was a significant increase in the mean value (p ≤ 0.05). the possible role of the associated microbiota is of special involvement in this present project. alterations in the make-up of the gut microbiota have been linked with an array of diseases, including cardiovascular diseases (cvd)37-38. the advancement of the growth of specific probiotics is believed to have preventative effects on heart complications due to the influence of these bacteria on human physiology, including the ability to reduce total serum low density lipoprotein-cholesterol and inflammation39-40. according to mach41, many diseases at present are as a result of lack of probiotic bacteria in the gut flora. dietary substrates such as parkia biglobosa pass largely un-metabolized in the upper gastro-intestinal tract where they are selectively utilized for the benefit of the host42-43. as shown in table 1, the organisms isolated from the fecal sample of the fermented-locust beans treated rats were escherichia coli and bacillus subtilis. proteus vulgaris and citrobacter freundii were found as fecal microbiota of the unfermented locust beans treated group. the fecal samples collected from the glibenchamide treated rats had citrobacter freundii, proteus vulgaris, enterobacter aerogenes while staphylococcus aureus, bacillus subtilis and enterobacter aerogenes were isolated from the control group. a common organism, bacillus subtilis was observed following comparison of the microbiota between the fermented locust beans treated rats and the untreated control. although, the source of this microorganism is unknown in the control group of rats; its presence in the gut of the fermented locust beans treated animals is adequately explainable. this is due to the fact that the fermentation of locust beans seeds was expedited with the action of bacillus subtilis starter culture, hence, the microbial cells was consumed live with the milled seeds during treatment. a number of literatures have confirmed the persistence of bacillus subtilis in the gut for as long as 18 days and more44-45. members of the bacillus spp have been associated with numerous probiotic properties such as production of extracellular enzymes, bile, salt and ph tolerance, bio-film formation, cellular aggregation and cell surface hydrophobicity46 as well as sensitization of the immune system, synthesis of antimicrobials such as bacteriocins, regulation of the composition of gut microbiota and anti-inflammatory effects40,47,48. in a particular study, purified exopolysaccharide from bacillus subtilis expressed therapeutic effects on cardiovascular diseases-related parameters such as reduction in blood sugar level, troponin, total cholesterol, ldl-c, and vldl as well as suppression of icam and vcam expression in streptozotocin induced diabetic rats37, 40. also, zouari et al.49 previously explored the hypoglycemic and antilipidemic properties of biosurfactants produced by bacillus subtilis spb1 strain in alloxan-induced diabetic rats. in their study, the biosurfactant reduced the plasma alphaamylase activity and rendered protection to pancreatic beta cells. apart from the hyperglycemic effects, biosurfactants controlled lipid level by promoting hdlcholesterol and inhibited the absorption of ldlcholesterol and triglycerides. this corroborates our study result which confirms that the presence of bacillus subtilis as part of the gut microbiota that may have contributed to lowering of blood glucose level of the rats; hence the general hypoglycemic effect of fermented locust beans. hyperglycemia remains a major clinical feature of poorly controlled diabetes which is associated with protein glycation (non-enzymatic glycosylation). a number of proteins including albumin, hemoglobin, collagen, and ldl undergo glycation in diabetes. the significant decrease in albumin levels in the fermented locust beans treated rats when compared to control indicates the hypoglycemic efficacy of fermented locust beans33. asides regulating carbohydrate metabolism, insulin acts a crucial role in lipid metabolism. similar to diabetes mellitus, insulin insufficiency is linked with hypercholesterolemia and hypertriglyceridemia. these conditions have been reported to occur in diabetic rats29. high level of cholesterol could cause a relative molecular ordering of the residual phospholipids, resulting in a decrease in membrane fluidity50-51. also, elevation in the levels triglycerides is one of the leading risk factors in heart disease. abnormalities in lipid profiles concentration have been shown to play a major role in the pathogenesis and progression of several disease conditions52. in this present work, the concentrations of total cholesterol (fig. 2) and triglycerides (fig. 3) significantly decreased (p < 0.05) when animals treated with fermented locust gut microbiota hypolipidemic modulating role with parkia biglobosa (fabaceae) seeds vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 108 r a d s j . b i o l . r e s . a p p l . s c i . 108 beans were examined with the untreated control group. this implies that fermented locust beans may be capable of preventing the progression of coronary heart diseases. the two of the main groups of plasma lipoproteins that are involved in lipid metabolism and the exchange of cholesterol, cholesterol ester and triglycerides between tissues are high density lipoproteins (hdl) and low density lipoproteins (ldl)29,53-54 studies have shown that increased concentrations of total cholesterol and/or ldls in the blood are important risk factors for coronary heart disease55-56. while hdl is usually termed ‘good cholesterol’, ldl is known as ‘bad cholesterol’. in this study, treatment of the rats with fermented locust beans resulted in a significant increase in the serum level of hdl-cholesterol (fig. 4) when compared with the diabetic control animals while it significantly decreased the level of ldl-cholesterol (fig. 5). the increased in hdl/ldl ratio in the animals treated with fermented locust beans when compared with the control diabetic rats indicates that a diet of fermented locust beans can reduce the risk of developing heart diseases, because a high hdl/ldl ratio has been confirmed to be beneficial and cause lower risk of coronary heart diseases34,54,57-59. in this study, treatment with parkia biglobosa seeds restored the derangements in diabetic rats. however, the fermented seeds showed the best efficacy in hypoglycemic and antidiabetic activities. this performance could be possibly attributed to the prebiotic property of parkia biglobosa as well as the probiotic activities of the starter culture bacillus subtilis and the gut beneficial microbiota of the rats. c o n c l u s i o n the findings of this study shows that african locust beans fermented with bacillus subtilis is capable of producing modulation in the gut microbiome composition of albino wistar rats, thereby leading to reduction of lipid levels as well as amelioration of blood glucose levels and the subsequent alleviation of diabetic state in the rats. the positive results obtained from this study suggests that well controlled diets with use of preferably fermented locust bean as prebiotics can serve as a major nonpharmacologic option for the treatment of diabetes. c o n f l i c t s o f i n t e r e s t the authors declare no conflicts of interest regarding the publication of this paper and no part of this manuscript has been submitted for publication to another journal. f u n d i n g s o u r c e the present project was partly funded by tetfund research intervention years 2017-2018. (ad/ord/44/tet/vol.3/128). a c k n o w l e d g e m e n t s the authors hereby appreciate the encouragement and support of prof. rocio campos-vega, autonomous university queretaro, mexico during this work. l i s t o f a b b r e v i a t i o n s hdl high density lipoprotein ldl low density lipoprotein tc total cholesterol tg triglyceride r e f e r e n c e s 1. boroni map, de cássia g, alfenas r. the influence of endotoxemia on the molecular mechanisms of insulin resistance. nut hosp. 2012; 27(2):382-90. 2. ada. american diabetes association: diagnosis and classification of diabetes mellitus. diabetes care. 2013; 36(1):67-74. 3. sule oj, godwin j, abdu ar. preliminary study of hypoglycemic effect of locust bean (parkia biglobosa) on wistar albino rat. j sci res rep. 2015; 4(5):46772. 4. murray hj, young mj, hollis s, boulton ajm. the association between callus formation, high pressures and neuropathy in diabetic foot ulceration. diabetic med. 1996; 13:979-82. 5. joh t. trans glucosidase improves the gut microbiota profile of type 2 diabetes mellitus patients: a randomized double-blind, placebo-controlled study. bmc gastroenterolitis. 2013; 13:81-7. 6. chan m, baxter h, larsen n. impact of botanical fermented foods on metabolic biomarkers and gut microbiota in adults with metabolic syndrome and gut microbiota hypolipidemic modulating role with parkia biglobosa (fabaceae) seeds vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 109 r a d s j . b i o l . r e s . a p p l . s c i . 109 type 2 diabetes: a systematic review protocol. bmj open. 2019; 9:1-12 7. reusch, je. diabetes, microvascular complications, and cardiovascular complications: what is it about glucose? j. clin. investig. 2003; 112: 986-8. 8. belayneh, ym, birhanu z, birru em, getenet g. evaluation of in vivo antidiabetic, antidyslipidemic, and in vitro antioxidant activities of hydromethanolic root extract of datura stramonium l. 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pharmacol. 1997; 7(29):162-7. 51. toya t, corban mt, marrietta e., horwath ie, lerman lo, murray ja. coronary artery disease is associated with an altered gut microbiome composition. plos one 2020; 15(1):1-13. 52. rotimi os, david ao, olusola at, regina nu, elizabeth ab, oladipo a. amoxillin and pefloxacingut microbiota hypolipidemic modulating role with parkia biglobosa (fabaceae) seeds vol. 11 (2), december 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 111 r a d s j . b i o l . r e s . a p p l . s c i . 111 induced cholesterogenesis and phospholipidosis in rat tissues. lip heal dis. 2015; 14:13-30. 53. riddell jb, gallegos aj, harmon l, mcleod kr. addition of a bacillus based probiotic to the diet of pre ruminant calves: influence on growth, health and blood parameters. int j appl res vet med. 2010; 8:78-84. 54. reetta s. modulation of gut microbiota for health by current and next-generation probiotics. nutr. 2019; 11:1921-8. 55. law mr. lowering heart disease risk with cholesterol reduction: evidence from observational studies and clinical trials. europ heart j. 1999; 1:s3-s8. 56. salonen a, de vos wm. impact of diet on human intestinal microbiota and health. ann rev food sci technol. 2014; 5:239-62. 57. castelli l. epidemiology of coronary heart disease. amer j med. 1984; 76:4-12. 58. grigorescu i, dumitrascu dl. implication of gut microbiota in diabetes mellitus and obesity. acta endocrinologica (buc). 2016; 2:206-14. 59. magdalena d, pieczynska y, yang s, petrykowski s, olaf kh, atanas ga, et al. gut microbiota and its metabolites in atherosclerosis development. mol. 2020; 25:5-15. 60. mduduzi pm, taurai m, ademola oo. perspectives on the probiotic potential of lactic acid bacteria from african traditional fermented foods and beverages. food nutr res. 2016; 60:29630-7. 61. aderiye b, laleye s. relevance of fermented food products in south west nigeria. plant foods hum nutr. 2003; 3:116-9. 62. bhagavathi ss, periyanaina k, mani ip, chaiyavat, ca. mini review on antidiabetic properties of fermented foods. nutr. 2018; 10(12):1973-9. 63. marco ml, heeney d, binda s, cifelli cj, cotter pd, foligné b, et al. health benefits of fermented foods: microbiota and beyond. curr opin biotechnol. 2017; 44:94-102. awareness of ebola virus disease vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 43 op e n ac c e s s f u l l l e n g t h a r t i c l e awareness of ebola virus disease among the medical and non-medical personnel in lahore waqas iftikhar1, farah javed2, maria nazir3, mussarat rafiq4,*, nimra afzal5, muhammad babar khawar6,* 1department of pharmacology and toxicology, university of veterinary and animal sciences, lahore, pakistan. 2department of food sciences and human nutrition, university of veterinary and animal sciences, lahore, pakistan. 3ruhr universitat, bochum nrw, germany. 4institute of zoology, university of punjab, lahore, pakistan. 5department of zoology, faculty of science and technology, university of central punjab, lahore, pakistan. 6department of zoology, university of narowal, narowal, pakistan. a b s t r a c t background: ebola hemorrhagic fever, also called ebola virus disease (evd), is caused by a member of filoviridae family known as ebola virus (ebov). the incubation period of this virus is 2 to 21 days and initial symptoms may include fever, chills, headaches, muscle aches, and loss of appetite. objectives: the study was aimed to assess the effectiveness of awareness sessions about evd among university population. methodology: a cross sectional study was carried out post awareness sessions at different universities of lahore and data was compared with data of medical personnel. results: our study indicated that 75% of university population have gained the knowledge about ebola virus disease after the awareness session. conclusion: the present study concludes that awareness sessions about evd are highly effective in spreading basic knowledge about the disease, therefore, it is recommended to use multidimensional approaches such as seminars, awareness campaigns, presentations and social media etc. to increase awareness of diseases so that the possible outbreak of this disease in pakistan could be prevented. keywords awareness, cross-sectional study, ebola virus, filoviridae, medical personnel, prevention. *address of correspondence babar.khawar@uon.edu.pk mussaratrafiq369@gmail.com article info. received: may 28, 2021 accepted: june 01, 2022 cite this article iftikhar w, javed f, nazir m, rafiq m, afzal n, khawar mb. awareness of ebola virus disease among the medical and non-medical personnel in lahore. 2022; 13(1):43-49. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n ebola virus is a fellow of the filoviridae viral family of rna viruses, which are recognized by their thin and long filaments. it is named after the ebola river from where the virus was originated. ebov is the causative agent of ebola hemorrhagic fever. it damages the endothelial cells lining of the blood vessels and lead to difficulty in coagulation of the blood cells. due to failure of platelets coagulation and damaged blood vessels, patients suffer from hypovolemic shock or decrease in blood pressure. ebola can have up to a 90% mortality rate1. many different types of ebola virus infect humans. currently these are zaire, sudan, and ivory coast ebola virus, named for the respective areas in africa where the strains were first found. reston ebola virus is a type of ebola that only infects animals. it was first discovered ebola outbreak in reston, virginia2. o r i g i n a l a r t i c l e awareness of ebola virus disease vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 44 zaire and sudan strains were discovered in 1976 when outbreak of ebola hemorrhagic fever occurred in zaire and sudan. the zaire ebola virus belongs to the group of human affecting viruses that have reportedly highest fatality rate. in the 1976 outbreak, the 88% of patients died, 81% in 1995, 73% in 1996, 80% in 2001-2002, and 90% in 2003. sudan ebola virus has a lower fatality rate, yet very dangerous, with the reported fatality as 53% in 1976, 65% in 1979, 53% in 400 patients infected in 2000, and 41% in 20042. fruit bats of the pteropodidae family are natural ebola virus hosts. ebola enters in the humans through close contact with blood, secretions, organs or other body fluids of infected animals such as chimpanzees, gorillas, fruit bats, monkeys. ebola can also spreads through human-tohuman contact or direct contact (through broken skin or mucous membranes) with the blood, secretions, organs or other body fluids of infected people, and with surfaces and materials (e.g. bedding, clothing) contaminated with these fluids3. the recovery period from ebola virus infection is up to 7 weeks. while during incubation period virus do not transmits. a person shows symptoms within 21 days after having contact with ebola. ebov transmission occur through following ways; (i) having slept in the same household with a case, (ii) has had direct physical contact with the case (dead or alive) during the illness, (iii) has had direct physical contact with the (dead) case at the funeral, (iv) has touched his/her blood or body fluids during the illness, (v) has touched his/her clothes or linens and (iv) has been breastfed (baby) by the patient4. the incubation period of the ebola virus is 2 to 21 days. human are not infectious until they show symptoms. initial symptoms that appear are sore throat, fever, muscle pain, fatigue, and headache, while other symptoms are diarrhea, vomiting, rash, symptoms of decreased liver and kidney functioning, and in some circumstances, both internal and external bleeding (e.g. oozing from the gums and blood in the stools). in the analytical way there is a reduction in white blood cell and platelets and elevation in liver enzymes has been reported. it is very problematic to isolate evd from other infectious diseases such as malaria, meningitis, and typhoid fever5. according to timeline of infection the different diagnostic tests are described in table 16. table 1. diagnostic test on the basis of infection timeline. timeline of infection diagnostic tests available within a few days after symptoms begin • antigen-capture enzyme-linked immunosorbent assay (elisa) testing • igm elisa • polymerase chain reaction (pcr) • virus isolation later in disease course or after recovery • igm and igg antibodies retrospectively in deceased patients • immunohistochemistry testing • pcr • virus isolation awareness of ebola virus disease vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 45 moreover, careful-rehydration with oral or iv fluids treatments are given according to specific symptoms. no proper treatment and vaccine for this virus is available. disease can be prevented by taking the standard precautions such as safe discard of sharp instruments, hygiene of hand, use of personal protective equipment (ppe) according to the risk evaluation, clean environment and sterilized clinical tools4. by assuring some safety measures inside ebv units of hospitals the spread of ebov can be reduced, following are the safety measures, (i) limit number of staff allocated to patient care, (ii) bound the number of visits (iii) retain log books to record staff caring for the patient as well as for visitors (iv) use of ppe by both health care personnel’s and visitors (v) correct use of surgical masks and goggles and (vi) eliminate ppe before leaving the isolation area. special precautions should be taken when eliminating ppe to prevent contact with eyes and mucous membranes7. similarly, health-care personnel should always follow the standard protective measures when handling the patients. these include basic safety measures such as respiratory hygiene, hand hygiene, use of personnel protective equipment. moreover, health-care personnel treating the ebola positive patients should avoid contact with the patient’s blood, body fluids and contaminated places or things such as clothing and bedding. when in close contact (within 1 meter) of ev patients, health-care personnel should wear medical mask and goggles8. world health organization (who) has warned pakistan about the possibility of ebola outbreak in pakistan. recent deadliest spread of evd in west africa has become alarming for pakistan as large number of people travel to pakistan from africa9, 10. a cross sectional study by salman et al., (2017) involving participants from various universities of lahore have reported that a large number of university population (91.8%) had inadequate knowledge of evd. this study had suggested that there is an immense need to increase awareness about evd among university population because universities hosts large number of national students and even international students11. therefore, we have designed the two step study, where we first aimed to increase the knowledge of university population, and then we have carried out a questionnaire base study to check the effectiveness of evd awareness sessions by comparing the results of post awareness session among university participants with medical personnel. m a t e r i a l s a n d m e t h o d s a cross sectional study was carried out to evaluate awareness about evd among two study groups, medical and non-medical professionals from different universities and hospitals of lahore. a predesigned and selfadministered questionnaire was used to collect data from study participants. non-medical study participants were selected from hajvery university lahore, university of lahore, lahore leeds university, on the other medical participants were included from various hospitals like shalamar hospital, services hospital, mayo hospital. questionnaire was distributed after taking consent from each participant and both study groups have equal number of sample size, 500 participants in each study group. seminars, presentations and broachers’ distribution was conducted for providing information to the people regarding the disease. we went to various groups and after giving awareness provided them with the questionnaires regarding the causes, incidences, risk factors, transmission, adverse effects, epidemiology, laboratory findings, and all other related knowledge about ebola virus disease. this observational research was independent of any variable manipulation. the purpose of our study was to assess the level of knowledge, find the percentage of the people who are well-aware about the disease and need to analysis the epidemiological features of ebola virus disease (evd), as well as our current thoughtful of the transmission dynamics and the effect of control interferences against ebola transmission. statistical analysis data was analyzed using ibm spss software version 22.0. data mining was performed after initial data coding. variables were recorded into binary variables by assigning code (0) to the correct option and all the wrong options were coded (1). recoded relevant items were aggregated into the variables i.e. knowledge of evd, precautionary knowledge of evd, overall awareness of evd of two (yes/no, aware/unaware) and three categories (low, medium, high). after categorizing variables, cross tabulations was performed between independent variable (medical vs. non-medical personnel) and dependent awareness of ebola virus disease vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 46 variables. in addition, the pearson chi-square test was used to check the association between study variables r e s u l t s questionnaire was distributed among different individuals, 1000 questionnaires were solved, compared and evaluated. the questionnaires were distributed among medical and nonmedical personnel (as shown in table 2) and evaluated afterwards. equal number of males and females participated in the study, involving students and working people (table 3). table 4 presents cross tabulation results between medical and non-medical personnel with the knowledge of evd. in addition, it also includes chi-square results at significant value 0.05. cross tabulation results indicated that 45% of medical and 35% of non-medical personnel know about ebola virus. the value of pearson chi-square test indicates that the difference between the knowledge of two groups about ebov was non-significant. similarly, awareness among participants of both groups about ebov diagnostic test, severity of disease and non-hygienic conditions that leads to evd was equal. while the knowledge about the vaccination of disease, air as its route of transmission and evb effects on liver enzymes was higher among medical personnel compared to non-medical personnel. moreover, both groups were well aware about treatment of disease, precautionary measures and transmission from mother to babies and from person to person through needles and body fluids. overall, the baseline results of our survey indicated that 75% of university population have knowledge about ebola virus disease. table 2. number of questionnaires of medical and nonmedical personnel. medical personnel 500 non-medical personnel 500 table 3. total number of participants of the study. total no. of participants 1000 male 500 female 500 students 700 working people 300 table 4. cross tabulation and pearson chi-square results of the knowledge of evd. knowledge of evd pearson chisquare aware unaware do you know what ebola virus is? medical non-medical 45% 35% 5% 15% 6.250 does it have any vaccination? medical non-medical 45% 30% 5% 20% 12.000* which test is preferred to diagnose evd? medical non-medical 33% 25% 17% 25% 2.627 contd…. awareness of ebola virus disease vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 47 is it treatable? medical non-medical 40% 30% 10% 20% 4.672 any non-hygienic condition can lead to the disease? medical non-medical 40% 38% 10% 12% 0.233 ebola virus disease is also called? medical non-medical 38% 25% 12% 25% 7.250* does ebola virus disease has severity? medical non-medical 25% 20% 25% 30% 1.010 the incubation period of ebola virus is medical non-medical 38% 33% 12% 17% 1.214 does ebola virus transmit through air? medical non-medical 43% 30% 7% 20% 8.574* does ebola virus affect the liver enzymes? medical non-medical 45% 30% 5% 20% 12.000* the level of liver enzymes are? medical non-medical 38% 33% 12% 17% 1.214 the ebola virus disease patients are placed in? medical non-medical 35% 33% 15% 17% 0.184 ebola virus is transmitted through? medical non-medical 45% 38% 5% 12% 3.473 can ebola virus transmit to babies from affected mother? medical non-medical 44% 35% 6% 15% 4.882 is ebola virus transmitted from? medical non-medical 30% 25% 20% 25% 1.010 is ebola virus is transferred from one country to another? medical non-medical 45% 38% 5% 12% 3.473 *= significant value at 0.05 awareness of ebola virus disease vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 48 d i s c u s s i o n findings of our study reveals that awareness sessions were very effective and significantly contributed in spreading awareness among university students. previously, a study by salman et al, reported that 91.8% of university population had inadequate knowledge about ebola virus disease. this study suggested that there is an immediate need to increase the knowledge and spread awareness among university population11. our study determined that use of multidimensional approaches such as seminars and awareness campaigns are highly effective in increasing knowledge of general public about evd. the results of our study are in accordance with the results reported by rehman and colleagues (2019), according to which the basic training session regarding evd are highly effective in increasing basic knowledge about ebola virus disease. the results of our study have suggested that about 75% of participants gave the correct responses which suggested that the awareness session significantly improved the knowledge of university participants12. c o n c l u s i o n the findings of our study concludes that evd awareness sessions are highly effective in improving understanding and knowledge of ebola virus disease. to halt the possible outbreak of evd in pakistan, it is mandatory that all organizations at different levels of the health care system and all health care personnel such as clinical, public health professionals, laboratory, etc. must be informed about the characteristics and modes of transmission of the disease. similarly, it is recommended to communicate with the public about the evd via social media, awareness campaigns, and seminar etc. as our study had proved their effectiveness. national health authorities are encouraged to control evd by the community cooperation. national authorities should advice travellers who travel to those countries where transmission of evd is documented, to get tested. media should spread information about the modes of transmission and prevention of evd. e t h i c a l a p r o v a l the cross section study was conducted following ethical approval guidelines. c o n f l i c t o f i n t e r e s t the author declares no conflict of interest. f u n d i n g s o u r c e the study has no internal or external funding source to declare. a c k n o w l e d g e m e n t s the author is grateful to the concerned institution for providing facilities to conduct this study. l i s t o f a b b r e v i a t i o n s elisa enzyme-linked immunosorbent assay ebov filoviridae family known as ebolavirus ppe personal protective equipment pcr polymerase chain reaction rna ribonucleic acid who world health organization elisa antigen-capture enzyme-linked immunosorbent assay r e f e r e n c e s 1. leroy em, kumulungui b, pourrut x, rouquet p, hassanin a, yaba p, et al. fruit bats as reservoirs of ebola virus. nature. 2005; 438(7068):575-6. 2. pourrut x, kumulungui b, wittmann t, moussavou g, délicat a, yaba p, et al.. the natural history of ebola virus in africa. microb infec. 2005; 7(7-8):1005-14. 3. weingartl hm, embury-hyatt c, nfon c, leung a, smith g, kobinger g. transmission of ebola virus from pigs to non-human primates. sci rep. 2012; 2(1):1-4. 4. siegel jd, rhinehart e, jackson m, chiarello l. 2007 guideline for isolation precautions: preventing transmission of infectious agents in health care settings. am j inf cont. 2007; 35:65-9. 5. sodhi a. ebola virus disease: recognizing the face of a rare killer. postgrad med. 1996; 99(5):75-8. 6. towner js, rollin pe, bausch dg, sanchez a, crary sm, vincent m, et al.. rapid diagnosis of ebola hemorrhagic fever by reverse transcription-pcr in an outbreak setting and assessment of patient viral load as a predictor of outcome. j virol. 2004; 78(8):433041. 7. poel wh, lina ph, kramps ja. public health awareness of emerging zoonotic viruses of bats: a awareness of ebola virus disease vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 49 european perspective. vector-borne zoo dis. 2006; 6(4):315-24. 8. garner js, hospital infection control practices advisory committee. guideline for isolation precautions in hospitals. inf cont hosp epidemiol. 1996; 17(1):54-80. 9. cdc a. outbreaks chronology: ebola virus dis. 2014; 1-9. 10. the nation newspaper. pakistan at risk of deadly ebola virus: who spokesperson. 2014; 23-9. 11. salman m, shehzadi n, hussain k, saleem f, khan mt, asif n, et al. knowledge of ebola virus disease among a university population: a cross-sectional study. american j inf cont. 2017; 45(2):e23-5. 12. rehman h, ghani m, rehman m. effectiveness of basic training session regarding the awareness of ebola virus disease among nurses of public tertiary care hospitals of lahore. jpma. 2020; 10-8. breeding potential of pakistan’s national bird vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 70 op e n ac c e s s f u l l l e n g t h a r t i c l e breeding potential of pakistan’s national bird chukar partridge: captivity vs natural habitat saima qadeer1,*, rabea ejaz2, asma u husna3, asima azam2, muhammad a khan4 1department of zoology, division of science & technology, university of education, lahore, pakistan. 2department of zoology, shaheed benazir bhutto women university peshawar, pakistan. 3department of zoology, university of haripur, khyber pakhtunkhwa, pakistan. 4punjab wildlife and parks department, punjab, pakistan. a b s t r a c t background: chukar partridge (alectoris chukar), the national bird of pakistan, is present in the himalayan ranges of balochistan, the hillside of punjab and sindh provinces. objective: the aim of this study was to compare the aspects of breeding biology of chukar partridge (alectoris chukar) viz nest structure, breeding season, clutch size, hatching success, and incubation period in natural habitat and captivity. methodology: as study areas, chinji national park (for natural habitat) and a private farm (for captive conditions) were selected for this purpose. during breeding season, field observations were taken to record the data. results: the breeding season of chukar partridge extends from mid-february to the end of may in natural habitat while in captive conditions, it extends from february to september. in the natural habitat, all of its nests were located on ground and were formed with rocks, stones and straws of dry vegetation. in captivity, eggs were laid on ground in pens especially made according to the comfort of the birds as in natural conditions. egg laying period for birds is extended in captivity (from mid of march till september) compared to natural habitat (from mid of march to the start of april). the clutch size of chukar partridge was observed to be greater in captivity (12.4 ±1.932) than in the natural habitat (9.33 ±1.59). however, mean incubation period was the same for both in natural conditions and captivity i.e., 20.5 ±0.70 days (range 18-23 days). the hatching rate of eggs per clutch was greater in captivity (10.7 ±1.70) compared to natural habitat (7.3 ±1.88). conclusion: in conclusion, chukar partridge has a greater breeding potential in captive conditions compared to natural environment, which supports its healthy population. keywords breeding potential, captivity, chukar, clutch size, egg laying, natural habitat. *address of correspondence saima.qadeer@ue.edu.pk article info. received: july 06, 2021 accepted: march 24, 2022 cite this article qadeer s, ejaz r, husna a, azam a, khan ma. breeding potential of pakistan’s national bird chukar partridge: captivity vs natural habitat. 2022; 13(1):70-75. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n pakistan has some of the world’s rarest animals and plants due to its diverse topography and geographic location i.e., between three zoogeographical regions: the palearctic, oriental and the ethiopian. pakistan has a variety of ecosystems and has diverse avian fauna1. five partridge species are found in pakistan; the chukar partridge (alectoris chukar), see-see partridge (ammoperdix griseogularis, snow partridge (lerwa lerwa), black partridge (francolinus francolinus) and grey partridge (francolinus pondicerianus)2. the chukar partridge is a game bird placed in the order galliformes, in the family phasianidae. the genus alectoris consists of seven o r i g i n a l a r t i c l e breeding potential of pakistan’s national bird vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 71 species and 24 subspecies worldwide, including 16 subspecies of alectoris chukar3. alectoris chukar is the national bird of pakistan. here, it is adaptable to various environments like rocky, arid, hilly, arid hillside and himalayan ranges. it prefers to live in open, dry, rocky mountain slopes, and hillsides4, 5. it is also found on barren plateaus and deserts with sparse vegetation4, 6. it is distributed in punjab, baluchistan, sindh, kohistan, chitral, salt range, swat, gilgit4, 7, 8 and ajk9. in natural, breeding behavior of this bird change according to altitude and latitude. in alpine pastures it does not start nesting till late june. however, at low altitude, it start breeding earlier. the main breeding season is from april to may, with a normal clutch size of 6 to 9 eggs, but in areas with high rainfall, the clutch size can vary between 15 and 19 eggs. most nests in balochistan were found in late apriljuly2. as the environmental conditions get unfavorable, only a few pairs breed. the male chakoor takes away his female bird and defend her from other male attraction. the couple begins to make calls and practices of stepping on the ground and poking various small things. initially the calls were slow from both partners, then transformed into more louder that was easy to hear even from a distance2. so far, only a few research studies has been conducted about the distribution, population, presence and habitat utilization on the alectoris chukar in pakistan10-14. the present study is a step forward in this connection and also explores the breeding potential of a. chukar in captive conditions. the study is aimed to compare the breeding potential of a. chukar in natural and captive habitats. m a t e r i a l s a n d m e t h o d s study area the study was conducted at chinji national park (natural habitat; 33°0'36.87"n, 72°29'30.98"e; 6076ha) and a private farm (for captive conditions) during the breeding season jan-oct, 2020. the park is a protected area of international union for conservation of nature category ii (national park) established in 1987, located in tehsil talagang, district chakwal, punjab, pakistan. average annual rain in this area is 537mm, out of which 308mm is during the monsoon season (july-sep). usually in this area, 27ºc is the maximum temperature (in june) and 2.2ºc is the minimum temperature (in january). the relative humidity during monsoon is up to 80%. the eroded land of chinji national park contains igneous rocks, sandstones and rock salt3 (fig. 1). figure 1. study area map of chinji national park: a natural habitat of alectoris chukar. breeding potential of pakistan’s national bird vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 72 study design a reconnaissance survey was used to select different study sites. the study surveys were conducted for ten months (january 2020-october 2020). field observations were taken twice a week, during breeding season, to record the data on breeding aspects of alectoris chukar including the onset of breeding season, clutch size, nest structure, hatching success and incubation period in the natural and captive conditions. for data collection of these breeding aspects, line transect surveys were conducted before the commencement of breeding season of alectoris chukar. chukar nests were traced by following the bird carrying food and nesting material, or by viewing the behavior cues. standard protocols were followed during monitoring to reduce disturbance15. when an active site was located it was marked and allotted a specific number by gps navigator. marked and numbered nests were regularly visited and data sheets were used to record information from egg laying till fledging from january 2020-october 2020. in captivity, the nest was tagged and visited regularly. statistical analysis before data analysis, the normality of the data was tested by using the shapiro-wilk test16. as the data was not normally distributed, a logarithm transformation log (x+1) was used. t-test was used to compare the clutch size, incubation period, and hatching success of a. chukar in natural habitat and captivity (α= 0.05). r e s u l t s the data on the comparison of the egg-laying period in natural and captive conditions is shown in table 1. in the natural habitat, the egg laying period was from mid of march to the start of april; while in captivity, it extended from mid of march till september. similarly, in the natural habitat, nests were formed with rocks, stones and straws of dry vegetation, while in captivity, eggs were laid on the ground in pens. the data on the comparison of clutch size, hatching success, and incubation period in natural and captive conditions are shown in fig. 2, 3 and 4, respectively. the clutch size in captive conditions (12.4 ±1.932 eggs per nest) was greater (p < 0.05) compared to natural habitat (9.33 ±1.59 eggs per nest). the incubation period remained the same (p > 0.05) for both natural habitat and captive conditions (20.53 ±1.76 days). however, the hatching rate was higher (p < 0.05) in captive conditions (10.2 ±1.79 eggs per clutch) compared to natural habitat (8.42 ±0.97 eggs per clutch). figure 2. comparison of clutch size of alectoris chukar in natural habitat and captive conditions. bars with different letters differ significantly (p < 0.05). table 1. comparison of egg laying period of alectoris chukar in natural habitat and captive conditions. types of habitat egg laying period starting period ending period natural conditions mid of march start of april captive conditions mid of march end of september breeding potential of pakistan’s national bird vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 73 figure 3. comparison of incubation period of alectoris chukar in natural habitat and captive conditions. figure 4. comparison of hatching rate of alectoris chukar in natural habitat and captive conditions. bars with different letters differ significantly (p < 0.05). figure 5. showing captive photograph of alectoris chukar. breeding potential of pakistan’s national bird vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 74 d i s c u s s i o n alectoris chukar, commonly known as chukar, is a game bird that belongs to the family phasianidae. chukar is a chunky bird with 32-35cm body length with an amazing color scheme like grey breast, brown back, and sandycolored belly with red legs and white face. the different populations have different shades of colors. chukar has rufous-streaked flanks, red legs, and coral red bill, and its face is white with a black gorget. the female is slightly smaller in size and lacks the spur3, 4 (fig. 5). as herbivores, the main source of food of chukar is annual and perennial grasses4. a few other sources of food incorporate seeds of weeds, wild natural products, leaves, and bulbs, which they are able to burrow up with their bills. since these birds live in drier situations, they have to have a satisfactory water supply accessible at all times, particularly in the summer months. the diet of chukar is different in the form of breeders. it primarily consists of soybean meal, yellow corn, limestone, wheat bran fat, and salt4. nesting habitat is also dry, having rocky slopes with open, brushy cover. in the present study, nests were found which were formed with rocks, stones, and straws of dry vegetation. the results of the present study suggested that the pairs are set in mid-march when a male performs a courtship display including a head tilt and a showing of his barred flanks5, 14. both start to call and take part in a "tidbit ting display" (pecking at various objects). in captivity, nests are laid on the ground in pens that are well-prepared to keep in mind the preference of material that is comfortable for birds and mostly similar to that of natural habitat. in captivity, the nests are also equipped with all needs required by birds i.e. food and water. egg laying occurs mainly in the months of mid of march and the start of april in the natural habitat, while in captivity it extends till september. this extension of the egg laying period is mainly because of the intensive care that is provided in captive conditions. moreover, in natural habitats, there is fear of predators i.e. snake, squirrel, mongooses, etc. furthermore, a pronounced increase in clutch size in captive conditions compared to natural habitat is also because of extra care that is provided in captive conditions. the incubation period is the same for both the habitats i.e. 18-23 days. in the present study, the hatching rate is increased in captive conditions compared to natural habitats. the probable reason for the increased hatching rate in captivity might be the availability of food, water, and human care to hatchlings especially the temperature of farms where hatching is well-adjusted. the protection from the predator in captive conditions is also the main reason for improved hatching success in captive conditions. the major threats to avian fauna include habitat destruction due to rapid forest cutting, use of land for agriculture, use of land for construction, land sliding, and disturbance by expanding human population17. major threats like egg poaching, illegal hunting, bird trapping, and habitat destruction particularly for chukar partridge are reported in pakistan14. more effective law enforcement, monitoring, and strong prosecution of lawbreakers should be undertaken on a serious note by authorities to restore our national bird population in its wild habitat. within the current political and socioeconomic situation, one cannot rely solely on the national conservation authorities to prevent the population decrease of this game bird from wild. a devoted, good-financed, and fully command body comprising of authorities, local governments, international and local ngos, and commercial breeders also needs to be established. captive breeding and reintroduction will inevitably have to play a vital role in the future of chukar partridge in the wild. c o n c l u s i o n the present study concludes that chukar partridge has a greater breeding potential in captive conditions compared to the natural environment. it is further recommended that coordinated reintroduction programs after captive breeding should be launched. this reintroduction will not only sustain the chukar population in the wild but also a good revenue could be generated with control permitted hunting of this game bird. c o n f l i c t o f i n t e r e s t the authors declare no conflict of interest. f u n d i n g s o u r c e none. breeding potential of pakistan’s national bird vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 75 a c k n o w l e d g e m e n t s the authors are thankful to the punjab wildlife and parks department for providing expertise in the field for finding active site of birds and data collection. l i s t o f a b b r e v i a t i o n s iucn international union for conservation of nature ngos non-governmental organizations r e f e r e n c e s 1. khan aa, khan r, ullah a, ali m, mahmood ja, sheikh km. conservation perspectives of the imperial aquila heliaca and steppe eagle aquila nipalensis in pakistan. in eagle studies world walking group on birds of prey (wwgbp), bu meyburg and rd chancellor, 1996. 2. robert tj. the birds of pakistan. vol. i. regional studies and non-passeriformes. oxford univ press karachi pakistan, 1991; 598-109. 3. hoyo dj, elliott a, sargatal j. handbook of birds of the world vol 2: new world vultures to guinea fowl. lynx edicons, barcelona discover life. fact sheet: chukar partridge, 1994. 4. degraaf r, scott m, virgil e, hamre rh. forest and rangeland birds of the united states: natural history and habitat use. agric handb 688. washington, dc: u.s. dep agri forest ser. 1991; 625-33. 5. leopold as, gutierrez jr, bronson mt. north american game birds and mammals. new york: charles scribner and sons, 1981; 198-205. 6. sibley cg, monroe bl. distribution and taxonomy of the birds of the world. new haven, ct: yale university press; 1990; 1111-9. 7. newton i. the contribution of some recent research on birds to ecological understanding. j anim ecol. 1995; 64(6):675-96. 8. wood mc. collins birds of the indian sub-continent, harper collins, london, uk; 1980. 9. anonymous. wildlife survey report. wildlife wing, forest department, azad government of the state of jammu and kashmir. 1985. 10. lateef m, rauf u, sajid ma. outbreak of respiratory syndrome in chukar partridge (alectoris chukar). j anim pl sci. 2006; 16:1-2. 11. awan ma, saleem mm, awan ms, basharat k. distribution, status and habitat utilization of alectoris chukar in machiara national park district muzaffarabad azad kashmir. j agric soc sci. 2006; 2(4):230-3. 12. shahabuddin sk, naveed a, adil k, waheed a, basit a. exploring the population status of family phasianidae in totalai game reserve, district buner, khyber pukhtunkhwa, pakistan. br j poul sci. 2016; 5:13-20. 13. yaseen, kq, ahmad m, naeem m, khan m, ali h, rehman hu, et al. distribution, population, and habitat utilization of alectoris chukar in district malakand khyber pakhtunkhwa, pakistan. j entomol zool stud. 2017; 5(2):964-6. 14. mahmood t, ahmad i, akrim f, hamid a, waseem m, hussain a, et al. breeding ecology of chukor partridge (alectoris chukar) in lower dir district, khyber pakhtunkhwa, pakistan. pak j zool. 2019; 51(1):26571. 15. martin te, geupel gr. nest-monitoring plots: methods for locating nests and monitoring success. j field ornith. 1993; 64:507-19. 16. shapiro ss, wilk mb. an analysis of variance test for normality. biometrika 1965; 52:591-611. 17. khalid s, awan ms, minhas ra, ashraf n, ahmed kb, shafi n, abassi s. distribution and habitat use of avian fauna of rawalakot city and its surroundings, azad jammu and kashmir, pakistan. pak j zool. 2017; 49(6):1-4. introduction the gastrointestinal tract is responsible for the digestion of the food that attributed through chemical and mechanical processes. it provides nutrients, fluids and electrolytes to the body and excretes out waste products. it consists of a group of organs that includes liver, pancreas and gi tract which metabolize nutrients, medicine and other exogenous materials (proctor, 2008). gastro esophageal reflux is a physiological action that consists of unwilling expulsion of stomach contents in the esophagus that causes heart burn. it is a common practice characterized by heart burn, acid regurgitation and with or without mucosal damage (kahrilas, 2003). usually it may not produce any damage to the mucosa. if there is any pathology involved, mucosal damage is sure. in 40% of the patients, the endoscopic findings are observed while 60% patients have no endoscopic results of heart burn (orlando, 2008). when there all the symptoms of the gastro esophageal reflux disease are present but the upper endoscopic results shows that there is no erosion or mucosal damage the condition is called non-erosive reflux disease (nerd) (dent et al., 1999; richter and castell, 1982; waring, 2001). anatomy of esophagus normally esophagus consists of 22cm to 25cm long tunnel and that its upper one third parts consists of skeletal muscles and lower two third part is smooth muscle. it initiates from the pharynx and ends at the stomach. it passes from behind the trachea and heart, right in front of the spine and passes through the diaphragm at the point of entrance in the stomach as shown in the figure 1. physiology of esophagus esophagus is an initial part of the digestive system that delivers food from oral cavity to stomach. it consists of two high pressure zones called lower esophageal sphincter and upper esophageal sphincter. the upper esophageal sphincter is controlled by the cricopharyngeus muscle and remains close all the times to prevent inspired air to esophagus and esophageal contents to oropharynx. during the swallowing process, the swallowing center in the introduction of gastro esophageal reflux disease and unani concepts allah nawaz1, 2, muhammad umar toseef 1, khan usmanghani 2,3*, aftab saeed 1, irshad ahmad 4 1faculty of eastern medicne, hamdard university, karachi. 2department of research and development, herbion pakistan (pvt) limited, plot 30, sector 28, korangi industrial area, karachi-74900, pakistan. 3department of pharmacy, jinnah university for women, karachi-74600, pakistan. 4department of pharmacy, the islamia university of bahawalpur. *corresponding author: ugk_2005@yahoo.com 36 figure 1: anatomy of the esophgus vol 4 (1), january 2013; 36-46 physiology in unani perspective al-tibri in his book firdos-al-hikmat fit tib state that the temperament of the esophagus is “cold and dry”. the stomach is the most sensitive internal organ of the body even from heart and liver therefore the stomach pain cause mental pressure that ultimately cause insanity. the lack of appetite is the result of if increased level of hotness in the stomach and increased appetite with slow digestive process refers that the temperament of the stomach goes towards the coldness. he also prescribed gastro esophageal reflux disease as “it is muscular disorders of the esophagus in which the tonicity of the muscles that controls the esophageal sphincter decreases and they remain relax, as a result the sphincter remains open” (tibri et al., 1994) gastro esophageal reflux disease mechanism gastro esophageal reflux is a physiological action that consists of unwilling expulsion of stomach contents in the esophagus that causes heart burn. it is a common practice characterized by heart burn, a c i d r e g u r g i t a t i o n a n d w i t h o r w i t h o u t mucosaldamage. the mechanism of acid reflux has prescribed in figure 3 (sperber et al., 2007) when food particles along with gastric juice having acidic ph reflux into the esophagus heart burn is developed. it happened due to the epithelial tissue lining that differs in esophagus than stomach, not suitable for the acidic environment (gerd, 2011). endoscopically it can be defined that which type of the material is present in the esophagus. the presence of the bilious material in esophagus is defined in the figure 4. jhonsson et al prescribed, usually it considered that gastro esophageal reflux disease related heart burn is caused by the acid reflux but studies shows that the cause of the intra-esophageal stimulus is not only acid reflux. it may be developed by the nonbrain located in the medulla oblongata passes impulses towards upper esophageal sphincter and lower esophageal sphincter’s skeletal muscles and smooth muscles through vagus nerve are carried by vagal postganglionic or pre-ganglionic cholinergic fibers, respectively, to pass the food. the acetylcholine contracts the skeletal muscles of the esophagus and smooth muscles are contracted by the vasoactive intestinal peptide and nitric oxide. there are three type of protecting mechanism systems are also involved during the process of swallowing to protect esophagus from the gastric acid reflux injury (orlando, 2008). these are; · antireflux barriers – frequency limitation of the reflux · luminal clearance mechanism –fasten the passing through the esophagus · tissue resistance – minimize epithelial contact to avoid damage 37 vol 4 (1), january 2013; 36-46 figure 2: normal endoscopic view of esophagus acidic stimuli that consist of increased volume due to over eating, non-acidic reflux or esophageal motor dysfunction etc. hence the myth “no acid, no heart burn” should be discarded, because acid reflux is not the only cause but one of the causes (johnsson et al., 1998). gastro esophageal reflux disease manifestations the different manifestations of gastro esophageal reflux disease are shown in the figure 5. initially the lower esophageal sphincter starts relaxing (a), in the case when the esophageal sphincter closes strictly instead of relaxing (b). patches developed in the esophageal walls (c). the indication of the mucosal damage of esophageal sphincter (d). relax sphincter along with the patches of esophageal wall (e). a big ulcerative patch in the wall of esophagus (f). relaxed sphincter with carcinoma (g, h). carcinoma causing complete contraction of the sphincter (i). the complete gastro esophageal reflux disease along with carcinoma and severe mucosal damage (figure-5j, k, l); in the world over both developed and developing countries, esophageal reflux is a common condition with different sign and symptoms but increasing rapidly around the globe (edwin, 1998). gastro esophageal reflux disease is not only the disease of the adults but it is also common in children. pediatric gastro esophageal reflux clinical practice guidelines have been issued by vendenplas y. a. compromise on the gastro esophageal reflux and gastro esophageal reflux disease’s diagnosis and treatment in the children are suggested that could be gainfully utilized for the diagnosis and treatment. these guidelines based on the literature search available evidence and bibliographies. this study reviews 600 plus articles. the evidence-based information provides guidelines for diagnosis and treatment of gastro esophageal reflux and gastro esophageal reflux disease in the children (vindelplas and rudolph, 2009). the patients having gastro esophageal reflux disease under the age of 12 years do not have heart bun while they have the symptoms of swallow difficulty, asthma and dry cough (nddic, 2007). jiang and co-workers reported an analysis on the role of proximal gastric acid reflux in causation of respiratory symptoms in children with gastro esophageal reflux. the correlation of gastric reflux and respiratory diseases is known but the mechanism of the two bringing the reflux activity is not clear. a 24-hour esophageal ph monitoring of proximal and distal esophagus was 38 vol 4 (1), january 2013; 36-46 figure 3: function of the lower esophageal sphincter figure 4. presence of bilious stomach material in the esophagus 5e. 39 vol 4 (1), january 2013; 36-46 5b. 5d. 5c. 5f. 5a. 5b. 5h. 40 vol 4 (1), january 2013; 36-46 5g. 5k. 5j. 5i. 5l. figure 5 (a-l). different stages of gastro esophageal reflux disease 41 vol 4 (1), january 2013; 36-46 observed in 23 and 31 children having gastric reflux with or without respiratory problems to find the onset. no significant differences in ph factors noted either in the proximal or distal esophagus in patients with gastric reflux without breathing. the proportion of patients with proximal gastric reflux in patients with respiratory symptoms was same from those without respiratory symptoms. ultimately the proximal esophageal acid reflux has no role in the increase or decrease of persistent respiratory symptoms in children. the distal esophageal acid reflux is the predominant form of reflux in children with gastric reflux irrespective of the frequency of respiratory symptoms and dependent on its own systemic disorders (jiang et al., 2007). reflux patient’s studies shows that the gastro esophageal reflux disease and age, both are the factors that have their effect on esophageal motility. the rate of peristaltic wave amplitude is significantly low as compared to non-gastro esophageal reflux disease patients and the ratio mimics in the old and young age. while dantas and associates supports esophageal wave amplitude progressive reduction but denies the age factor (gutscham et al., 2011). jayadevappa and associates analyzed the gastro esophageal acid-related disease, co-morbidity and medical care cost a retrospective cohort research which was carried out on six hundred patients with gastro esophageal reflux disease were randomly assigned and 600 patients without gastro esophageal reflux disease were assigned for age, gender, drug efficacy. the demographics, medical health care was a s s e s s e d f r o m o rg a n i z a t i o n f o r 3 y e a r s demographically were compared between gastro esophageal reflux disease and non gastro esophageal reflux disease group. random coefficient log linear regression models analyzed vis a vis costs and to determine its relationship with all type diagnoses. gastro esophageal reflux disease is a chronic disease that is mixed with forms of disorders and diagnoses that significantly treatment is low costs is high without gastro esophageal reflux disease. therefore in ultimate analysis new disease in the diseases with gastro esophageal reflux will cost more in health c a r e ( j a y a d d e v a p p a a n d c h h a t r e , 2 0 0 8 ) pereira has given detail account for the regression of gastro esophageal reflux disease symptoms using dietary supplement with melatonin, vitamins and amino acids and compared with omeprazole “gastro esophageal reflux disease is increasing in communities”. gastro esophageal exhibits symptoms of regurgitation, heartburn, cough, hoarseness, dysphagia, or chest pain. the study was conducted to find that dietary supplement containing vitamin b6, vitamin b12, melatonin, folic acid, l-tryptophan, betaine and methionine can help to increase energy and execute this malaise and compared with 20 mg of omeprazole. melatonin inhibits the secretion of gastric acid and synthesis of nitric oxide. however it is understood that nitric oxide significantly affect the relaxation of lower esophageal sphincter that is reflux in patients with gastro esophageal reflux disease. one randomized blind study was conducted in two groups, a176 patients that were treated as above, b175 treated with the dosage form of 20mg of omeprazole. symptoms were assessed, items and changes in severity of symptoms recorded. all patients in group a reported 100% worsening of symptoms after 40 days of treatment. on the other hand, 115 persons reported 65.7% worsening symptoms omeprazole. the significant difference between groups supports the withdrawal symptoms of gastro esophageal reflux disease and no side effects (pereira, 2006). dettmar et al gave a detail account on reflux and its consequences the laryngeal, pulmonary and esophageal manifestations, 30% of adult population is affected by the gastro esophageal reflux disease. the classical esophageal symptoms such as heartburn and acid reflux often overlap with atypical symptoms that affect the respiratory tract which is termed as non-esophageal reflux disease or laryngopharyngeal reflux that appears chronic cough, laryngitis, hoarseness, voice disorders and asthma manifestations. the clinical and non-clinical performa was preferred to find out the above mentioned condition. the refluxate of components such as acid, pepsin, bile acid and nonacid reflux, resulting from tissue damage and protection of the esophagus and laryngopharynx airways were analyzed. clinical symptoms of reflux, the cause, including asthma, chronic cough, respiratory disease, were examined at length. in addition to strategies for diagnosis and treatment of gastro esophageal reflux disease thereafter finding the results of nonacid reflux were proposed to be rendered as inclusion criteria (dettmar et al., 2011). symptomatology after reviewing the research articles and consulting books the symptoms that may be developed in gastro esophageal reflux disease are as follows; heart burn, chest pain, epigastric pain, acidic feeling in the mouth, regurgitation, sore throat, hoarseness of voice, wakeup during sleep, restless sleep, fatigue, decreased appetite, stress, discouragement, anorexia, unnecessary anxiousness, frustration and irritation lump in throat, swallow difficulty, vomiting, pain on swallowing, flatulence, belching/burping, nausea, tenesmus, constipation/diarrhea, general weakness (zeneca and molandal, 2005) cough, laryngitis (dettmar et al., 2011). increase in appetite, dyspepsia, breathlessness, taste usually goes bitter but there may be possibility of tastelessness also, dry mouth (ahmed), palpitation, abdominal cramps, abdominal pain, peripheral numbness, hematemesis, melena, dizziness (kabiruddin). causes of gastro esophageal reflux disease gastro esophageal reflux disease can be caused by the prolonged use of the following foods; ø spicy and fast food ø f r i e d a n d h e a v y f a t c o n t a i n i n g f o o d ø betel nut, pan, gutca, snuff ø alcoholic drinks ø chocolate ø coffee, tea, cola ø tomato, orange juice (high acidic contents) ø peppermint, onion, garlic ø hot in temperature and cold food simultaneously ø over eating along with these foods the following risk factors are also involved in the development of the gastro esophageal reflux disease this has been elaborated in figure 7 (lafullarde et al., 2001), ø self medication and excess medication like antidepressants, nsaids, corticosteroids, calcium channel blockers ø obesity and excess of abdominal fats ø age over 50 years ø smoking ø pregnancy ø asthma 42 vol 4 (1), january 2013; 36-46 figure 6. foods that cause gastro esphageal reflux desease 43 vol 4 (1), january 2013; 36-46 gastro esophageal reflux disease may be developed through following basic ways; 1. prolonged contact of gastric juice with esophageal epithelium 2. unusual damage of esophageal epithelium with acidic contents 3. frequent use of anti-acids and overflow of the stomach the lower esophageal sphincter remain relax in gastro esophageal reflux disease and the gastric material invade in the esophagus while the esophagus functions properly. hiatus hernia, an anatomical abnormality of stomach may develop gastro esophageal reflux disease. normally the muscular sheet of diaphragm generates pressure to close lower esophageal sphincter. in case of hiatus hernia, the supporting pressure decreases or may be lost, such conditions provide ease to reflux. hiatus hernia may be developed in any age but it is common in the age group of more than fifty years (nddic, 2007). the therapy for gastro esophageal reflux disease follows the continuous medication. when the medication stops the reflux relapse and its ratio goes up to 90%. medication along with lifestyle changes bring up the results more than three fourth of the patients. the patients that do not tolerate medicine and fed up with long term medication goes for surgery. frequently used surgical procedure is called laproscopic nissen fundoplication. the response rate is very encouraging in 5 year follow up i.e. 90%of the cases (lafullarde et al., 2001). pathology there may or may not be any pathology involved in the gastro esophageal reflux disease. the major pathology we are studying here in this study is helicobacter pylori (h. pylori). the easiest route of induction of the h. pylori infection is; · oral to oral · fecal to oral history of helicobacter pylori helicobacter pylori is also known as h. pylori. it was firstly detected by the bany marshal and j. robin warren. they found an organism in the culture of the gastric biopsy tissues in 1982 and name it campylobacter pylori. its rate of infection, portal of exit and no animal reservoir has been found yet (jacquelyn, 2005). mechanism of helicobacter pylori h. pylori have highest ability of genetic diversity than other human pathogens. it is due to the genetic strain differences. so, its role in different type of stomach cancer is 89%. it is a bacteria that survives in the acidic environment of the stomach. it happens due to a specific quality that it utilizes uric acid and produces ammonia, which neutralizes the acidity and provides suitable environment for survival and reproduction around the bacterial cells. they colonize directly above the epithelial layre of the stomach and penetrate into it. their growth rate is slower in the laboratory culture due to the microaerophilic conditions. identification markers for h. pylori is directly from gastric biopsies, for the evaluation of the enzyme urease and culturing specimens an special media, serologically specific antibody against h. pylori test is used for identification (jacquelyn, 2005). in a susceptible host, h. pylori determine figure 7. risk factors of the gastro esophageal reflux disease 44 vol 4 (1), january 2013; 36-46 chronic active gastritis that may lead, in turn, to duodenal and gastric ulcer disease, gastric cancer, and maltomas. h. pylori infection causes chronic active gastritis, which is characterized by a striking infiltrate of the gastric epithelium and the underlying lamina propria by neutrophils, t and b lymphocytes, macrophages, and mast cells. mast cells, usually responsible for the immune response balance, may be important effectors cells in the pathogenesis of gastritis. however, h. pylori do not seem to invade the gastric mucosa, although evidence suggests that the mucus creates a niche wherein the germ is protected from gastric secretions (santacroce and bhuttani, 2011). pathogenesis marshall et al. first of all cultured helicobacter pylori in 1982 from gastric biopsy tissues and name it campylobacter pylori. it survives in the highly acidic environment of the stomach because it develops ammonia around it from the uric acid that neutralizes the acidity around the bacterial cells and carried out the survival and reproduction by providing suitable environment. they colonize direct above the epithelial layer of the stomach and penetrate into it. during culture in laboratory their growth is slow because of the requirement of the microaerophilic conditions and an enriched medium. h. pylori have highest ability of genetic diversity than most of the other human pathogens because of its strain differences. so, its role is 89% indifferent types of the stomach cancers. no animal reservoir has been found yet (strapoli, 2010). h. pylori invade the body through food, fluids and contaminated utensils. its identification marker is directly from gastric biopsies for the evaluation of enzyme urease and culturing specimens on special media. a sophisticated urea breath test is used to measure the quantity of the carbon dioxide developed in the stomach after oral presentation of urea. serologically specific protein antibody test against h. pylori is used for identification. the final and the easiest approach that provides the accurate results is fecal antigen test (zarling, 1998). the therapy for gastro esophageal reflux disease follows the continuous medication. when the medication stops the reflux relapse and its ratio goes up to 90%. medication along with lifestyle changes bring up the results more than three fourth of the patients. the patients that do not tolerate medicine and fed up with long term medication goes for surgery. frequently used surgical procedure is called laproscopic nissen fundoplication that shows very encouraging response rate in 5 year follow up i.e. 90% (lafullarde et al., 2001). clinical presentation commonly the reflux may be found in any age, but it affects mostly the age group of 20 to 50 years. male and females are equally victimized, and both are hospitalized. but in men the endoscopic findings of esophageal damage are two to three fold more than females of china and west (kay et al., 1994; chang et al., 1997; liker et al., 2005; galmiche et al., 2006). esophageal reflux has basic symptoms of epigastric pain that starts after one hour eating and may lasts up to two hours, aggravates with the spicy food, citrus food, tomato, onion and alcohol etc. it starts with heartburn, refers to epigastric pain along with regurgitation. if it happens during sleep the symptoms of hyper salivation and bitter taste are also found (zarling, 1998). galmiche and coworkers have reported that functional esophageal disorders of the esophagus are associated with esophageal symptoms such as heartburn, chest pain, dysphagia, a globe. these could not view from the perspective of structural disorders, movement disorders based on histopathology or gastro esophageal reflux. gastro esophageal reflux is taken into account of the diagnosis of reflux esophagitis or esophageal acid exposure there, episodes of acid reflux or respond to anti reflux treatment. management methods that modulate the central symptom (perception, noxious stimuli) of the esophagus were excluded. all this has resulted in the understanding of the symptoms, the new strategies and give a way to diagnosis and treatment (zhang et al., 1996). references ahmad k. r. amraz-e-maida, so-e-mizag maida. sharah-e-asbab. ideal packages karachi. 2: 33245 vol 4 (1), january 2013; 36-46 338. chang c. s., poon s. k., lien h.c., et al. 1997. the incidence of reflux esophagitis among the chinese. am j gastrol., 92(4): 668-671 dent j., brun j., fendrick a., fennerty m., janssens j., kahrilas p., et al. 1999. an evidence-based appraisal of reflux disease managementthe genval workshop report. gut, 44(suppl 2): s1-s16. dettmar p. w. parikh s., orlando r. c., johnston n., allen j., tinling s. p., et al. 2011. reflux and its consequences – the laryngeal, pulmonary and oesophageal manifestations. aliment pharmacol ther, 33 (suppl. 1): 1–71 galmiche j. p., clouse r. e., balint a., cook i. j., kahrilas p. j., paterson w. j., et al. 2006. functional e s o p h a g e a l d i s o r d e r s . g a s t r o e n t e r o l o g y, 130:1459–1465. gerd. 2011. condition factsheets. the facts on gerd. available at: http://bodyandhealth. canada.com/condition_info_details.asp?disease_id =62 gutschow c. a. leers j. m., schröder w., prenzel k. l., fuchs h., bollschweiler e., et al. 2011. effect of aging on esophageal motility in patient with the gastro esophageal reflux disease. ger med sci, 9: doi: 10.3205/000145. jacquelyn g. b. 2005. peptic ulcer and chronic gastritis, oral and gastrointestinal diseases. in: microbiology: principles and explorations. john wiley and sons inc., pp. 659-661. jayadevappa r., chhatre s., weiner m. 2008. gastrooesophageal acid-related disease, co-morbidity and medical care cost. chronic illness, 4: 209-218. jiang m. z., wang t. l., yu j. d., zhou x. l., ou b. y. 2007. role of proximal gastric acid reflux in causation of respiratory symptoms in children with gastro esophageal reflux. indian pediatr, 44(8): 575-579. johnsson f. weywadt l., solhaug j. h., hernqvist h., bengtsson l. 1998. one week omeprazole treatment in the diagnosis of gastro-oesophageal reflux disease. scand j gastroenterol, 33(1):15–20. kabir-ud-din m. varm-e-maidah, tarjma-e-kabir (sharah-e-asbab ka tarjmah) dafter al-maseh qarool bagh delhi 1936. jamal printing works delhi. 2: 844-850. kahrilas p.j. 2003. diagnosis of symptomatic gastroesophageal reflux disease. a m j gastroenterol, 98: s15–s23. kay l., jorgensen t., jensen k. h. 1994. epidemiology of abdominal symptoms in a random population: prevalence, incidence, and natural history. eur j epidemiol, 10(5):559-566. lafullarde t., watson d. i., jamieson g. g., myers j. c., game p. a., devitt p. g. 2001. laparoscopic nissen fundoplication: five-year results and beyond. arch surg,136(2):180-184. liker h., hungin p., wiklund i. 2005. managing gastro esophageal reflux disease in primary care: the patient perspective. jabfp; 18(5), oct. 2005. n a t i o n a l d i g e s t i v e d i s e a s e i n f o r m a t i o n clearinghouse (nddic). 2007. heartburn, gastro esophageal reflux, and gastro esophageal reflux disease. publication no. 07-9882. orlando rc. 2007. diseases of the esophagus. in: goldman l, ausiello d, eds. cecil medicine. 23rd e d . p h i l a d e l p h i a , p a : s a u n d e r s e l s e v i e r. pereira r. c. 2006. regression of gastroesophageal r e f l u x d i s e a s e s y m p t o m s u s i n g d i e t a r y supplementation with melatonin, vitamins and aminoacids: comparison with omeprazole. j. pineal res, 41:195–200 proctor dd. 2008. approach to the patient with gastrointestinal disease. in: goldman l, ausiello d, eds. cecil medicine. 23rd ed. philadelphia, pa: saunders elsevier. 46 vol 4 (1), january 2013; 36-46 richter j.e., castell d.o. 1982. gastroesophageal reflux: pathogenesis, diagnosis, therapy. ann intern med, 97:93–103. santacroce l. and bhutani m. s. 2010. helicobacter pylori infection. medscape. available from: http://emedicine.medscape.com/article/176938overview#aw2aab6b2b5aa. sperber a.d. halpern z., shvartzman p., friger m., freud t., neville a., fich a. 2007. prevalence of gerd symptoms in a representative israeli adult population. j clin gastroenterol, 41: 457-461. strapoli a.a. 2010. helicobacter pylori and gastritis. greenwich village gastroenterology. available at: http://www.starpoli.com/helio/. tibri a. h. a. 1994. al-muqalah-tul-sadisah min al-nao-e-rabe. firdos-al-hikmat fit-tibb. shaikh muhammad bashir & sons, urdu bazar, lahore. pp. 563-585. vandenplas y., rudolph c. d. 2009. pediatric gastroesophageal reflux clinical practice guidelines: joint recommendations of the north american society of pediatric gastroenterology, hepatology, and nutrition and the european society of pediatric gastroenterology, hepatology, and nutrition. j. pediatr. gastroenterol. nutr. 49:498–547. waring j.p. 2001. nonerosive reflux disease.bsemin gastroenterol dis.,12:33–37. zarling e. j. 1998. a review of reflux esophagitis around the world. world j gastroenterol, 4(4): 280284. zhang t. c., zhu m. z., geng x. c., li y. j., cao t., zhang l. p. 1996. endoscopic study of reflux esophagitis. jama southeast asia, 12(2suppl):2224. detection of carbapenem resistant acinetobacter: from clinical samples naheed afshan1*, huda fatima1 1department of microbiology, jinnah university for women, karachi. abstract acinetobacter has appeared from an organism of uncertain pathogenecity towards an infectious agent. among nonfermenting bacterium a. baumannii is the second-most-commonly-isolated organisms in human. the fast intensify of their resistance to antibiotics, especially global emergence and extend of acinetobacter strains resistant to carbapenem more restricted the therapeutic alternatives. in this study we evaluate the % of resistivity of acinetobacter against carbapenem antibiotics at jinnah university for women, karachi. total 439 isolates of acinetobacter were collected from different clinical samples of hospitalized patients from january to december 2013, identified by standard microbiological methods. antibiograms were done on mueller-hinton agar plates with disk diffusion method (kirby bauer method). disc tested: meropenem (10 µg/disk). the results were interpreted according to the guidelines of the clinical and laboratory standards institute. (clsi). among 439 samples, 300 (68.3%) samples were resistant to meropenem and the remaining that is 139 (31.7%) showed sensitivity to the drugs. in underdeveloped countries including pakistan the contentment of multidrug resistance and their dissemination in acinetobacter species is not a simple task. while multiple drug resistance is increasing in this pathogen, and carbapenem conflict is quickly spreading which may become a major threat in future. so in pakistan needs detail and organized data about carbapenem resistant acinetobacter in order to understand the existence of acinetobacter in our community and to manage almost certainly outbreaks because we have less information according to resistance trends of acenetobacter. keywords: antibiogram, carbapenem, resistant, outbreaks. introduction species of genus acinetobacter are gram-negative belongs to the class gammaproteobacteria. species are non-motile, coccobacili in shape appear in pairs, oxidase-negative (peleg et al., 2008). acinetobacter consist of 27 authentically named and 11 unknown (genomic) species. species names, including: a. baumannii, a. calcoaceticus, a. haemolyticus, a. johnsonii, a. junii, and a. lwoffii (manchanda et al., 2010). widely dispersed in environment they can be alive on dry and moist surfaces, as well as in hospital surroundings (maragakis and perl, 2008). patients in the intensive care unit (icu), including burn patients, trauma patients, and patients requiring mechanical ventilation are mainly affected by acinetobacter baumannii (bassetti et al., 2008). also some strains have been found from foodstuffs, water, and associate with low occurrence of allergies. several species of acinetobacter can cause serious infections in immunocompromised patients (peleg et al., 2008). they also show comparatively wide scale of antibiotic resistance (towner, 2009). infections together with skin and wound added complication as well as bacteremia, and meningitis (choi et al., 2008). the bacteria have the ability to colonize in medical utensils e.g. catheters (doughari*corresponding author: naheedafshan7@hotmail.com 09 vol 5 (1), january 2014; 09-13 et al., 2011). plasmid-borne markers which made the acinetobacter resistant to various antibiotics are capable of transferring to new pathogenic bacteria through horizontal gene transfer (juni, 1978). ‘mdr acinetobacter spp.’ defined as the isolate resistant to at least three classes of antimicrobial agents — all penicillins and cephalosporin (including inhibitor combinations), fluroquinolones, and amino glycosides. ‘xdr acinetobacter spp.’ that is resistant to the three classes of antimicrobials described above (mdr) and shall also be resistant to carbapenems (kurcik-trajkovska, 2009). the clinical isolates of acinetobacter spp. were generally susceptible to gentamicin, minocycline, nalidixic acid, ampicillin, or carbenicillin, singly or in a combination therapy, throughout the early 1970s (bergogne-bérézin and towner, 1996). but, since 1975, growing resistance started to appeare in almost all groups of drugs including the first and second generation cephalosporins. primarily, they retained at least partial susceptibility against the third and fourth generation cephalosporins, fluoroquinolones, semi synthetic aminoglycosides, and carbapenems, with nearly 100% isolates holding susceptibility to imipenem (vila et al., 2007). however, during late 1980s and 1990s, worldwide emergence and spread of acinetobacter strains resistant to imipenem further limited the therapeutic alternatives. by the late 1990s, the only useful agents that fight many severe infections caused by acinetobacter sp. were carbapenems (cunha, 2013). moreover, the therapeutic options are decreasing due to the emergence of carbapenem resistance in the strains of a. baumannii (spl, 2011). various mechanisms have been found to be accountable for the resistance to carbapenems in a. baumannii (cuh, 2013). the resistivity mechanismsusually fall into 3 categories: 1. antimicrobial-inactivating enzymes, 2. reduced access to bacterial targets, or 3. mutations that change targets or cellular functions (camp and tatum, 2010). treatment is difficult for healthcare-acquired infection caused by a. baumannii resistant to imipenem (smith et al., 2007). carbapenems still represent the treatment of choice. a. baumannii is competent to grow at different temperature and ph because it does not have fastidious growth requirements. the versatile organism exploits a variety of both carbon and energy sources. these qualities explain the ability of acinetobacter species to persist in either moist or dry conditions in the hospital environment, thereby contributing to transmission. this hardiness, combined with its intrinsic resistance to many antimicrobial agents, contributes to spread in the hospital setting. materials and methods setting: department of microbiology, jinnah university for women karachi. duration of study: from january 2013to december 2013 sampling technique: non-duplicate consecutive sampling. inclusion criteria: all acinetobacter colonies isolated from different clinical samples of patients. exclusion criteria: sample showing no growth or growth of gram positive bacteria, growth of gram negative bacteria other than acinetobacter and yeast. repeat and duplicate samples from the same patient were also being excluded. study design: descriptive study. 10 vol 5 (1), january 2014; 09-13 figure 1: outbreaks of acinetobacter in united states between 2002 and 2007 (chuang et al., 2011). data collection: all clinical samples were collected in sterilized container according to samples from patients of different hospitals and platted right after the collection. identification will be taken by standard microbiological methods. inoculation of clinical samples was done on standard media such as sheep blood agar (sba) mackonkey agar and chocolate agar. antibiograms were done on mueller-hinton agar plates with disk diffusion method according to kirby bauer method. disc tested: meropenem (10 µg/disk) (oxoid ltd., england) the results was interpreted according to the guidelines of the clinical and laboratory standards institute (clsi). results discussion we have performed our studies on 439 isolates. we hereby observed the sensitivity and resistivity pattern of meropenem on different samples including pus, urine, tracheal aspirates, sputum, blood and body fluids which were loaded with acinetobacter. we observed that among 439 samples, 295(67%) samples were resistant to meropenem the remaining i.e. 144(33%) showed sensitivity to the drug. out of 439 samples 195 were tracheal aspirates, 34 were blood samples, 30 were urine, 101 were pus and swabs, 41 were sputum samples and 38 were fluid samples. this increased resistivity of acinetobacter is considered as an important health problem due to considerable clinical impact of this resistivity on the management of health care associated infections. in intensive care unit, the serious concern of high morbidity and high mortality rates of a.baumanii is their nosocomial outbreaks. clinical threats scored by acinetobacter are its adaptation to the environment, versatile metabolism and its ability to develop resistance against antibiotics that are used in clinical settings. this danger has caused an noticeable and alarming decline in the available chemotherapeutic resources, that includes drugs like, carbapenem antibiotics that inhibits peptidoglycan biosynthesis that was considered the first-rate standard for acinetobacter treatment until recently (guerrero et al., 2010). infections due to acinetobacter frequently involve organ systems that have a high fluid content (eg, respiratory tract, csf, peritoneal fluid, urinary tract), manifesting as nosocomial pneumonia, associated with continuous ambulatory peritoneal dialysis (capd), or catheter-associated bacteruria. the presence of acinetobacter isolates in respiratory secretions in incubated patients nearly always represents colonization. acinetobacter pneumonia occur in outbreaks and are usually associated with colonized respiratory-support equipment or fluids. nosocomial meningitis may occur in colonized neurosurgical patients with external ventricular figure 3: sensitivity & resistivity % of meropenem among different clinical samples. 11 vol 5 (1), january 2014; 09-13 figure 2: sensitivity & resistivity % of meropenem of total samples. table i: susceptibity pattern of acinetobacter sp. from different samples. drainage tubes. a. baumannii is a multiresistant aerobic gramnegative bacillus sensitive to relatively few antibiotics. multidrug-resistant acinetobacter is not a new or emerging phenomenon, but a. baumannii has always been an organism inherently resistant to multiple antibiotics. since the past decade, antimicrobial resistance among acinetobacter sp. is a rising concern. the species are equipped with extensive antimicrobial resistance due to the presence of the porin channels, efflux mechanisms and the non static behaviour of the bacteria in hot and humid conditions (davies and rubin, 2007). conclusion in underdeveloped countries including pakistan , india and bangladesh the contentment of multidrug resistance and their dissemination in acinetobacter sp. is not a simple task. while multiple drug resistance is increasing in this pathogen, and carbapenem conflict is quickly spreading which become a major threats in future because patient-to-patient transmission in hospitals through contaminated hands of healthcare workers and fomites is the main factors w h i c h i n c r e a s e s t h e s p r e a d i n g o f m d r acenetobacter and cure should be followed. the increased resistivity of the pattern of the organism has become a serious threat for health in pakistan. references bassetti m, righi e, et.al 2008. drug treatment for multidrug-resistant acinetobacter baumannii infections. future microbiol.;3(6):649-660. bergogne-bérézin e and k j towner.1996. acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological f e a t u r e s . c l i n . m i c r o b i o l . r e v. , 9 ( 2 ) : 1 4 8 . camp c, tatum ol.2010. a review of acinetobacter baumannii as a highly successful pathogen in times of war callie camp. lab med.;41(11):649-657 choi ch, jun sik lee1, et.al . 2008. acinetobacter baumannii invades epithelial cells and outer membrane protein a mediates interactions with epithelial cells. bmc microbiology, 8:216 doi:10.1186/1471-2180-8-216 chuang yc et.al.2011. influence of genospecies of acinetobacter baumannii complex on clinical outcomes of patients with acinetobacter bacteremia. clin infect dis, 52(3):352-360. cuh. 2013. acinetobacter. cambridge university hospitals. infectious control. http://www.cuh.org. uk/cms/addenbrookes-hospital/infection-control/wellknown-infections-faqs/acinetobacter c u n h a b a . 2 0 1 3 . a c i n e t o b a c t e r. http:// e m e d i c i n e . m e d s c a p e . c o m / a r t i c l e / 2 3 6 8 9 1 overview#a0199 davies jc, rubin bk.2007. emerging and unusual gram-negative infections in cystic fibrosis. semin. r e s p i r. c r i t . c a r e m e d . , 2 8 ( 3 ) : 3 1 2 3 2 1 . doughari hj, lois pa et.al. 2011. the ecology, biology and pathogenesis of acinetobacter spp. an overview. microbes environ. 26(2). doi:10.1264/ jsme2.me10179 guerrero dm et.al.2010. acinetobacter baumanniiassociated skin and soft tissue infections: recognizing a broadening spectrum of disease. surg. infect. (larchmt), 11(1): 49–57. juni, e. 1978. genetics and physiology of acinetobacter. ann. rev. microbiol. 32:349-371. kurcik-trajkovska b. 2009. acinetobacter spp. – a serious enemy threatening hospitals. macedonian j o u r n a l o f m e d i c a l s c i e n c e s , 2 ( 2 ) : x x xx.doi:10.3889/mjms.1857-5773.2009.0043 12 vol 5 (1), january 2014; 09-13 13 vol 5 (1), january 2014; 09-13 manchanda v, et.al. 2010. multidrug resistant acinetobacter. j glob infect dis., 2(3): 291–304. maragakis ll, perl tm. 2008. acinetobacter baumannii: epidemiology, antimicrobial resistance, and treatment options. clin infect dis., 46(8):125463. doi: 10.1086/529198. peleg ay, harald seifert, et al .2008. acinetobacter baumannii: emergence of a successful pathogen. clin microbiol rev. july; 21(3): 538–582 smith, mg et.al. 2007. new insights into acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis. doi:10.1101/gad.1510307 genes & dev.. 21: 601-614 spl. 2011. acinetobacter fact sheet. http://www. specialpathogenslab.com/spl-advantage/factsheet acinetobacter. pdf towner kj. 2009 ‘’ acinetobacter: an old friend, but a new enemy’’. j hosp infect.; 73: 355-363. vila, j.; marti, s.; sanchez-cespedes, j. 2007. porins, efflux pumps and multidrug resistance in acinetobacter baumannii. 59(6):1210-1215. xiao yh et.al.2011. epidemiology and characteristics of antimicrobial resistance in china. drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy, 14(4–5):236-250. yang ys et.al.2013. comparison between bacteremia caused by carbapenem resistant acinetobacter baumannii and acinetobacter nosocomialis. bmc infectious diseases, 13:311 doi:10.1186/1471-233413-311 introduction protozoan intestinal parasites are transmitted when anyone comes in contact with infected feces through contaminated soil, food or water. the most common protozoa are giardia and entamoeba histolytica. giardia lamblia is a flagellated protozoan parasite that colonizes and reproduces in the small intestine, causing giardiasis as the trophozoid of giardia lamblia attaches itself on to the convex surfaces of the epithelial cells of the intestine and cause disturbance of intestinal function, leading to malabsorption of fat due to which patient suffers persistent looseness of bowels, mild steatorrhoea, harm by its toxic effect, traumatic and irritative effect. amoebiasis is also termed “amoebic dysentery” signifies a condition in which the infection is confined to the intestinal canal and is characterized by the passage of blood and mucus in the stool. initially the infection is limited entirely to the large intestine but secondarily the parasite migrates to extra colonic areas such as liver, lungs and brain. e. histolytica is the third leading parasitic cause of death in humans after malaria and schistosomiasis (sebastiaan, 2007). it is also a serious helath threat in tropical and subtropical developing areas. (ohnishi, 2004). the diseases caused by these intestinal protozoan parasites are known as giardiasis, amoebiasis and they are associated with diarrhea. ashok et al., (1995), studied children’s stool sample from islamabad hospital for intestinal parasites. kaur et al., (2002) investigated intestinal parasites of children in delhi. waqar (2003) observed the intestinal infection in children of northern areas of pakistan. al-hindi et al., (2005) found protozoan infection in children of gaza. benetton et al., (2005) conducted epidemiological study in brazil. chandrashekhar et al., (2005), studied intestinal parasitic prevalence in school children of kaski, nepal. haque et al., (2006) observed e. histolytica in children. karaman et al., (2006) recorded intestinal infection in municipal sanitary workers in malatya. al harthi and jamjoom (2007) studied infection due to entamoeba in makkah al mukarramah. barnawi et al., (2007) detected e. histolytica dispar in population of jeddah city. fotedar et al., (2007) observed e. histolytica e, dispar and e. moshkouskii among the people of sydney, australia. hamze et al., (2009) observed intestinal infection in north lebanon. sehgal et al., (2010) a record of intestinal protozoan infection from different hospitals of karachi pakistan rukhsana talat1, farzana ibrahim1, zaira1 and qurat ul ain1 1department of biological sciences jinnah university for women 5c nazimabad karchi4600 abstract the prevalence of human intestinal protozoan parasites with special reference to entamoeba histolytica and giardia lamblia was studied in different area of karachi city. a total 234 cases were recorded from 7 different hospitals of karachi during january to december 2011. the total patients of entamoeba histolytica were 136 including 69 male with 50.73% and 67 female with 49.26%, while the total no of patients suffering from giardia lamblia were 98 from which 51 were male with 52.04% and 47 were female with 47.95%. key words: protozoan, infection, hospitals, entamoeba histolytica, giardia lamblia *corresponding author: rukhsanatalat4@gmail.com 12 vol 3 (2), july 2012; 12-17 studied intestinal parasites in pregnant women and school children of chandigarh, north india. zahida (2010) recorded the distribution of e. histolytica among humans. talat et al., 2012 studied protozoan i n t e s t i n a l i n f e c t i o n i n k a r a c h i , p a k i s t a n . material and method the present study was carried out from january to december 2011. information for the present investigation was collected from the pathological laboratories of seven different hospitals, selected from six towns of karachi city, as nazimabad town including ’saifee hospital’, ‘khokrapar malir’ town including ‘sindh govt. hospital’, ‘usman memorial hospital’ from f. b area, ‘ibne-sina hospital’ from gulshan-e-iqbal town, and ‘haleem hospital’ located in sakhi hasan north nazimabad while ‘khizar hospital’ and ‘the hospital’ situated in shah faisal town. results and discussion human intestinal protozoan parasites are more common in developing countries and cause high mortality rate of the world, usually transmitted when someone comes in contact with infected feces through contaminated soil, food and water. the most common protozoa are giardia and entamoeba. children and elderly are more likely to get infection and weaken the immune system. pregnant women may become more serious (sehgal et al., 2010). present research work was based on the collection of human intestinal protozoan parasites among the people of different regions of karachi city reported from 7 different hospitals, including ‘saifee hospital’, ‘sindh govt. hospital’, ‘usman memorial hospital’, ‘ibne-sina hospital’, ‘haleem hospital’, ‘khizar hospital’ and ‘the hospital’, located in different areas such as nazimabad, khokrapar malir, fedral b. area, gulshan e iqbal, north nazimabad and shah faisal town respectively, during january to december 2011. total 234 patients including male and female of all age were found positive for protozoan intestinal infection due to entamoeba histolytica and giardia lamblia. amoebiasis due to entamoeba histolytica was found in highest intensity rates i.e. total 136 cases including 69 with 50.73% male and 67 with 49.26% female while giardiasis caused by giardia lamblia observed in 98 patients showing 51 male with 52.04% and 47 females with 47.95% in overall summarized record (table: 3) the overall combined infection cases of both giardia and entamoeba were recorded in highest incidence in ‘saifee hospital’ i.e. 70 patients having 29.9 % infection including 30 males with 42.85% and 40 female with 57.14% (table:3) intensity of infection on second number found in ‘usman memorial hospital’ i.e. 49 patients with 20.94% including 27 male with 55.10% and 22 females with 44.89% while the lowest intensity found in ‘haleem hospital’ i.e. 15 patients with 6.41% including 10 males with 66.6 % and 05 females with 33.33%, the remaining hospitals as ‘sindh govt. hospital’ showed 45 patients with 19.23 %, ‘ibne sina hospital’ having 22 patients with 9.40%, ‘khizar hospital’ have 17 patients with 7.26% and ‘the hospital’ f o u n d 1 6 p a t i e n t s w i t h 6 . 8 3 % ( ta b l e : 3 ) . the incidence of protozoan intestinal infection was found most common in the area of nazimabad than on 2nd no in f.b. area and on 3rd no khokrapar malir while the lowest incidence found in north nazimabad. distribution of both the protozoan parasites showed high percentage in males than females except in ‘saifee hospital’ and ‘the hospital’ (table 1 and 2) it was verified by the results of klein (2000 a and b), that prevalence of e. histolytica is higher in males than females because males are more susceptible than females and generally exhibit reduced immune responses and increased intensity of infection compared to females. shakya et al., (2006) evaluated that male had slightly higher infection i.e. 43.8% than females which was 40.4%. ozyurt et al., (2007) found 76% in males and 33 % in females. talat (2012) reported 52.6% in males and 47.40 % in females. intestinal infections are worldwide and have the greatest single worldwide cause of illness and disease. intestinal parasitic 13 vol 3 (2), july 2012; 12-17 14 infection constitute a global health burden causing clinical morbidity in 450 million people (sehgal, et al. 2010) to minimize the risk for getting intestinal parasites must adopted the sanitary laws and hygiene, use of boil water for drinking, thorough wash green vegetables and fruits before eating, and also wash hands properly before eating meal, always eat fresh and well cooked food. avoid simple carbohydrates, such as these found in refined foods, fruits, juices, dairy products and all sugars, except honey. eat more raw garlic, pumpkin seeds, pomegranates, beets and carrots, all of which have been used traditionally to kill parasites, researchers found that a mixture of honey and papaya seeds cleared stools of parasites in 23 out of 30 subjects. drink a lot of water to help flush out system. eat more fiber, which may help get rid of worms, and keep restore tract healthy. digestive enzymes will help restore intestinal tract to its normal state, which makes it inhospitable to parasites. papain is an enzyme from the papaya plant that may help kill worms when taken 30 minutes before or after meals. vitamin c and zinc supports the immune system. uses of herbs are generally a safe way to strengthen and tone the body’s systems. (carr and frei., 1999) vol 3 (2), july 2012; 12-17 table 1: over all patients infected with entamoeba histolytica diagnosed from seven different hospitals of karachi city during january to december 2011. months saifee hospital sindh govt. hospital usman memorial hospital ibne-sina hospital haleem hospital khizar hospital the hospital 15 vol 3 (2), july 2012; 12-17 table 3: overall summarized record of the patients of giardia lamblia and entamoeba histolytica collected from seven different hospitals of karachi during january to december 2011. table 2: over all patients infected with giardia lamblia diagnosed from seven different hospitals of karachi city during january to december 2011. saifee hospital sindh govt. hospital usman memorial hospital ibne-sina hospital haleem hospital khizar hospital the hospital grand total hospitals names 16 references al-harthi sa and jamjoom mb. 2007. diagnosis and differentiation of entamoeba infection in makkah al mukarramah using microscopy and stool antigen detection kits. w. j. med. sci. 2:15-20 al-hindi a, shubair me, marshal i, ashford rw, sharif fa, abed aa and kamel eg. 2005. entamoeba histolytica or entamoeba dispar among children in gaza, gaza strip. j. egpt. soc. parasitol. 35:59-68. ashok kt, shamim aq, koji h and mushtaq ak., 1995. intestinal parasites in stool samples from children at the childrens hospital laboratory, islamabad. pak. pediatr. j. 19: 61-64. barnawi ab, tonkal am, fawad ma and al-braiken fa., 2007. detection of entamoeba histolytica/dispar in stool specimens by using enzyme-linked immunosor-bent assay in the population of jeddah city. saudia arabia. j. egypt. soc. parasitol., 37:143150 benetton ml, goncalves av, meneghini mef, silva ef and carneiro m. 2005. risk factors for infection by the entamoeba histolytica/e. dispar complex: an epidemiological study conducted in outpatient clinics in the city of manaus, amazon region, brazil trans. r . s o c . tr o p . m o d . h y g . , 9 9 : 5 3 2 5 4 0 . carr ac 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medi. net. j., 12: 73-77. zahida t, kausar s and lashar mh. 2010. prevalence of entamoeba histolytica in humans. pak. j. pharm. sci., (23) (3): 344-348. call for papers publication is free of cost rads journal of biological research and applied sciences welcomes the submission of research papers, review articles and short communications in the following subject fields: t biochemistry t biotechnology t botany t chemistry t environmental science t medical & health sciences t microbiology t molecular biology t pharmacy t physics t zoology t clinical research note:all manuscripts should be sent by e-mail to the editor with your e-mail address : rads@juw.edu.pk or deanresearch@juw.edu.pk our aim is to provide a platform for budding scientists researchers, research scholars, academicians etc. to present their research findings in the field of biological and applied sciences. prevalence of plasmodium spp. in gulshan-e-iqbal, sindh, pakistan vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 163 op e n ac c e s s f u l l l e n g t h a r t i c l e prevalence of plasmodium spp. in gulshan-e-iqbal, sindh, pakistan aly khan1,*, iqra sheikh2, nasira khatoon2, samina waheed1, s. shahid shaukat3, adnan khan4 1cdri, pakistan agricultural research council, university of karachi, karachi-75270, pakistan 2department of zoology, university of karachi, karachi-75270, pakistan 3institute of environmental studies, university of karachi, karachi-75270, pakistan 4department of microbiology, university of karachi, karachi-75270, pakistan a b s t r a c t background: malaria is caused by malaria parasite of genus plasmodium. it is transmitted by the bite of an infected anopheles mosquito. four species of plasmodium namely p. malariae, p. falciparum, p. vivax and p. ovale are responsible for causing malaria in humans. the fifth one, p. knowlesiare is responsible to cause zoonotic infection in humans. severe malaria may lead to death in humans. objectives: to study the prevalence of plasmodium spp. and to evaluate the percentage of infection in males, females and children of gulshan-e-iqbal, karachi, sindh, pakistan. methodology: samples were collected from different laboratories and hospitals of gulshan-e-iqbal, karachi, sindh, pakistan. chisquare test for frequency data was performed. results: a total of 411 cases were found to be positive out of 2096 suspected cases. p. vivaxwas more prevalent than p. falciparum. out of 411 patients, 296 cases were of p. vivax, 112 were of p. falciparum, 2 were of p. malariaewhile one was of p. ovale. the frequency of different species was found to be highly significant (p<0.001). in age class <1-15 yrs, 16-40 yrs and 41->80 yrs the frequency differed significantly as indicated by chi-square test (p<0.001). our results show that infection of malaria is more frequent in males as compared to females. conclusion: present survey shall be beneficial for the control of plasmodium infection. screening of patients who have malaria is important in other areas of karachi as well. additional studies with large sample sizes in other localities are required to fully understand malaria pathology in detail. the presence of p. ovaleand p. malariaecould be due to travel of subjects to african countries or srilanka. keywords malaria, plasmodium spp., gulshan-e-iqbal, karachi, pakistan. *address of correspondence aly.khan@hotmail.com article info. received: november 05, 2021 accepted: november 21, 2022 cite this article: khan a, sheikh i, khatoon n, waheed s, shaukat ss, khan a. prevalence of plasmodium spp. in gulshan-e-iqbal, sindh, pakistan. 2022; 13(2):163-167. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n malaria parasite (genus plasmodium) is a single celled microorganism which in human population inflicts huge burden in terms of complications leading to sudden unexplained death1. plasmodium has many species out of which four species are responsible for inflicting humans (causing malaria) namely plasmodium falciparum, p. malariae, p. ovaleand p. vivaxwhile the fifth one p. knowlesicauses zoonotic infection in humans2. the death rate due to malaria ranges between 1.5 and 2.7 million deaths each year3. it is endemic in 109 countries and widespread in the tropics and sub-tropics4. in africa, papua, new guinea and haiti p. falciparum is the most o r i g i n a l a r t i c l e prevalence of plasmodium spp. in gulshan-e-iqbal, sindh, pakistan vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 164 common species while in parts of s. america, middle east, north africa and indian subcontinent p. vivaxis endemic. in pakistan it is a big public health problem as both p. vivaxand p. falciparum occur simultaneously. poor sanitation, drug resistance, climate change and improper development activities are the main causes of spread of malaria5. in a previous study from agha khan university hospital from 1997 to 2001 it was observed that percentage of p. vivaxwas 51.8 while that of p. falciparum was 46.5 with 1.5-2% mortality6. almost ninety five million people here live in areas with high malaria incidence7. cerebral malaria may cause sudden death, it is common when p. falciparum infects an individual. sequestration of infected rbc within cerebral vessels is an important pathological reason of this disease. in places with endemic malaria patients with delirium and malaria must have at least three negative blood smears 8-12 days apart with microscopy in order to be considered fully recovered, although pcr test has shown to be more excessively affected than microscopy but does not provide any information of parasite load and is very expensive8,9. in the present study in gulshan-e-iqbal, karachi 411 infected cases were studied for the type of palsmodiuminfection besides infection rate in three different age groups <1-15, 16-40, 41->80, both male and female individuals was recorded. m a t e r i a l s a n d m e t h o d s a cross-sectional survey was carried out in karachi to determine the prevalence of plasmodium spp. targeting population in gulshan-e-iqbal, karachi. a total of 411 cases were considered in this study out of 2096 suspected cases from gulshan-e-iqbal, karachi, sindh, pakistan. gulshan-e-iqbal is in district east of karachi, most of its population is working class having residential and commercial neighbourhood. the clinics and hospitals visited were namely darulsehat hospital, lnh laboratory service, dow university of health sciences, sindlab clinical laboratory, chughtai laboratory, ibn-e-sena hospital, dr. essa laboratory and patel hospital over a period of 1 year from february 2019 to january 2020. the patients name, sex, age, details of clinical examination findings, antimalarial treatment if previously taken, history of blood transfusion were recorded in requisition form. after acquiring consents, 3 ml of blood specimen were collected from the antecubital vein from all the patients by using disposable syringe. both thick and thin smears were prepared. the remaining blood was stored in a deep freezer at each laboratory (8±2ºf). the thick smear was dehaemoglobinized, slide stained with leishmans stain. after fully drying the slides were observed under oil-immersion lens of microscope in parasitology section, department of zoology, university of karachi. if at least one asexual form of parasite was detected in 100 microscopic field in thick film it was considered positive otherwise the report was termed negative. while the thin blood smears were thoroughly examined for malarial parasite. in positive cases the plasmodium spp. was identified by a well trainedparasitologist. the study was approved by the chairperson of parasitology section, department of zoology, university of karachi, pakistan. chi-square test for frequency data was performed10. r e s u l t s a total of 411 cases were found to be positive out of 2096 suspected cases. four species of plasmodium were identified. p. vivaxwas more prevalent than p. falciparum. out of 411 patients, 296 cases were of p. vivax, 112 were of p. falciparum, 2 were of p. malariaewhile one was of p. ovale. maximum number of cases 191 (46.47%) were observed in the age group 16-40 years, 123 (29.92%) in 1-15 years and 97 (23.61%) in the age group 41-80 years. the overall number of infection in the males was 246 out of which 175 (59.13%) were p. vivax, 69 (61.61%) were p. falciparum, one was p. malariaeand one was p. ovale. the overall number of infection in the females was 165 out of which 121 (40.87%) were p. vivax, 43 (38.39%) were p. falciparum and 1 was p. malariae. table 1.number of male and female infection of different species of plasmodium in gulshan-e-iqbal, karachi. total cases gender p. vivax p. falciparum p. malariae p. ovale male female total male female total male female total male female total male female 411 246 165 296 175 121 112 69 43 2 1 1 1 1 0 chi-square=504.1, df=3, p<0.001 prevalence of plasmodium spp. in gulshan-e-iqbal, sindh, pakistan vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 165 table 2.percentage of infection in both male and female of three different age groups. number of positive cases gender age group percentage % 123 ♂ ♀ <1-15 yrs 29.92 191 ♂ ♀ 16-40 yrs 46.47 97 ♂ ♀ 41->80 yrs 23.61 chi-square=80.2, df=2, p<0.001 the frequency of type of malaria namely plasmodium ovale, p. malariae, p. vivaxand p. falciparum was found to be highly significantly different as disclosed by chisquare test (chi-square=504.1, df=3, p<0.001) (table 1). with respect to age classes <1-15 yrs, 16-40 yrs and 41>80 years of patients (malaria cases) the frequency differed significantly as indicated by chi-square test (chisquare=80.2, df=2, p<0.001) (table 2). d i s c u s s i o n malaria is endemic in the province of sindh, pakistan. more than five lac cases are reported only from two provinces of pakistan namely balochistan and sindh province11. all four type of malaria namely plasmodium ovale, p. vivax, p. falciparum and p. malariaewere found in gulshan-e-iqbal, karachi, sindh with respect to age classes of malaria patients the frequency differed significantly. screening of patients in karachi revealed the presence of p. vivaxto be two time higher than p. falciparum12 similar to the study conducted at ayub teaching hospital, abbotabad13. one year data from korangi creek area, karachi was collected and found among 481 infected individuals 82.32 percent p. vivaxand 17.6 percent p. falciparum infection5. in a cross sectional study of swat of malakand division and district lower dir (k.p) both thin and thick films were observed for plasmodium infection. overall positive percentage was 12%, p. vivax(99.07%) and p. falciparum (0.92%). no other plasmodium species or mixed infection was recorded. both chloroquine and artemether were used for treatment14. it was recorded that the infection rate in children (5-15 years) p. vivaxwas more frequent (2.69%) than p. falciparum (0.35%) in rural areas of bannu15 which was in agreement to the findings where was of the total of 11,353 malaria suspected samples studied by microscopy 1829/11353 (16.11 percent) samples were positive amongst which p. vivaxwas observed in 1825 subjects while p. falciparum was recorded only in 4 cases (0.2 percent)16. it was stated that in bangladesh malaria exhibited highly seasonal and hypodermic transmission in geographic hotspots. chittagong hill tracts remained malaria hotspot for a period of four years examined17. it was reported that at rural health center, sinawan, muzzafargarh, district punjab, amongst 10,023 suspected malaria cases 208 were confirmed as p. falciparum cases18. 135 cases of p. vivaxand 108 cases of p. falciparum amongst 241 cases of children visiting tertiary care hospital, karachi was recorded19. it is stated that not more than 20 percent population is availing any government health facilities and many programmes lack facilities as have no microbiologist/parasitologist to detect occurrenece of plasmodium is available11. karachi being the biggest city of pakistan with half the population of sindh has very low annual parasite index (api)20. it was suggested that chloroquine drug has become inaffective for the treatment of p. falciparum in many countries including aligarh, india due to development of resistance by the parasite which could be due to overexposure to improper therapeutic regimes, over the counter availability of drugs and pressure by improper prescribing practices by private doctors21. forty seven positive patients were recorded for plasmodium out of 210 suspected cases in blood smears. out of 28, 59.57% were identified as p. vivaxand the rest 40.43% were p. falciparum in district malakand, khyber pakhtunkhwa, pakistan22. the recommended drug for both p. falciparum and p. vivaxis artemisinin, other treatments for malaria include chloroquine, quinine, amodiaquine and doxycycline. c o n c l u s i o n frequency of malaria causing species of plasmodium was observed in total four species including p. vivax, p. falciparum, p. malariaeandp. ovalein gulshan-e-iqbal, karachi, sindh, pakistan. the presence of p. ovaleand p. malariaecould be due to travel of subjects to african countries or srilanka. the frequency of type of malaria prevalence of plasmodium spp. in gulshan-e-iqbal, sindh, pakistan vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 166 and age classes of patients differed as well, so it is suggested that screening of patients who have malaria is important in other areas of karachi. additional studies with large sample sizes in other localities are required to fully understand malaria pathology in detail. e t h i c a l a p p r o v a l ethical approval for cross-sectional survey was obtained from the chairperson of parasitology section, department of zoology, university of karachi. c o n f l i c t s o f i n t e r s t the authors declare no conflict of interest. f u n d i n g s o u r c e none. a c k n o w l e d g e m e n t s none. l i s t o f a b b r e v i a t i o n s spp. species ml mili liter % percentage df degrees of freedom p probability value r e f e r e n c e s 1. menezes rg, kanchan t, rai s, rao ppj, naik r, shetty bsk, et al. an autopsy case of sudden unexplained death caused by malaria. j forensic sci. 2010; 55(3):835-838. 2. lee k-s, divis pcs, zakaria sk, matusop a, julin ra, conway dj, et al.plasmodiumknowlesi: reservoir hosts and tracking the emergence in humans and macaques. plospathog. 2011; 7(4):e1002015. doi: 10.1371/journal.ppat.1002015. 3. soyemi s, faduyile a, lawal o, benebo a, obafunwa j, mordi v. sudden death due to cerebral malaria in a nigerian adult: a rare post mortem finding. world j pathol.2013;2:48-52. 4. world health organization (who). world malaria report 2008. 2008. p. 190. 5. khatoon n, malik r, khan a, shaukat ss, saifi zs, khan a. incidence of malaria in the population of korangi creek area, karachi, pakistan. pak j pharm sci.2018; 31(6):2575-2578. 6. zubairi abs, nizami s, raza a, mehraj v, rasheed af, ghanchi nk, et al. severe plasmodium vivaxmalaria in pakistan. emerg infect dis.2013; 19(11):1851-1854. 7. suwonkerd w, ritthison w, ngo ct, tainchum k, bangs mj, chareonviriyaphap t. vector biology and malaria transmission in southeast asia. in: manguin s, editors. anopheles mosquitoes new insights into malaria vectors, vol. 10. intech. 2013. doi: 10.5772/56347. 8. idro r, jenkins ne, newton crjc. pathogenesis, clinical features, and neurological outcome of cerebral malaria. lancet neurol. 2005; 4(12):827-840. 9. olumese pe, gbadegesin ra, adeyemo aa, brown b, walker a. neurological features of cerebral malaria in nigerian children. ann trop paediatr. 1999; 19(4):321-325. 10. zar jh. biostatistical analysis. 6th ed. prentice hall, englewood cliffs, n.j. 2008. 11. nizamani ma, kalar na, khushk ia. burden of malaria in sindh, pakistan: a two years surveillance report. j liaquatuniv med health sci.2006; 5(2):7683. 12. mahmood kh. malaria in karachi and other areas in sindh. pak armed forces med j.2005; 55(4):345348. 13. idris m, sarwar j, fareed j. pattern of malarial infection diagnosed at ayub teaching hospital, abbottabad. j ayub med coll abbottabad. 2007; 19(2):35-36. 14. zaman n, haq fu, khan z, ullah w, ualiyeva d, waheed y, et al. incidence of malarial infection and response to antimalarial drugs at districts of lower dir and swat of khyber pakhtankhwa, pakistan. dialogues in health. 2022; 1:100035. http://dx.doi.org/10.1016/j.dialog.2022.100035. 15. awan z, shah hta, shah ah, khan ma, suleman m. malaria among the students of religious schools of bannu district khyber pakhtunkhwa, pakistan. pakistan j zool.2012; 44(4):959-962. 16. khan mw, khan mn, khan ra. epidemiology and parasitological survey of malarial parasites in khyber pakhtunkhwa, pakistan. j pak med assoc. 2018; 68(1):145-146. 17. noé a, zaman si, rahman m, saha ak, aktaruzzaman mm, maude rj. mapping the stability of malaria hotspots in bangladesh from 2013 to 2016. malar j.2018; 17:259. doi: 10.1186/s12936-0182405-3. prevalence of plasmodium spp. in gulshan-e-iqbal, sindh, pakistan vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 167 18. sahar s, akhtar t, bilal h, rana ms. prevalence of plasmodium falciparum, malarial parasites in muzaffargarh district, punjab-pakistan: a two year study. pak j sci.2012; 64(1):64-66. 19. maheshwari n, shaikh m, chand r, maheshwari h, yasir m. malarial hepatopathy in children visiting a tertiary healthcare hospital in karachi. cureus.2020; 12(1):e6696. doi: 10.7759/cureus.6696. 20. prybylski d, khaliq a, fox e, sarwari ar, strickland gt. parasite density and malaria morbidity in pakistani punjab. am j trop med hyg.1999; 61(5):791-801. 21. shujatullah f, khan hm, khatoon a, khan pa, ashfaq m. in vitro chloroquine resistance in plasmodium falciparum isolates from tertiary care hospital. malar res treat. 2012; 538481. doi: 10.1155/2012/538481. 22. imran m, ahmad i, kalim m. identification of plasmodium vivax and plasmodium falciparum in the northern areas (district malakand) of khyber pakhtunkhwa, pakistan. psm microbiol. 2017; 2(3):59-62. utilization of probiotics targeting lactose intolerant individuals vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 8 op e n ac c e s s f u l l l e n g t h a r t i c l e utilization of probiotics for the development of non-dairy (oat and barley based) milk products targeting lactose intolerant individuals saniya ramzan1,*, mian kamran sharif1, rebia ejaz2 1national institute of food science and technology, university of agriculture, faisalabad, pakistan. 2department of food technology, government university for women, faisalabad, pakistan. a b s t r a c t background: lactose intolerance is a condition in people with inability to digest and breakdown the lactose sugar for metabolism. this disaccharide sugar is present in milk, and its products can be broken down into monosaccharide units by lactase enzyme, but its absence genetically or environmentally can make it difficult to consume the lactose containing products. objectives: the objective of this study is to develop and evaluate non-dairy products for consumer acceptance as milk alternate. oats and barley milk can be the alternatives with addition of soluble and in-soluble fibers. moreover, introduction of probiotics helps to improve nutritional characteristics and formulation of the product. methodology: the present study was designed to develop non-dairy milk from oats and barley, further fermented by using probiotics, namely lactobacillus acidophilus and streptococcus thermophilus to develop the drinking type yogurt product with better acceptability. moreover, the milk products were examined through physical and chemical characteristics and the statistical analysis including ph, acidity, total soluble solids, specific gravity, and proximate analysis was conducted. likewise, the fermented products were investigated for proximate, syneresis, color, texture, and sensory assessment to obtain best fit for milk substitute. results: the statistics for the prepared products showed that the color (using color meter) among the product range was l = 65.03379.16; a* = -3.916 to -6.556 and b* = 13.847 to 23.0, while moisture was 87.233% to 97.713%. furthermore, fat was 0.0143% to 2.3533%, protein 0.5433% to 3.286%, ash 0.095% to 0.4233%, texture 0.065 to 0.408, and syneresis range was 54.33 to 82.67 among the products. conclusion: after sensory evaluation and the analysis, the best treatment t1 (100% oat milk) is considered fit-to-substitute cow milk and its products, with higher consumer acceptability. keywords non-dairy product, lactose intolerance, probiotics, syneresis, substitute. *address of correspondence saniyaramzan8@gmail.com article info. received: january 24, 2021 accepted: february 28, 2021 cite this article ramzan s, sharif mk, ejaz r. utilization of probiotics for the development of non-dairy (oat and barley based) milk products targeting lactose intolerant individuals. rads j biol res appl sci. 2021; 12(1):8-16. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n lactose is a sugar (carbohydrate) that is present in dairy products. for its proper digestion and absorption in the body, lactose is hydrolyzed in the intestine by an enzyme β-galactosidase, which is usually called as lactase. due to some reasons, there is a lack of lactase in humans and without it, there is difficulty in lactose digestion inherently. even in normal conditions, lactose approaches the distal small intestine without being absorbed. up to 8% of lactose o r i g n a l a r t i c l e utilization of probiotics targeting lactose intolerant individuals vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 9 enters the ileum area of intestine without being processed. fermentation of unabsorbed lactose occurs by the colonic bacterial flora in the colon area of the intestine, which results in the symptoms of lactose intolerance1. lactase deficiency is present in 15% northern europeans approximately, while 100% of american indians & asians and up to 80% of blacks and latinos2. lactose malabsorption is prevalent in about 75% of the global population3. primary adult lactose malabsorption in pakistan was found to be 60%4. oat (avena sativa) belongs to family poaceae and genus avena. it has many unique properties that makes it diverse from other cereal grains, such as oat hull is separate from endosperm and contains more fat content in comparison with other cereal grains. it is also known for its high percentage of soluble dietary fibres i.e., β-glucan. moreover, phenolics and niacin are also present in appreciable quantities5. oat’s excellent lipid contents exist as lipid bodies, similar to emulsion droplets, surrounded by proteins and phospholipids which makes it a uniquely nutritious food. oat milk is extracted from oat by adding water and salt, which extract these lipid bodies along with proteins6. barley (hordeum vulgare) is also a member of family poaceae and genus hordeum. the most recently, barley based products are gaining popularity due to presence of β-glucan which has many physiological benefits. additionally, it is also high in phenolic compounds such as phenolic acids, tannins, pro-anthocyanidins, flavonols, flavones, flavanones, chalcones and amino phenolic compounds. it has nutritional benefits as well such as high amount of dietary fibres, minerals (molybdenum, manganese, chromium, phosphorus, selenium, copper and magnesium) and vitamins (vitamin b1, e, and niacin)7. plant-based milk alternatives are the extracts obtained from legumes, cereals, seed oil or pseudo-cereals that look like milk. these replacements are commonly prepared by the extraction of grains material in water. then the elimination of solid particles and product formulation is done. now-a-days, their trend is increasing due to peoples’ awareness about their diet, as a lifestyle choice or for medical reasons (e.g. cow’s milk allergy, lactose intolerance etc.)8. the cereal and grain milk are cholesterol and lactose free in comparison to bovine milk. so, for people, they are an attractive substitute of bovine milk9. plant-based milk alternatives are a growing trend and can serve as an economical alternate to low income group of developing countries and places with insufficient cow’s milk supply. majority of these milk alternatives lack nutritional balance when compared to bovine milk, but they contain functionally active components with health promoting properties which fascinate health conscious consumers3. also, in recent years, plant sources are accepted as functional food and nutraceuticals due to presence of health promoting components such as dietary fibres, minerals, vitamins and antioxidants10. for proper labelling, the legislation and labelling requirements allow names in accordance with the composition and the law and custom of the country, so that the product is sold and in a manner not to mislead the consumer. national legislation on food labelling varies from country to country, principle terminology in categorizing these plant based milk alternatives has been under debate at international level. in united states, the fda covers these plant based milk alternatives under the definition of imitation milk and imitation milk products3. interestingly, oat milk contains 1.033g/100g protein, 33.51g/100g carbohydrates, and 8.2mg/100g calcium11. probiotics are living microbes that impart beneficial effect on the host which helps to maintain the health and have preventive and curative effects on host. multiple researches have illustrated their health benefits on gastrointestinal tract infections, betterment in lactose absorption, antimicrobial activity, anti-mutagenic properties, decrease in blood cholesterol level, immune system stimulus, anti-cancer & anti-diarrheal properties, betterment in inflammatory bowel disease and many more12. usually, members of the lactobacillus and bifidobacterium genus are recognized as probiotics. nowa-days, probiotics are increasingly used in food products and development of new functional foods by the food industries13. furthermore, in terms of acceptability of any new product in pakistan, mothers generally will not feed their infants/children with foods to which they are not familiar, or which are not acceptable to them. so, there is a need to familiarize them with newly introduced products and their usage14. the present project has been designed to achieve the objectives including: development and evaluation of cereal-based fermented yogurt-like product and accessing consumer acceptability of the developed utilization of probiotics targeting lactose intolerant individuals vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 10 products through physico-chemical analysis and sensory evaluation. m a t e r i a l a n d m e t h o d s preparation of oat and barley milk oat (avena sativa) and barley (hordeum vulgar) were procured from ayub agriculture research institute, faisalabad. oat and barley grains were soaked in water for 12hours before extraction of milk following the modified method described by kljusric et al.15. the respective milk concentrates were diluted to get the oat and barley milk. analysis of cow, oat and barley milk for raw milk obtained from oat and barley cereals, they were tested for different proximate analysis as for moisture, ash content, protein, fat, and nitrogen free extract and other physical analysis for acidity, ph, specific gravity, total soluble solids and lactose content as described by association of official agricultural chemists (aoac)16. american association of cereal chemists (aacc)17 method was used to determine crude fiber. the color was measured by using colorimeter according to the modified method of rocha and morais18 by putting the sample against the colorimeter through which light waves of different wavelengths passes. introduction of probiotics for fermented product preparation oat milk and barley milk were used in various proportions (table 1) to formulate fermented products by utilization of probiotics lactobacillus acidophilus and streptococcus thermophilus as stated by bernat and colleagues19. inoculum strains were activated from their frozen forms (stored in 40g/100ml glycerol at -80°c) by transferring each one to its selective broth until optimal bacterial growth is assured. fermentation process was carried out by adding the corresponding amount of starter culture (prepared by mixing in a 1:1 volume ratio) to the formulated and sterilized oat milks. further, incubation at 40°c was carried out because it is the optimal growth temperature of the mixed culture. fermentation process was stopped when ph of samples reached 4.4-4.6 and cool the samples at 4°c (storage temperature) until the analyses were done. analysis of fermented product for moisture, total ash, fat, crude protein, and color determination; previously described methods were used. furthermore, texture of the treatments was determined by using ta-xt plus texture analyzer20. another physical test namely syneresis of all the developed products was accomplished by utilizing the method of li and guo21. sensory evaluation all the developed products were analyzed by the panel consisting staff, students, and faculty members of nifsat, uaf for sensory characteristics like appearance, texture, flavor, mouthfeel, consistency and overall acceptability by panel of judges following 9-point hedonic score system22. statistical analysis significant difference among obtained data for each parameter was analyzed statistically using analysis of variance technique p < 0.05. completely randomized design anova was further utilized to evaluate the level of significance for the data23. table 1. treatments prepared by different proportions of oat milk and barley milk. treatments cow milk (%) oat milk (%) barley milk (%) t0 100 t1 100 t2 80 20 t3 60 40 t4 40 60 t5 20 80 t6 100 t0 = 100% cow milk as control. utilization of probiotics targeting lactose intolerant individuals vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 11 r e s u l t s a n d d i s c u s s i o n cow, oat and barley milk analysis the grain milk was extracted from oat and barley, and then further subjected to the analysis that lead to the data obtained as mean square results of physical analysis of oat milk, barley milk, and cow milk ranges for different parameters, and shows a highly significant results for acidity, ph, total soluble solids (tss), lactose content, and color, while significant result was obtained for specific gravity. furthermore, for the average of the samples, minimum ph 6.38 was found in oat milk, while maximum ph 6.86 was observed in barley milk. furthermore, maximum acidity 0.13% was identified in cow milk while minimum acidity 0.03% in barley milk. specific gravity had highly significant results among the samples that contain minimum value 0.97 for barley milk and maximum result 1.02 for cow milk. total soluble solids among all the samples vary in extent and have the values (2.22 < 7.25 < 10.23⁰brix) in the manner having barley milk (tbm) least value and cow milk highest value15. lactose content was found to be 0% in oat milk and barley milk as compared to cow milk with 3.8% lactose. similarly, pereira et al.24 reported that cow milk contains 4.7% lactose sugar. the mean squares of the treatments for various parameters of chromaticity includes l, a* and b* that also showed highly significant results. a* and b* values are the four unique colors for human vision including red, green, blue and yellow. the values for the lightness denoted by l have the highest value 79.09 for cow milk while least score was found in barley milk that is 65.267. in terms of avalue the observed results for cow milk have maximum value with minimum value for barley milk -3.8967. furthermore, in terms of b value the highest score 22.753 was observed in oat milk (tom) while, lowest score 13.813 was of cow milk (tm). the results of means of all the analysis are present in the table 2. while, in case of the mean of the values (table 3), the highest percentage 97.55% in terms of moisture among all dairy and non-dairy milk samples was in tbm that is milk prepared from barley grain. the least value 85.67% was obtained by tm that is cow milk. results in terms of crude fat have highest value 3.40% that is presented by tm treatment referred to cow milk, while the least result 0.024% was obtained from tbm that is barley milk. results among the values for samples provided for protein indicates the highest value 3.27% for the cow milk. furthermore, the oat milk tom has the value for protein content 0.63%. the highest value among the samples for fiber is 0.22% that is represented by tom prepared from oat milk. the lowest value is for milk that is 0% as milk does not contain any fiber in it. in case for ash content, the maximum ash percentage 0.70% was found in tm treatment that is 100% cow milk with the least score 0.22% obtained from tbm treatment that is barley milk. the highest value for nitrogen free extract (nfe) is 6.97% represented by cow milk and the least value observed was 1.07% obtained by barley milk. these results are similar to studies done by butt25 and makinen26. the results of means of the values are prominent in table 3. table 2. average of physical analysis of cow, oat, and barley milk. samples tm tom tbm acidity % 0.13±0.0 0.11±0.0 0.03±0.0 ph 6.63±0.0 6.38±0.0 6.86±0.0 specific gravity 1.02±0.0 1.01±0.0 0.97±0.1 lactose % 3.8±0.0 0.00±0.0 0.00±0.0 tss (⁰brix) 10.23±0.3 7.25±0.1 2.22±0.0 color l value 79.090±0.17 72.593±0.75 65.267±0.45 a* value -6.533±0.05 -5.25±0.24 -3.8967±0.02 b* value 13.813±0.02 22.753±0.05 19.127±0.11 *values are mean + sd for samples analyzed in triplicate, tm = cow milk, tom = oat milk, tbm = barley milk. l value = lightness, a* value = red/green coordinate, b* value = yellow/blue coordinate. utilization of probiotics targeting lactose intolerant individuals vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 12 table 3. average for the chemical analysis of the dairy and non-dairy milk. treatments tm tom tbm moisture % 85.67±1.5 97.45±0.0 97.55±0.1 fat % 3.40±0.1 0.09±0.0 0.024±0.0 protein % 3.27±0.3 0.63±0.0 0.77±0.0 fiber % 0.00±0.0 0.22±0.0 0.14±0.0 ash % 0.70±0.0 0.32±0.0 0.23±0.0 nfe % 6.97±1.8 1.28±0.0 1.07±0.1 values are mean + sd for samples analyzed in triplicate, tm = cow milk, tom = oat milk, tbm = barley milk. table 4. means for the chemical analysis of the dairy and non-dairy milk fermented products. treatments moisture % ash % fat % protein % t0 87.233±0.92 0.4233±0.02 2.3533±0.09 3.2867±0.24 t1 97.067±0.20 0.1893±0.01 0.0717±0.00 0.5433±0.02 t2 97.143±0.22 0.1680±0.00 0.0653±0.00 0.5857±0.00 t3 96.90±0.45 0.1483±0.00 0.5233±0.03 0.6233±0.02 t4 97.510±0.05 0.1377±0.00 0.3567±0.02 0.6617±0.00 t5 97.713±0.17 0.1147±0.01 0.0227±0.00 0.6733±0.02 t6 97.533±0.35 0.0957±0.00 0.0143±0.00 0.6477±0.01 values are mean + sd for samples analyzed in triplicate. prepared products analysis products prepared from different concentrations of oat milk, barley milk and their blend along with the control treatment cow milk were subjected to moisture, fat, protein, ash, color, texture and syneresis analysis. the means of the results were observed (table 4) then it showed that most of the treatments for moisture content fall into similar category and have only a little deviation from each other. as the highest result 97.713% was obtained by t5 treatment prepared from 80% barley and 20% oat milk. while, the highest value for ash 0.4233% was found in t0 (100% cow milk product) followed by t1 with result 0.1893% that is 100% oat milk product. the least amount of ash 0.095% was found in 100% barley milk. the highest percentage for fat was presented 2.353% by cow milk product t0 and the least value 0.0143% was obtained by t6 (100% barley milk product). for protein the highest value 3.2867% was found in t0 treatment with 100% cow milk followed by the 0.6733% of t5 80% barley and 20% oat milk composition. the lowest percentage 0.5433% was obtained by t1 (100% oat milk). the results are similar to the findings of amanze and amanze27. such findings show that there is increased effect of the physical as well as chemical parameters on the products that may affect its quality based on time duration or the storage facilities. by the results of color, texture and syneresis of the products and statistics applied to them showed the highly significant results. the mean results for the analysis performed on the treatments for color, texture and syneresis are stated in table 5. by the studies of these parameters, it was found that treatment t1 prepared from 100% oat milk (table 1) is the best suited to substitute cow milk, followed by t2 prepared from 80% oat milk and 20% barley milk as second-best option. for syneresis, the least score 54.33 was recorded by t1 100% oat milk composition that means it has best water holding ability followed by t2 80% oat milk and 20% barley milk having value of 61.83 even at high centrifugation rate rather than other utilization of probiotics targeting lactose intolerant individuals vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 13 treatments and even the control treatment t0 (100% cow milk) with value 82.67. so, it is better at maintaining its shape for more time rather than the other treatments. furthermore, t3 (60% oat milk and 40% barley milk) have shown the least stability during centrifugation with value 63.13. a study by amanze and amanze27 on the oat flakes, non-dairy beverages showed the syneresis ranged from 48-67. for texture, the highest value 0.4087 was obtained by t3 (60% oat milk and 40% barley milk). this is followed by treatments t1 (100% oat milk) with value 0.3867 and t0 100% cow milk with 0.0813. least score 0.0650 was obtained by t6 (100% barley milk). the different parameters for color analysis are referred by l, a* and b* values. in case of l value, there seem to be a regular pattern among the treatments. the highest value 79.16 was for t0 (100% cow milk) value followed by 71.53 for t1 treatment (100% oat milk). the lowest value 65.033 was obtained by t6 having 100% barley milk. as for a-value, the treatments got the values as: -6.5567 for t0,-5.0100 for t1 while least value was obtained -3.9167 for t6. for the bvalues of the fermented products, the highest result 23.0 was obtained by t1 followed by t2 with value 19.79. while, the least value 13.847 was presented by t0 (100% cow milk). such findings are in agreement with the previous studies done by kljusric15 and luana28. sensory evaluation these treatments were subjected to sensory evaluation by sensory panel to check its acceptability in the market by consumers. these six treatments along with control treatment were analyzed for appearance, texture, flavor, mouthfeel, consistency and overall acceptability by the sensory panel based on 9-point hedonic scale (table 6). the analysis of variance of the results shows highly significant results for all the parameters. the mean values for overall appearance including color, shape of the treatments, the t1 treatment (100% oat milk) was found to have the highest value 6.7 after t0 7.7 that is the control treatment prepared from cow milk. furthermore, t2 (80% oat milk: 20% barley milk) is next best option after t1 having score about 6.5. the lowest value for the appearance is t6 that is 3.9 due to its color deviation from milky white to pinkish white. moreover, it was not in such a good shape as the standard had. t1 and t2 was more near to the standard in appearance other than any treatment. the mean values for the texture of different treatments showed the highest score 7.5 for the t1 treatment (100% oat milk) that was prepared with 100% oat milk. the score for t0 treatment is 6.9 that is at 2nd place followed by t2 with 6.4. the least score 4.5 is for treatment t6 having 100% barley milk. these results showed that the texture in terms of hardness, firmness and shape stability is best suited to the treatments having higher content of oat milk that also exceeded to the control treatment. while there is a decreasing trend of texture maintenance among the treatments having higher ratio of barley milk. the mean values for flavor showed the results for the treatments among them the highest value is for t1 (100% oat milk) that is 7.5 followed by the 7.1 for t2 treatment. these two values gave better consumer acceptability than the control treatment that has score on 3rd place as t0 with score 6.8. the lowest value for the flavor is 4.4 for t6 prepared with 100% barley milk. the mean values for mouthfeel of the treatments showed highest score 7 was found in product prepared from 100% oat milk after the score of control treatment with score 7.8 prepared with 100% cow milk while lowest value was 4.1 showed by t6. the mean values for consistency of treatments showed highest score 7.2 was found in treatment t1 that was prepared from 100% oat milk. this value even exceeded the score of control treatment t0 that is 6.8. t2 treatment score is equal to the value of t0. t6 is the treatment with 100% barley milk showed the least value 4.4 for consistency. as in case of overall acceptability, t1 (100% oat milk) got highest the score 7.6 followed by t0 that has value 7.3 with least acceptability of t6 that is 4.6. the results showed that t1 prepared from 100% oat milk is the best suited to substitute cow milk followed by t2 prepared from 80% oat milk and 20% barley milk as second best option. both these treatments were not only found to be best among the treatments but they also outcast the preference of control treatment that is conventional yogurt prepared from cow milk. the mean values for the sensory parameters are available in table 6. utilization of probiotics targeting lactose intolerant individuals vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 14 table 5. means for texture, syneresis and color of dairy and non-dairy milk fermented products. treatments texture syneresis color l value a* value b* value to 0.0813±0.00 82.67±2.5 79.160±0.07 -6.5567±0.04 13.847±0.02 t1 0.3867±0.01 54.33±2.1 71.530±0.11 -5.0100±0.2 23.0±0.22 t2 0.2053±0.00 61.83±1.6 70.470±0.01 -4.7867±0.08 19.790±0.17 t3 0.4087±0.01 63.13±2.7 69.517±0.04 -4.4067±0.11 18.997±0.04 t4 0.3253±0.01 66.83±1.6 68.627±0.02 -4.4867±0.08 19.090±0.21 t5 0.1253±0.01 66.83±2.0 68.940±0.12 -4.3100±0.09 19.067±0.28 t6 0.0650±0.00 68.83±1.3 65.033±2.25 -3.9167±0.01 19.190±0.06 *values are mean + sd for samples analyzed in triplicate. table 6. means for sensory attributes of dairy and non-dairy milk fermented products. treatments appearance texture flavor mouthfeel consistency overall acceptability to 7.7±0.7 6.9±0.7 6.8 ±0.8 7.8±0.8 6.8±0.8 7.3±0.7 t1 6.7±0.7 7.5±0.7 7.5 ±0.7 7±0.5 7.2±0.6 7.6±0.7 t2 6.5±0.5 6.4±0.5 7.1 ±0.7 6.9±0.9 6.8±0.9 6.9±0.3 t3 5.8±0.6 6.2±0.6 6.6 ±0.5 6.3±0.8 6.4±0.7 6.3±0.7 t4 5.2±1.1 5.8±1.1 6.2 ±0.6 6.1±0.6 5.8±0.8 6±0.5 t5 4.7±0.9 4.9±0.9 5.6 ±0.7 4.8±0.8 5.3±0.5 5.3±0.5 t6 3.9±1.3 4.5±1.3 4.4 ±0.7 4.1±1.1 4.4±1.0 4.7±0.7 values are mean + sd for samples analyzed in triplicate. c o n c l u s i o n for the purpose of combating lactose intolerance, oats and barley milk was prepared and their products were utilized as substitute for the cow milk and its products. among all the treatments t1 and t2 were best suited to substitute conventional cow milk. both these treatments were not only found to be best among the treatments but they also outcast the preference of control treatment that is conventional yogurt prepared from cow milk. as cereal grains are major food constituents of daily food in pakistan, it is staple food for the people, so people tend to like the flavor and unique properties of fermented product prepared from oat milk. they are more comfortable with the product prepared rather than the conventional dairy product. as oat and barley have no lactose sugar, so there are no chances of any lactose intolerance prevalence among the consumers. study about non-milk derivatives or plantbased milk products is very vast and will be of main concern for the scientist and researchers in the near future. not only its compositional analysis but more ways will be open in r & d section for innovative products development. c o n f l i c t s o f i n t e r e s t none. f u n d i n g s o u r c e none. utilization of probiotics targeting lactose intolerant individuals vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 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https://www.google.com.pk/search?tbo=p&tbm=bks&q=inauthor:%22anam+aman%22&source=gbs_metadata_r&cad=2 https://www.google.com.pk/search?tbo=p&tbm=bks&q=inauthor:%22dr+hayati+mohd+yusof+aab%22&source=gbs_metadata_r&cad=2 https://www.google.com.pk/search?tbo=p&tbm=bks&q=inauthor:%22norhayati+abd+hadi%22&source=gbs_metadata_r&cad=2 https://www.google.com.pk/search?tbo=p&tbm=bks&q=inauthor:%22dr+adzim+mohd+khalil+rohin+amkr%22&source=gbs_metadata_r&cad=2 utilization of probiotics targeting lactose intolerant individuals vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 16 24. pereira pc. review: milk nutritional composition and its role in human health. nutr. 2013; 30:619-27. 25. butt ms, nadeem mt, khan mki, shabir r, butt ms. oat: unique among the cereals. eur j nutr. 2008; 47:68-79. 26. mäkinen oe, lowe tu, o’mahony ja, arendt ek. physicochemical and acid gelation properties of commercial uht-treated plant-based milk substitutes and lactose free bovine milk. food chem. 2015; 168:630-8. 27. amanze ko, amanze jo. quality evaluation of yoghurt from cowmilk, soymilk and cow/soymilk. jorind. 2011; 9:44-7. 28. luana n, rossana c, jos´e antonio c, kaisa p, marco g, giuseppe rc. manufacture and characterization of a yogurt-like beverage made with oat flakes fermented by selected lactic acid bacteria. int j food microbiol. 2014; 5:1-40. rads journal of biological research & applied sciences vol 3 (2), july 2012 issn : 2225-62229 original articles sodicity control by gypsum & fym with reference to green gram (vigna radiate) growth saima ibrahim and shakila bano ......................................................................................... 07 cell phones: carrier for microbes in women’s university fasiha saeed, anum nayyer , shazia tabassum hakim, sayyada ghufrana nadeem ...11 a record of intestinal protozoan infection from different hospitals of karachi pakistan rukhsana talat, farzana ibrahim, zaira1 and qurat ul ain ......................................17 teaching undergraduates students through connectivity: part 3 iftikhar imam naqvi, ghazala kanwal and anam shafi ...............................................20 diagnostic significance of mcv, mch and nestroft in thalassemia minor individuals naheed afshan and maryam hussain ...................................................................24 review articles pharmaceutical quality assurance & control ghulam sarwar and m. shakil siddiqui ....................................................................... 27 the global polio eradication initiatives: from past to presentr shazia tabassum hakim, sayyada ghuffrana nadeem, shaista malik, urooj javed2, abdul basit khan............................................................................. 34 instruction to authors introduction soil salinity severely affects agricultural productivity by causing salt accumulation on the superficial layer of agricultural land (jaleel. et al. 2007). the term salinity relates to the total concentration of dissolved inorganic ions, i.e. na, ca+2, mg+2, k+, hco3, so2-4 and clin groundwater (epstein and rains, 1987). excessive uptake of certain ions often results in reduced uptake of some essential plant nutrients causing nutrient imbalances and deficiencies. thus, crops grown on such saline/sodic soils suffer nutritional disorders, resulting in inhibited growth and low yields of growing crops. na+ is the dominant cation in saline soil due to which salt affected soils suffer deterioration in their physical properties. soluble source of ca+2 is essential for reclamation of such soils. for this purpose, gypsum (caso4.2h2o) is commonly used to reclaim the sodic soil (gupta, et al. 1985). it reacts with na2co3 and adsorbed sodium. when it applied to soil, it dissolves somewhat slowly and separates into the ca+2 and so4-2 ions (dissociation). the calcium from gypsum replaces the sodium associated with the soil clay particles. on the application of gypsum to the sodic soil, sufficient and good quality of water must be added to leach the displaced sodium beyond the root zone. make sure drainage is adequate prior to amending the soil. this research was conducted to evaluate the plant response to gypsum application alone/along sodicity control by gypsum & fym with reference to green gram (vigna radiate ) growth saima ibrahim1, shakila bano1 1department of botany, jinnah university for women. karachi, pakistan. abstract sufficiently present sodium ions (na+) of sodic soil adversely affect the growth of growing plant. sodic soil reclaimation requires ca+2 ions supply for the replacement of exchangeable na+ ion. gypsum as an effective ca+2 source used to reclaim sodic soil and improve soil ground water infiltration. addition of fym improves the sodic soil permeability by adsorbing insoluble salts. a pot experiment was conducted on artificial sodic soil for its reclaimation by combining different approaches i.e. application of gypsum and fym. two sets of experiments were designed to check the effects of salt and applied reclaimatives (alone or in combination) at different rates with respect to the growth of green gram. the treatments were: t0(control), t1(saltna2co3), t2(salt+gypsum1), t3(salt+ gypsum1+fym1), t4(salt +gypsum2), t5(salt+gypsum2+fym2). in the given research, gypsum was applied at the rate of 1.5ton/ha (gyp1) and 2.5ton/ha (gyp2) alone or in combination with fym at the rate of 2.5ton/ha (fym1) and 5ton/ha (fym2) respectively. the soil was artificially sodify by adding 0.15% na2co3 and 0.25% na2co3 separately in two sets of experiments. thus, the gypsum provided the best treatment when applied alone it both rate. however, the combination of gypsum and fym at low concentration was more effectives for all growth parameters than gypsum alone. high rate of fym along with high gypsum rate required maximum irrigation to flush soluble salt which highly restricted growth if not leached down or drained out properly. at both sodicity levels an increase in soluble carbohydrate, total protein and proline contents was observed. key words: sodic soil; sodicity; farmyard manure; reclaimatives corresponding author: ibrahim4sept@hotmail.com 01 vol 3 (2), july 2012; 01-07 with fym, growing under na2co3 conc. proper reclaimation and fertilization of sodic soil should serve to supplement nutrients and to reduce excess toxic ion by improving ion exchange, leaching and flashing of insoluble and soluble salt respectively. the main objective of current work is to analyzing the salinity effects exerted by sodic soil on growth parameters and physiological processes of green gram. materials and methods a pot experiment was conducted to check the inhibitory effect of sodic soil on green gram and to evaluate its reclaimation through gypsum alone or together with fym. na2co3 salt was supplied in soil @ 0.15% and 0.25% to induce sodicity. the experiment was performed in 2-sets of treatments to check the efficiency of reclaimatives at different rate (low and high) under same salt concentration. ten healthy seeds of gram were sown per pot. pots were placed in random blocks with 3-replicates and irrigated with dw. both sets of experiment were and maintained the setup for about 12-15 days in order to get the treatment effects up to plant maturity. the effects of treatment were recorded by observing shoot and root length, fresh wt. and dry wt. separately. the dry weight was obtained after oven drying at 65oc for 48hours. basic biochemical tests were also performed to check the effects of treatment on physiological processes of plants. carbohydrates and protein were determined by the method of e. w. yemm and a. j. willis (1954) and lowry (1951), proline was determined by bates (1973) method. inhibitory percentage (i): the percentage of inhibitory on growth (length, fr.wt, dry wt.) in comparison to control was calculated by surendra and pota, (1978) formula: i = 100t/c x 100 where, i is percentage of inhibition, t is treatment, reading and c is control plant reading. data analysis: the data were subjected to analysis of standard deviation statistically to evaluate the treatment effect through duncan’s multiple range test (dmrt) results a) morphological parameter result: table 1 and 2 indicated that, high conc. of salt (0.25% na2co3) more drastically reduced all growth parameters of green gram including sl, rl sfw, sdw, rfw and rdw than low conc.(0.15% na2co3). similarly high dose of gypsum (alone) i.e. 2.5tons/ha has more promoting effect on all growth parameters than low dose i.e. 1.5tons/ha at both sodic concentrations. shoot length (cm): table1 & 2 showed that, both concentration of salt (i.e. 0.15% and 0.25%) significantly reduced shoot length in gram. this inhibitory effect on shoot length was efficiently overcome by supplying gypsum at two different rates. the rate of gypsum also played an important role in controlling shoot length reduction i.e. high dose of gypsum (2.5tons/hec) has more promotive effect than low dose (1.5tons/ha). similarly addition of fym also supports gypsum positive effect as a substitution reclaimatives. the amendment of soil with gypsum and fym positively increased shoot length more than gypsum alone. both crops showed the same positive effect in shoot length, when supplied with gypsum alone and together with fym, growing under both salt concentrations. table1 showed that, -54.2% shoot length reduction at low salt concentration (i.e. 0.15%) highly reduced to 26.1% at gypsum + fym treatment while in table2, -43.50% reductions at high salt concentration (i.e. 0.25%) overcome to -27.92% at low dose of gypsum and fym. root length (cm): table 1 & 2 showed that both concentrations of salt (0.15% and 0.25%) had inhibitory effect on root length of gram. gypsum high dose alone and together with fym was more efficiently work with root length than low dose. -69.04% root length reduction at low salt concentration reduced to -18.8% with high gypsum (alone) treatment. the addition of fym at this high gypsum dose was slightly more inhibitory 02 vol 3 (2), july 2012; 01-07 03 vol 3 (2), july 2012; 01-07 * value in parenthesis indicate percent increase (+) or decrease (-) over control. ** means followed by different letters show significant result at the level of standard deviation. * value in parenthesis indicate percent increase (+) or decrease (-) over control. ** means followed by different letters show significant result at the level of standard deviation table-1: effect of salinity reclaimatives (gypsum and farm yard manure) on growth parameters of green gram growing under 0.15% na2co3 table-2 effect of salinity reclaimatives (gypsum and farm yard manure) on growth parameters of green gram growing under 0.25% na2co3 (-20%) may be due to increasing osmotic content in soil which create stress in root that restrict its growth. at high salt, 70.11% root length reduction s i g n i f i c a n t l y o v e r c o m e t o 3 9 . 1 0 % w i t h gypsum+fym at high dose. shoot fresh weight (gm): table1 & 2 showed that, -69.04% root length reduction as low salt concentration reduced to-18.8% with gypsum (alone) treatment. the addition of fym at this high gypsum dose was slightly more inhibitory (-20%) may be due to increasing osmotic content in soil which create stress in root that restricted its growth. at high salt concentration 70.11% root length reduction significantly overcome to -39.10% with gypsum + fym at high dose. shoot dry weight(cm): both concentration of salt greatly reduced shoot dry wt. in gram , addition of gypsum balanced the dry wt. of shoot by decreasing the inhibitory effect of salt, this effect was significantly improved with the application of fym along with gypsum at both sodic conditions. maximum controlling result appeared at high dose of gypsum along with fym in both crop. table 1 & 2 showed that -37.93% and -55.44% shoot dry weight reduction at low and high sodic conditions were significantly reduced with the addition of low gypsum rate at low dose up to 13.79% in low salt and -23.83% at high salt treatment. addition of fym along with gypsum significantly improved crop growth by increasing shoot dry weight up to +6.89% to +29.31% over control at low salt concentration while at high salt addition of fym wan only supportive with low gypsum dose and reduces the salt inhibitory effect of dry weight accumulation in gram from -23.83% to -14.50%. at high dose of gypsum and fym shoot dry wt. showed -34.19% inhibitions over control this may be due to the inhibition of several physiological processes at high salt concentration that restricted biomass production. root fresh weight (gm): root fresh wt. was directly influenced by the presence of external solute which restricted water absorption. gram showed tolerance toward osmotic gradient and absorbed much water under external solute stress 04 vol 3 (2), july 2012; 01-07 table 3: effect of sodicity reclamative (gypsum and fym) on carbohydrate, protein and free proline of green gram growing under 0.15% and 0.25% na2co3 * value in parenthesis indicate percent increase (+) or decrease (-) over control. especially when gypsum was applied along with fym at low dose at both sodic conditions. table 1 & 2 showed that, low concentration of salt increased root fresh wt. i.e. 2.85% over control while high concentration inhibits water absorption and decreased root fresh wt. i.e. -15.33%. addition of gypsum at both sodic conc. was favorable for root fresh wt. especially at high concentration and increased it to +13.08% and +143.48% at low and high dose respectively. fym application was useful only with low gypsum dose i.e. + 14.2% at low salt and +123.90% at high salt concentration. root dry weight (gm): table showed that, reduction was maximum at root dry weight level at both sodic conditions (i.e.-51.51% & -60.71% in gram at low and high sodic condition respectively). this inhibitory effect of both sodic soils was significantly controlled by gypsum addition especially at low rate that reduced -51.51% inhibitions at 0.15% salt and -60.71% at 0.25% salt to -24.24% and -4.08% respectively. addition of fym was also favorable at both sodic conditions with both gypsum rates. b) biochemical parameter result: table-3 showed an effect of sodicity on biochemical parameters of treated plants, both levels of sodicity (i.e. 0.15% and 0.25% na2co3,) induced a significant decreased in protein content while, the soluble carbohydrate and free proline content was remarkably increased with increasing salt concentration. carbohydrate content: table-3 showed that, accumulation of carbohydrate was higher at low salt concentration (i.e. +89.28%) than high salt concentration (i.e. +4.73%) that proved the maximum absorption of salt at 0.15% na2co3, treatment while at 0.25% na2co3, high salt content at rhizosphere increased osmotic gradient that restricted salt absorption along with water. addition of gypsum reduced some degree of stress and showed less carbohydrate accumulation than salt treated plant. application of fym reduced carbohydrate contents from all above treatments due to interaction with chlorophyll content and net photosynthesis. protein: table-3 showed inhibitory effects of sodicity on total protein content of gram. compared to control, the percentage of reduction was between -9.69% and 16.9% in green gram in both sodic concentrations respectively. addition of gypsum at high concentration significantly improved protein contents in gram at both salt concentrations. this positive effect was also supported by fym application that enhanced protein accumulation from +19.21% in low and +64.90% to +159.61% at high salt concentration respectively. free proline contents: table-3 showed that salt stress significantly increase free proline content in gram. the percentage of increase gradually enhanced along with sodicity concentration increment. the percentage of increase raised from +16.78% to +156.10% in green gram in low and high salt concentrations respectively. addition of gypsum also adds a solute stress in rhizosphere thus increased proline synthesis in gram at both rate. the content of free proline at low and high gypsum rate were +186.8% to +326.21% in low salt concentration and +209.76% to +358.53% in high salt concentration in green gram in compared to control plant. addition of fym decreased salt stress also suppressed proline synthesis due to its high phosphoric content, resulted in less proline accumulation than gypsum and salt treatments at both rate at both salt concentration. discussion a) morphological parameters: soil salinity affects many physiological processes and resulted in reduced growth. this negative effect increases with elevated salt concentration. in saline soils, the inhibitory effect of soluble salt on plant growth are associated with (a) low osmotic potential of soil solution resulting in water stress (b) nutritional imbalance, and (c) specific ion effect. these factors cause adverse effect on plant growth and development at physiological and biochemical level (ashraf and harris, 2004). the expected causes of the reduction in growth parameters i.e. shoot length, root length 05 vol 3 (2), july 2012; 01-07 could be the shrinkage of the cell contents, limiting of cell wall elasticity, reduced development and differentiation of tissues, unbalanced nutrition, damage of membrane and disturbed avoidance mechanism. in a given research reduction in fresh and dry weight under saline conditions were also due to reduced water uptake, toxicity of sodium and chloride in the shoot cell as well as reduced photosynthesis. the addition of gypsum significantly effects alone or along with farmyard manure to overcome sodicity effect. chaudhry et al. (1982) reported gypsum @ 50% as the best treatment following by gypsum @ 50% + 50t fym ha-1 > h2so4 @ 10% gypsum added to saline/sodic soils can improve permeability due to both electrolyte concentration and cation exchange effects. m. anwar et al. (2005) also indicates that combined application of manure and fertilizer helps to increase crop quality and productivity and also maintain soil fertility. b) biochemical parameters i) effect of salt (0.15% & 0.25% na2co3) on metabolic products of green gram: biotic and abiotic stresses cause plants to increase the production of metabolic compounds i.e. proline sugar and other organic solutes to increase stress tolerance (el-darier and youssef, 1998). the increase of organic metabolites content in plant has been widely reported as a response of salinity stress (ahmad and jhon, 2005). this is due to the conservative strategy of plant to face external stress condition. ii) soluble carbohydrate content: carbohydrate contents increased with increasing levels of sodicity. in the present study, na2co3 stress caused a significant increase in soluble sugar in green gram at both level (i.e. 0.15% and 0.25% na2co3). the contents of carbohydrate in shoot tissue tend to increase with elevating salt level. many plant, which are stressed by salinity, accumulated starch and soluble carbohydrate (rathert, 1984) this accumulation has been attributed to impaired carbohydrate utilization (munns and termat, 1986). due to abiotic stress from salt, the plant tries to lope with the situation by increasing its carbohydrate content as a conservative strategy. the accumulation of organic solute (soluble and insoluble carbohydrates) might play an important role in increasing the internal osmotic pressure (zidan and al-zahrani, 1994). this has been widely regarded a s r e s p o n s e t o s a l i n i t y s t r e s s c o n d i t i o n . iii) soluble protein content: in present work a significant decrease in protein contents were found in green gram at both sodic concentrations. yurekli et al,.(2004) reported that nacl stress severely reduced leaf protein content in phaseolus vulgaris plant. similarly the decline in total soluble protein content was showed in lycopersicum esculentum, oryza sativa, vicia faba, amaranthus tricolor and brugiera purviflora plants under nacl stress. (parida and das, 2005; parvaiz and satyavati, 2008; wang and nill, 2000). yurekli et al,. (2004) reported that total soluble protein content significantly decreased in salt sensitive phaseolus vulgaris. iv) free proline accumulation rose with increasing level of soil salinity. the accumulated proline due to salt stress might have substituted for sugars as a respiratory substrate leading to a reduction in proline content at later stages of crop growth. the increase in proline content under salt stress is mainly due to the breakdown of proline-rich protein and fresh synthesis of proline amino acid. mishra and gupta (2005) also reported an increase in proline content of green gram ( p h a s e o l u s a u re u s ) u n d e r n a c l s t r e s s . reference ashraf m and harris pj. 2004. potential biochemical indicators of salinity tolerance in plants. plant sci., 166: 3-16. bates is, waldren rp and teare id. 1973 rapid determination of free proline for water stress studies. plant & soil 39, 205-208. chaudhry mr and ullah t. 1982. role of inorganic and organic amendments in the reclamation of saline 06 vol 3 (2), july 2012; 01-07 sodic soils. mrep pub. no. 124. epstein e & rains dw. 1987. advances in salt tolerance. plant & soil 99,17-29. gupta rk, bhumbla dr and abrol ip. 1985. release of exchangeable sodium from a sodic soil upon amendment application role of variable charge and exchangeable cation hydrolysis. soil sci., 139:312317. jaleel ca, manivannan p, sankar b et al. 2007. calcium chloride effects on salinity-induced oxidative stress, proline metabolism and indole alkaloid accumulation in catharanthus roseus.c r biologies, 330: 674-683. anwar m, patra dd, chand s, alpesh k, naqvi aa, khanuja sps. 2005. effect of organic manures and inorganic fertilizer on growth, herb and oil yield, nutrient accumulation, and oil quality of french basil., volume, issue, 1737 1746 munns r and termaat a .1986. whole plant responses to salinity. aust. j. plant physiol. 13, 143–160. parida ak and das ab .2005. salt tolerance and salinity effect on plants: a review. ecotoxicol. environ. saf., 60: 324-349. rathert g .1984. sucrose and starch content of plant parts as a possible indicator for salt tolerance of crops. aust. j. plant physiol. 11, 491–495. surendra mp and pota kb. 1978. the allelopathic potentials from root exudates from different ages of celosia argenta linn. natural acd. sci. letters, 1: 56-58. wang y and nil n. 2000. changes in chlorophyll, ribulose biphosphate carboxylase-oxygenase, glycine betaine content, photosynthesis and transpiration in amaranthus tricolor leaves during salt stress. j. hortic. sci. biotechnol., 75: 623-627. willis aj and yemm ew. 1954. new phytol. (in the press). yurekli f, porgali zb, turkan i. 2004. variations in abscisic acid, indole-3-acetic acid, gibberellic acid and zeatin concentrations in two bean species subjected to salt stress. acta biol. cracov. bot., 46: 201-212. zidan ma and al-zahrani hs. 1994. effect of nacl on germination seedling and some metabolic changes in sweet basil (ocimum basilicum). pak. j. sci. ind. res.37, 541-543 07 vol 3 (2), july 2012; 01-07 management of gdm and trans-generational prevention vol.12 (2), december 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 146 op e n ac c e s s f u l l l e n g t h a r t i c l e management of gestational diabetes mellitus with risk factors and trans-generational prevention sabiha gul1, kiran rafiq2,*, shagufta nesar3, syed waleed ahmed bokhari1, muhammad azhar mughal4, hafiza tania naveel5, muhammad idrees1 1department of pharmacology, faculty of pharmacy, hamdard university, karachi, pakistan. 2department of pharmaceutical chemistry, institute of pharmaceutical sciences, jinnah sindh medical university, karachi, pakistan. 3jinnah college of pharmacy, sohail university, karachi, pakistan. 4department of pharmacology & therapeutics, jinnah sindh medical university, karachi, pakistan. 5department of pharmacology, faculty of pharmacy, jinnah university for women, karachi, pakistan. a b s t r a c t background: gestational diabetes mellitus (gdm) has become a pervasive health issue of today’s era, leading to be a complicated disorder globally. it has also been proved to be highly accountable for causing an undesired impact on maternal health of patient as well as progeny. the high number of reported cases with consequent complications need appropriate medical care and timely attention. objectives: the present study was intended to display the real figure of a high rise in gdm in pakistan, with all involved parameters, to rectify the real factors either socioeconomic or domestic, accountable for the jeopardizing of disease. methodology: the study was conducted in different maternity hospitals of karachi, pakistan. association between gdm with age, family history, co-morbid complications, fetal complications and others were analyzed by pearson chi-square test using spss. results: more than 50% of participants belong to the age bracket of 31-45 years and 31.2% having a positive family history of diabetes. no known risk factor regarding gdm was present in 40.6% of women. gdm was observed prevalent and associated with poor health management of mother, and found to increase with elderly mothers (i.e. age of mother at the time of conception) in pakistan. conclusion: gdm should be considered as primary health care for the trans-generational prevention of diabetes and needs to be addressed as a public health issue in order to cure the mother and fetus during pregnancy and to prevent long term effects of this disease. keywords fetal complication, gestational, hyperlipidemia, insulin resistant, overweight, gdm. *address of correspondence kiranrafiq@hotmail.com article info. received: january 14, 2021 accepted: july 04, 2021 cite this article gul s, rafiq k, nesar s, bokhari swab, mughal ma, naveel ht, idrees m. management of gestational diabetes mellitus with risk factors and trans-generational prevention. rads j biol res appl sci. 2021; 12(2):146-153. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n pregnancy and parenthood are the state of emotional wellbeing and account for satisfaction and sense of worth. the creation of new life gives courage and happiness to the marital relations, however, during all the period of pregnancy mother is on the front line during all the state of creation1, 2. as a matter of fact, from the beginning of conception till birth and even after birth, a mother faces numerous experiences regarding health, socio-economic burdens, family pressures, health issues etc3. pregnancy is a cluster of physical and psychological alterations, and o r i g i n a l a r t i c l e management of gdm and trans-generational prevention vol.12 (2), december 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 147 interestingly both may affect the health of fetus and mother in either positive or negative manner4. according to a research, approximately 20% women face mood swings and anxiety disorders, while those having history of psychiatric illness are even more susceptible for such conditions that cannot be treated by any psychotropic medicine, since as fda has not approved any such therapy during pregnancy. the handling of pregnancy is a highly sensitive matter therefore, because of having teratogenic effects, neonatal toxicity, and risk of long-term neurobehavioral consequences, the medicines are carefully prescribed during pregnancy5, 6. besides all the possible complications during pregnancy, gestational diabetes itself is a complex matter7 that is highly accountable for making the pregnancy handling more difficult for mothers8, 9. simply, diabetes has been found to be highly associated with stress and depression with a fact that this anxiety disorder is also accompanied with the pregnancy either early or late. hence, various factors like social, economic and health related factors enhance the level of stress that may in turn be responsible for the onset of hyperglycemic state during the gestational period. the disorder is thus the state of glucose intolerance10. according to the data, the prevalence of hyperglycemia associated with pregnancy is found one in six globally, and among that 84% are gdm. the prevalence frequency of gdm is high among asian women than white women as in asia, the prevalence of gdm is increasing over years11, 12. the gdm has been found with the progression of type ii diabetes in the future. however, due to gdm, a transitory condition is induced by the metabolic stress of pregnancy which causes carbohydrate intolerance, and can be treated by diet control and by insulin therapy during the period of pregnancy. but the condition may or may not be complicated if it persists after childbirth. this abnormality in glucose metabolism may or may not be normalized after delivery, for a reason that there are 40% chances of gestational diabetics to develop into non-insulindependent diabetes mellitus (niddm) within fifteen years after childbirth13, 14. the physiological changes during pregnancy correlate the future programming about the metabolism and health in upcoming life, hence childbearing is believed as a window of maternal health in upcoming life. adequate health care guidance and complete nutrition develop an intrauterine environment that creates an impact on the growth of the baby, and for this purpose, women during pregnancy require medical care and guidance. however, in spite of all significant and essential cure, there is one unavoidable and unpredictable physiological change that happens during pregnancy i.e. hyperglycemia, referred to as gestational diabetes mellitus (gdm) or glucose intolerance. this may also occur due to hormonal imbalances, dysfunctioning of pancreatic β-cells responsible for releasing insulin, and distressing insulin sensitivity which normally works as an anabolic hormone, thus enhances glucose uptake by peripheral tissues and maintain glucose equilibrium by controlling the production of glucose from the liver, while antagonizing the adipose tissue to release lipids15-17. insulin resistance is the defined cause of gdm, as a state of decline in insulin concentrations, however, placental hormones trigger the insulin resistance to assure receiving sufficient nutrients by the fetus for vigorous growth. in such situation, the β-cells release more insulin to normalize the maternal blood glucose levels and to maintain glucose homeostasis. despite insulin resistance, the maternal βcells create a balance by increasing insulin synthesis and secretion, leading to maternal hyperglycemia18-22. gdm is in compliance with various genetic and environmental factors, as family history has a significant impact on the occurrence of diabetes, and as a highrisk factor for the development of gdm23-25. moreover, variations of dna for diabetes in gene polymorphisms is also responsible for transgenerational inheritance of obesity and glucose intolerance in the offspring from both mother and father. however, the types of genetic variation contribute to genotypic and phenotypic characteristics differently in different ethnicities such as asian women have the highest gdm rate26-28. this associated fact also identify the accountability of climate conditions for gdm, for e.g.: the extremity of weather and cold to hot temperature situations influence the physiologic mechanisms, hormonal balance, fats and lipid regulation, therefore the higher prevalence of gdm has been observed at high temperature regions29-32. along with regional and climate factors33-35 different socio-economic status, awareness about the disorder, and health care measures significantly distress the occurrence of gdm in a direct or indirect manner, which if not addressed, results management of gdm and trans-generational prevention vol.12 (2), december 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 148 in adverse maternal and neonatal outcomes that will increase drastically. this demands an early antenatal screening irrespective of the presence of risk factors for gdm and thus, it can be controlled by further promoting awareness of gdm and other pregnancy complications through educational sessions with dietician, diabitician or healthcare provider in order to prevent maternal and fetal complications. m a t e r i a l a n d m e t h o d s study design and study period: this cross-sectional observational study was conducted in different maternity/tertiary care hospitals and clinics of karachi, pakistan from october-december, 2020. all the participants were recruited from primary health care clinics and hospitals. study population and sample size: sample size calculations were performed using open epi software to determine the size of sample with 95% confidence interval. total 1000 pregnant women of fertile age (16-45 years old) were selected for this study. exclusion criteria for study population: women pre-diagnosed with type 1 diabetes were excluded from the study. study tool for the purpose of current study, a questionnaire was designed according the standard dipsi (diabetes in pregnancy study group india) criteria33 that was designed under the guidelines of world health organization (who) and international association of diabetes and pregnancy study groups (iadpsg) criteria for the diagnosis of gdm. accordingly, the study covered following aspects to analyze the frequency of diabetes among the pregnant women in pakistan along with the associated factors. the survey questionnaire comprises important objectives including: 1. socio-demographic details (four items) 2. risk factors for gdm 3. fetal complication associated with gdm 4. disorder association with gdm 5. treatment choice for gdm statistical analysis: data were analyzed using spss-20 and the results were expressed in the form of frequency and percentages. the association of gdm with different parameters was analyzed by pearson chi-square test. the prevalence of gdm was compared in different groups made with respect to age, ethnicity, trimester, family history, co-morbid disease, maternal complications and fetal complications. r e s u l t s the present study was designed and proceeded through a survey based questionnaire according to the dipsi criteria to analyze the frequency and related factors for gestational diabetes among pakistani women33. according to the information obtained from pakistan fertility and family planning survey (pffps) for analyzing the health issues of women during pregnancy, the majority of women faced diabetes during pregnancy, and in recent years, a high jeopardy has been observed. according to the reported data, the different big cities of pakistan like karachi, peshawar, and lahore have gdm cases ranging from 8% to 26% and interestingly, no regular data was reported from rural areas, that indicates the negligence about this disorder. during the analysis, different aspects influencing the gdm were included like age of mother matters for gdm. similarly, a significant correlation was recorded between age of pregnant woman and gdm. moreover, in women with age bracket of 16-30 years, 45.2% population had diabetes while, in women with age group from 31-45 years, 54.8% were observed having diabetes during pregnancy. the outcome revealed that the women of older age has more chances to suffer with gdm according the significant estimated p-value. as per weeks of gestation, 8.2% patients were in first trimester, 38.9% patients were in second trimester and 52.9% patients were in third trimester, with increased incidence noted in third trimester (table 1). the data showed that maternal age is a traditional risk factor for gestational diabetes mellitus (gdm), as according the american diabetes association the lowest cut off is 25 management of gdm and trans-generational prevention vol.12 (2), december 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 149 years, however no such harmony of the age is stated above which there is significantly high risk of gdm. regarding ethnicity in the gdm group, 12.0% participants were punjabi, 7.9% pukhtoon, 13.8% balochi, 6.9% sindhi speaking and 59.5% participants were mohajir. as ethnicity relates different demographic facts, cultural background, and socioeconomic differences and consequently affects the behavior and approach towards health and education, and sometimes gender discrimination is observed in some races. all this may also lead to cause unhealthy pregnancy with certain complications. the family history also found to matter as more cases were of patients who had diabetes, likewise heart diseases and thyroid were also reported with gdm. however, 40% population were reported with no family history but developed gdm. complicated cases having diabetes and thyroid along with heart problems were observed in 8% pregnant women. according to the study, the most accountable factor for gdm was family history. furthermore, pregnancy is complicated due to gdm in a variety of manner as like most of the infants of gdm mothers were overweight as compared to the non-gdm mothers (table 2). table1. socio-demographic data of women having gdm. socio-demographic characteristics age in years gestational diabetes mellitus (gdm) (n) gestational diabetes mellitus (gdm) (%) p-value age group of women 16-30 452 45.2% 0.000 31-45 548 54.8% ethnicity punjabi 120 12.0% >0.05 pukhtoon 78 7.8% balochi 138 13.8% sindhi 69 6.9% mohajir 595 59.5% trimesters first 61 6.1% >0.05 second 339 33.9% <0.0001 third 600 60.0% table 2. gdm and associated fetal / maternal complications. pregnancy-related fetal, and maternal complications pearson chi square value asymptotic significance family history of pregnant women 1.777 0.000 diabetes heart disease diabetes + thyroid + heart disease thyroid disease no risk factor fetal complication 17.283 0.000 large size (more than 9 pound) pre-term labor no complications pre mature birth small size down syndrome contd… management of gdm and trans-generational prevention vol.12 (2), december 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 150 maternal complications (comorbidity) 2.336 0.000 obesity hypertension + obesity abnormal blood lipids liver disease kidney disease hypertension depression thyroid disease no disease treatment choice for gdm 1.000 0.000 insulin metformin diet controlled only exercise glyburide d i s c u s s i o n pregnancy, a distinctive normal physiological condition, is a feeling of completion and happiness and a new life is created. however, normally during pregnancy the body passes through various changes including both physiological and psychological, and most of them normalize after delivery whereas, some account to induce long lasting effects throughout the life as like diabetes. as during the period of pregnancy, fetus development is completely reliant on mother, and to keep the growth of fetus and mother healthy, there are certain pregnancy induced metabolic changes considered as normal. but sometime these disturbances may lead to some pathological conditions like impaired glucose tolerance, leading to gdm34, 35. with the progression of gestation, fasting glucose drops off gradually with advancing gestation however, the mechanism is not well defined. but there are some accountable factors like increased plasma volume in the beginning of gestation, high feto-placental glucose utilization, and uptake of carbohydrates (glucose) by mother in second and third trimester. interestingly, more production of hepatic glucose regardless of a decline in fasting glucose in gdm leads to a simultaneous increase in fasting insulin and decrease in fasting glucose and this may aggravate with prolonged fasting36. the hepatic disturbance causes increased glucose concentrations in blood despite high insulin concentrations, and that sustains discrepancy between tissue insulin demands for glucose monitoring and the ability of the pancreatic β-cells to produce the required insulin accordingly. the increasing frequency of diabetes mellitus among pregnant women needs the development of preventive strategies. different socio-economic factors like cast, region, diet intake, stress affects the prevalence of this disorder. therefore, the present study was designed to identify and focus on the facts and figures with associated features of gdm among pakistani women. unfortunately, the disorder is unnoticed in pakistan due to unavailability of appropriate data and uncertain data collection, as the major part of population lives in rural areas. hence, cases form underprivileged areas are not reported or treated, and most pregnancies and deliveries are handled by inexperienced or non-institutional and uncertified experienced obstetricians at home or at local unregistered clinics and small hospitals settings, devoid of basic emergency facilities. further, lack of facilities for antenatal care and childbirth, and unwanted pregnancies in married women are more accountable in developing complications during management of gdm and trans-generational prevention vol.12 (2), december 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 151 pregnancy, leading to different disorders37. the reality states that the awareness and information related to reproductive health including infertility, abortions, and pregnancy handling is inadequate. in current study, gestational diabetes mellitus risk is higher in women with age group of 31-45 years, as compared to the women aged between 16-30 years, with a highly significant p-value. the high risk of gdm in old age is due to the fall in pancreatic beta cells performance with age, while in elderly age, the inadequate pancreatic beta cell response to stimulation develops more insulin resistance as compared to younger age. one of the strong factor of gdm is family history. statistical analysis showed highly significant ratio between family history and gdm. different socio-economic aspects like joint family system, load of responsibilities, and week physical health are most common in indo-pak that hinders to live healthy and happy life, and these are the important reasons of antenatal disorders. consequently, medicines are required to be prescribed to manage the glycemic level in pregnancy and to avoid possible unhealthy effects on both baby and mother before and after delivery38. the current study revealed that gdm is managed through diet control, exercise, insulin and medicine including metformin and glyburid, however the insulin therapy was found to be adopted by majority of the population whereas, second option was by medicine at high frequency and metformin was established at higher rate for treatment. management of gdm through diet control was observed relatively at low level may be due to the ground reality of pregnancy cravings, for fulfilling the nutritional need of the woman and fetus; a principal biological reality. the exercise and workout like aerobics, yoga, and walk are considered as a best remedy for curing or controlling the diabetes and cholesterol, and also help to reduce stress and depression normally, and in pregnancy significantly. but unfortunately the adaptation of this safe management was observed at very low level as gestating is a lengthy, tiring, and uncomfortable situation and sometime this laborious work induces laziness that does not allow the physical activity and women are more prone to rest39. the obesity in pregnancy are inter-related with gdm and according to royal college of obstetricians and gynecologists (rcog) moderate exercise like yoga and walk for 30-60min three times a week is safe during pregnancy with gdm, and it also significantly reduces the occurrence of gdm and gestational obesity, along with associated hypertension and preeclampsia40. the present study was also focused on the other health conditions and disorders already present and account for aggravating the hyperglycemia leading to the gdm. the obtained data revealed that obesity is a prominent cause of gdm at high frequency, and more prevalence and comorbidity was observed of obesity with hypertension. however, hypertension, lipid disorder, kidney and thyroid diseases were found to be co-morbid with diabetes in pregnancy at equal rate. nonetheless, the handling and care of pregnancy is essential right of every woman and immediate relations should be responsible and should play positive role for saving mother and baby both. firstly, proper meal plan and supplements are basic need for healthy pregnancy. healthy diet with low glycemic index help to reduce the risk of gdm and hypertension. various social and economical factors influence the diet and healthy environment for a pregnant women. unfortunately, in pakistan, high poverty rate, low income, and more dependence on one income hinders in providing ideal conditions during pregnancy. thus, the present study showed that majority of the population is deprived of healthy and good diet and furthermore no essential supplements were consumed after conception, and women are being consulted with gynecologists in second or third trimester. all these factors are cumulative cause to lead to high gdm rate in pakistani women. c o n c l u s i o n the outcomes of this study showed an increasing incidence of gdm at older age pregnancy because of more susceptibility towards hypertension and type 2 dm. additionally, different socio-economic factors were also observed to influence gdm rate including family income, lifestyle, stress etc. that directly or indirectly affects the health during and after the pregnancy. to overcome the associated risks during pregnancy, health care programs, plans, and campaigns should be arranged and healthcare professionals should work to give awareness about maternal and fetus health care. management of gdm and trans-generational prevention vol.12 (2), december 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 152 e t h i c a l a p p r o v a l all procedures were performed in accordance with the ethical principles, and the study approved by the faculty research committee, department of pharmacology, jinnah sindh medical university, karachi, (ref. no. jsmu/pharma/151/2020). after verbal consent, the data was collected and firmly preserved for the privacy of all information given by participants. ethical issues (informed consent, misconduct, data assembly, etc.) have been fully observed by the authors. c o n f l i c t s o f i n t e r e s t none. f u n d i n g s o u r c e none. a c k n o w l e d g m e n t s none. l i s t o f a b b r e v i a t i o n s dm diabetes mellitus gdm gestational diabetes mellitus ogtt oral glucose tolerance test spss statistical package for social sciences r e f e r e n c e s 1. deave t, johnson d, ingram j. transition to parenthood: the needs of parents in pregnancy and early parenthood. bmc pregnancy childbirth. 2008; 8:30-9. 2. mcleish j, redshaw m. peer support during pregnancy and early parenthood: a qualitative study of models and perceptions. bmc pregnancy childbirth. 2015; 15:257-63. 3. stoyles bj. the value of pregnancy 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occasion fleeting; experience fallacious, and judgment difficult. the physician must not only be prepared to do what is right himself, but also to make the patient, the attendants, and externals cooperate." thus, hippocrates, the father of modern medicine, epitomized the art of the physician. "to do what is right" in terms of quality control and prevention has far-reaching implications, not only for physician, but for "the patient, the attendants, and externals." in keeping with this sublime philosophy, this article focuses on the assurance of quality as a part of regulated pharma industry. the pharmaceutical quality control laboratory serves one of the most important functions in pharmaceutical production and control. a significant portion of the cgmp regulations (21 cfr 211) pertain to the quality control laboratory and product testing. similar concepts apply to bulk drugs. quality assurance quality assurance covers all activities from design, development, production, installation, servicing and documentation. this introduced the rules: "fit for purpose" and "do it right the first time". it includes the regulation of the quality of raw materials, assemblies, products and components; services related to production; and management, production, and inspection processes. one of the most widely used paradigms for qa management is the pdca (plan-do-checkact) approach, also known as the shewhart cycle quality control is a process employed to ensure a certain level of quality in a product or service. it may include whatever actions a business deems necessary to provide for the control and verification of certain characteristics of a product or service. the basic goal of quality control is to ensure that the products, services, or processes provided meet specific requirements and are dependable, satisfactory, and fiscally sound. essentially, quality control involves the examination of a product, service, or process for certain minimum levels of quality. the goal of a quality control team is to identify products or services that do not meet a company's specified standards of quality. if a problem is identified, the job of a quality control team or professional may involve stopping production temporarily. depending on the particular service or product, as well as the type of problem identified, production or implementation may not cease entirely. usually, it is not the job of a quality control team or professional to correct quality issues. typically, other individuals are involved in the process of discovering the cause'of quality issues and fixing them. once such problems are overcome, the product, service, or process continues production or implementation as usual. quality control can cover not just products, services, and processes, but also people. employees are an important part of any company. if a company has employees that don't have adequate skills or training, have trouble understanding directions, or are misinformed, quality may be severely diminished. when quality control is considered in terms of human beings, it concerns correctable issues. however, it should not be confused with human resource issues. often, quality control is confused with quality assurance. though the two are very similar, there pharmaceutical quality assurance & control ghulam sarwar1 and m. shakil siddiqui1 1faculty of pharmacy jinnah university for women karachi pakistan *corresponding author: g-sarwaar@hotmail.com 25 vol 3 (2), july 2012; 21-23 are some basic differences. quality control is concerned with the product, while quality assurance is process-oriented. even with such a clear-cut difference defined, identifying the differences between the two can be hard. basically, quality control involves evaluating a product, activity, process, or service. by contrast, quality assurance is designed to make sure processes are sufficient to meet objectives. simply put, quality assurance ensures a product or service is manufactured, implemented, created, or produced in the right way; while quality control evaluates whether or not the end result is satisfactory. quality control: the term quality control refers to the sum of all procedures undertaken to ensure the identity and purity of a particular pharmaceutical. such procedures may range from the performance of simple chemical experiments which determine the identity and s c r e e n i n g f o r t h e p r e s e n c e o f p a r t i c u l a r pharmaceutical substance (thin layer chromatography, infrared spectroscopy, etc.), to more complicated requirements of pharmacopoeial monographs. activities extend to the area of quality control laboratories (good laboratory management practices, models, e.g. for certificate of analysis and lists of laboratory equipment, and an external assessment scheme. it is defined as all measures to be taken during manufacturing designed to ensure the uniform outputs of pharmaceutical products conforming to established specifications of identity, strength, purity and other characteristics such as potency, safety, and toxicity. quality assurance: qa is defined as a procedure or set of procedures intended to ensure that a product or service under development (before work is complete, as opposed to afterwards) meets specified requirements. qa is sometimes expressed together with qc as a single expression, quality assurance and control (qa/qc). quality assurance, or qa for short, is the activity of providing evidence needed to establish quality in work, and that activities that require good quality are being performed effectively. all those planned or systematic actions necessary to provide enough confidence that a product or service will satisfy the given requirements for quality. for products, quality assurance is a part and consistent pair of quality management offering supposedly factbased external confidence to customers and other stakeholders that a product meets needs, expectations, and other requirements. qa claims to assure the existence and effectiveness of procedures that attempt to make sure in advance that the expected levels of quality will be reached. failure testing a valuable process to perform on a whole consumer product is failure testing, the operation of a product until it fails, often under stresses such as increasing vibration, temperature and humidity. this exposes many unanticipated weaknesses in a product, and the data is used to drive engineering and manufacturing process improvements. often quite simple changes can dramatically improve product service, such as changing to mould-resistant paint or adding lock-washer placement to the training for new assembly personnel. statistical control many organizations use statistical process control to bring the organization to six sigma levels of quality, in other words, so that the likelihood of an unexpected failure is confined to six standard deviations on the normal distribution. this probability is less than four one-millionths. items controlled often include clerical tasks such as order-entry as well as conventional manufacturing tasks. traditional statistical process controls in manufacturing operations usually proceed by randomly sampling and testing a fraction of the output. variances of critical tolerances are continuously tracked, and manufacturing processes are corrected before bad parts can be produced. total quality control total quality control is the most necessary inspection 26 vol 3 (2), july 2012; 21-23 control of all in cases where, despite statistical quality control techniques or quality improvements implemented, sales decrease. the major problem which leads to a decrease in sales was that the specifications did not include the most important factor, "what the customer required". the major characteristics, ignored during the search to improve manufacture and overall business performance were: 1. reliability 2. maintainability 3. safety as the most important factor had been ignored, a few refinements had to be introduced: 1. marketing had to carry out their work properly a n d d e f i n e t h e c u s t o m e r ' s s p e c i f i c a t i o n s . 2. specifications had to be defined to conform to these requirements. 3. conformance to specifications i.e. drawings, standards and other relevant documents, were introduced during manufacturing, planning and control. 4. management had to confirm all operators are equal to the work imposed on them and holidays, celebrations and disputes did not affect any of the quality levels. 5. inspections and tests were carried out, and all components and materials, bought in or otherwise, conformed to the specifications, and the measuring equipment was accurate, this is the responsibility of the qa/qc department. 6. any complaints received from the customers were satisfactorily dealt with in a timely manner. 7. feedback from the user/customer is used to review designs. 8. consistent data recording and assessment and documentation integrity. packaging material testing where a drug product is presented in an inadequate package, the entire effort put into the initial research, product development and manufacturing control is wasted. drug quality is directly dependent on packaging quality. in many cases (e.g., metereddose aerosols), packaging quality is critical to the overall performance and effectiveness of the drug product. faults in the packaging and labelling of a drug product continue to be a major cause of drug recalls. packaging materials are required to be tested or examined prior to their use in a packaging operation to ensure that materials of acceptable quality are used in the packaging of drugs required to be tested or examined prior to their use in a packaging operation to ensure that materials of acceptable quality are used in the packaging of drugs 27 vol 3 (2), july 2012; 21-23 effect of different sowing methods on wheat vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 33 op e n ac c e s s f u l l l e n g t h a r t i c l e effect of different sowing methods on the growth and yield of wheat (triticum aestivum l.) shafqat ali bhatti1, muhammad nawaz kandhro1, muhammad saleem chang2,*, jay kumar sootaher3,*, kashif ali buriro1, nadia mangrio4, piar ali shar3, mukesh kumar soothar5, mohsin wadho1 1department of agronomy, sindh agriculture university, tandojam, sindh, pakistan. 2department of agronomy, subcampus umerkot, sindh agriculture university, tandojam, sindh, pakistan. 3department of plant breeding and genetics, sindh agriculture university, tandojam, sindh, pakistan. 4sugar research institute, tandojam, sindh, pakistan. 5department of soil science, sindh agriculture university, tandojam, sindh, pakistan. a b s t r a c t background: an appropriate choice of a planting method is very vital for the fixation of seeds on their position to grow and develop well without any obstacles. in pakistan, drilling and broadcast sowing methods are largely applied, but the most usable method is broadcast on a large land area. however, drilling method is better than broadcast method for many reasons as crop cultivation is greatly impacted by the seed rate. drilling method needs lower seed rate and still gives good plant population. objectives: the aim of this work was to conduct the assessment of different sowing methods to observe their effects on the growth and yield of wheat to highlight the best method that can be utilized for the improvement and development of agriculture in the future. methodology: this research work was carried out on the field of agronomy section, ari, tandojam sindh, pakistan (gps coordinates 25° 25’ 35.58” n and 68° 31’ 29.568” e) for assessing the effect of different sowing methods on wheat yield and other traits during 2017-2018. the research was carried out using three replications in split plot design with treatments in which factor a consisted of sowing methods and factor b comprised of varieties. results: the collected data on parameters indicated that maximum tillers (385m-2), spike length (14cm), spikelets spike-1 (21), grains spike-1 (58), seed index (77g), biological yield (11822kg.ha-1) and grain yield (7751kg.ha-1) were recorded in triple dwarf (td-1) wheat variety of sindh. however, the maximum plant height (87cm) was recorded in sindhu. in case of drilling sowing methods, maximum tillers, plant height, spike length, spikelets spike-1, grains spike-1, seed index, biological yield and grain yield were found to be of 340m2, 92cm, 13cm, 18, 48, 63g, 10132kg.ha-1 and 6691kg.ha-1, respectively. however, low values for all these parameters were recorded under the broadcast method. conclusion: it was concluded from present findings that td-1 x drilling sowing method remained the most suitable interaction for getting more yield of wheat than other varieties and sowing methods. keywords broadcasting method, drilling, growth, sowing method, wheat, yield. *address of correspondence jaykumar3030@gmail.com; mschang@sau.edu.pk article info. received: july 06, 2021 accepted: march 24, 2022 cite this article bhatii as, kandhro mn, chang ms, sootaher jk, buriro ka, mangrio n, shar pa, soothar mk, wadho m. effect of different sowing methods on the growth and yield of wheat (triticum aestivum l.). 2022; 13(1):33-42. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n wheat is the world's most vital cereal without which no one can live. according to an estimates, 36% of the world's population consumes wheat. to be used as a staple food, the crop is grown in more than 40 nations on the planet1. o r i g i n a l a r t i c l e effect of different sowing methods on wheat vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 34 in comparison with other higher grain producing countries, pakistan is far behind in average yield competition. out of the total area under cultivation, 37% is occupied by this crop and it shares 70% in the rabi crops in pakistan2. wheat demand is mostly fulfilled by punjab and sindh in pakistan, with punjab ranking as the first in wheat production, followed by sindh. crop management factors such as sowing methods, seed rate, and genotypes, all have an impact3, 4. low production is caused by a wide range of reasons such as soil fertility, proper irrigation, resistance to weeds and sowing methods. choice of planting method is vital for fixation of seeds in their position to germinate for better growth and development in the future without any obstacles. according to the pakistan bureau of statistics, the most widely eaten crop in pakistan is wheat, which is produced in 25979.4 million tons over an area of 9199.3 million hectares5. seed rate is the only factor which governs planting density by affecting crop growth, nutrient uptake, tillering capacity and finally grain yield, through plant-to-plant competition. many researchers advocate drilling plantation as the seeds are phenotypically similar and can reach appropriate depths owing to this method6, 7. in the modern world of science, new techniques have been developed to increase the yield per unit area by sowing the wheat seed after utilizing different approaches such as bed planting introduced by turkey. further parameters include proper germination-free from the attack of weeds, and other biotic/abiotic constraints recommended for good wheat cultivation. cimmyt (internacional de mejoramiento de maíz y trigo) in mexico also used this bed method for the cultivation of maize and wheat and gave good results. after these experiments, many benefits have been notified regarding wheat cultivation on beds. developed countries put this method into practice and have greatly succeeded in wheat production8. apart from sowing methods, sowing time, and sowing of different desirable and high yielding cultivars are also of great importance because they also contribute significantly to better cultivation of the wheat crop. in some regions, crops are early sown while in others, they are sown late, but the most suitable time is early sowing especially for good cultivation of wheat crop. due to early sowing, uniform seedling emergence with vigorous growth and development take place. poudel et al. suggested that crop production and productivity must be augmented to feed the expected population of 9.1 billion up to 2050. henceforth, the research was planned to analyze the effects of a wide range of seed sowing methods on the growth and yield of wheat so that the best method can be utilized for the improvement and development of agriculture in the future. m a t e r i a l s a n d m e t h o d s experimental site this research work was carried out on the field of agronomy section, ari, tandojam sindh, pakistan (gps coordinates 25° 25’ 35.58” n and 68° 31’ 29.568” e) for assessing the effect of different sowing methods on wheat yield and other traits during 2017-2018. the bulk density ranged from 1.30 to 1.37g.cm-3 in the soil profile. the available water holding capacity of the soil was 85.2mm to 60cm-1. the water quality classification was determined as c2s1 according to the usa salinity laboratory graph system (ec=0.625dsm-1, sar=0.61). experimental design factorial split plot design was practiced with three replications. the details of treatments and factors are as follows: treatments = (two factors a and b) factor a: sowing methods (02) m1 = drilling m2 = broadcasting factor b: varieties (03) v1 = td-1 v2 = imdad-2005 v3 = sindhu land preparation the land was prepared by applying two dry ploughs. after the soaking dose, when the soil reached correct wetness level, two cultivator ploughs were applied to the seedbed thoroughly. three different kinds of chemical fertilizers were applied including urea, diammonium phosphate (dap) and sulphate of potash (sop) for the requirement of npk (nitrogen, phosphorus, potassium) (120:75:56kg.h-1) in which diammonium phosphate (dap) and sulphate of potash (sop) and one fourth of urea were effect of different sowing methods on wheat vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 35 applied as basel dose at the time of sowing with seed cum fertilizer drill. the remaining dose of nitrogen was applied in three splits, first at the seedling or emergence stage, second at tillering, and third at the dough stage of wheat crop for proper growth and development. cultural practices were also applied for different purposes like freeing the crop from weed and insect attack. to capture agronomic observations, five plants were chosen at random and labeled from each plot. manual calculation of values was done for the number of tillers, spikelets grains, and plant height (by ruler), however, the digital calculation of electric balance was used for seed index, biological yield, and grain yield. formula for biological yield (kg.ha-1) 𝐵𝑌 (𝑘𝑔. ℎ𝑎−1) = 𝐵𝑌 (𝑝𝑙𝑜𝑡 −1) 𝑃𝑙𝑜𝑡 𝑠𝑖𝑧𝑒 (𝑚−1) 𝑥 10,000 formula for grain yield (kg.ha-1) 𝐺𝑌 (𝑘𝑔. ℎ𝑎−1) = 𝐺𝑌 (𝑝𝑙𝑜𝑡−1) 𝑃𝑙𝑜𝑡 𝑠𝑖𝑧𝑒 (𝑚−1) 𝑥 10,000 statistical analysis anova and the least significant difference test was applied according to the methods developed by torrie et al., 19808 to compare treatments superiority. r e s u l t s a n d d i s c u s s i o n tillers (m-2) hexaploid ranks globally as a highly important staple food consumed all over the world at a time. results showed variable and desirable differences of wheat genotypes for tillers (m-2) at probability level p < 0.05 under two different seed sowing methods (table 1). the results of different wheat varieties indicated that the maximum tillers (385m-2) were recorded by td-1 variety, followed by imdad-2005 (336m-2), respectively, while the minimum tillers (280m-2) were observed in the sindhu variety. for sowing methods, maximum tillers (340m-2) were seen for the drilling sowing method and minimum tillers (327m-2) were noted in the broadcasting sowing method. nevertheless, the highest and the lowest values for this attribute (394m-2 and 275m-2) were articulated by variety x sowing methods under the interaction of td-1 x drilling-sowing method, and sindhu x broadcasting sowing method (table 1)9. sikander et al., 2003 demonstrated the same effects of sowing methods for wheat cultivars in his experiment. it was stated by sikander et al., 200310 that plant growth is affected by proper and improper method of seed sowing. they further noted that the highest plant emergence was observed in the drilling method. flat and bed sowing methods for wheat were also tested by mehmood et al., 201311 and it was concluded that triple-row bed-sowing method was the most suitable for proper seed setting. it was concluded by bakhsh et al., 202012 that the drilling method was more effective for high production of tillers which resulted in more grain yield per plant. table 1. tillers (m-2) of wheat genotypes under different sowing methods. varieties sowing methods mean for varieties drilling broadcasting td-1 394 375 385 a imdad-2005 342 331 336 b sindhu 284 275 280 c mean for methods 340 a 327 b varieties sowing method varieties x sowing method s.e ± 3.3015 2.6957 4.6690 l.s.d 0.05% 7.3562 6.0063 10.403 t1 s u b -c h a n n e l t6 s u b -c h a n n e l s u b c h a n n e l t3 t2 t5 t4 t3 t4 t1 t4 t3 t5 t5 t2 t6 t2 effect of different sowing methods on wheat vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 36 table 2. plant height (cm) of wheat genotypes under different sowing methods. varieties sowing method means of varieties drilling broadcasting td-1 76 67 78 c imdad-2005 88 79 84 b sindhu 98 90 87 a mean for methods 92 a 74 b varieties sowing method varieties x sowing method s.e ± 1.2168 0.9935 1.7208 l.s.d 0.05% 2.7111 2.2136 3.8341 table 3. spike length (cm) of wheat genotypes under different sowing methods. varieties sowing methods means of varieties drilling broadcasting td-1 16 12 14.a imdad-2005 13 10 11 b sindhu 10 9 10 c mean for methods 13 a 10 b varieties sowing method varieties x sowing method s.e ± 0.6824 0.5572 0.9651 l.s.d 0.05% 1.5205 1.2415 2.1503 plant height (cm) yield development and efficiency may contrast under various planting strategies and planting densities. the consequences of various wheat cultivars showed that the maximum plant height (87cm) was recorded for sindhu, followed by imdad-2005 (84cm) whereas, the minimum plant height (78cm) was verified in td-1. in case of sowing methods, the longest and shortest plants (92cm and 74cm) were found at the drilling and broadcasting method. on the basis of interaction between varieties x sowing methods, long and short plants (98cm and 67cm) were identified in the interaction of sindhu x drilling sowing and td-1 x broadcasting sowing method13 (table 2). dagash et al., 2014 likewise determined the impact of plant height in drilling practice when contrasted with broadcast approach. these outcomes were in agreement with dagash et al., 201414. it was also reported by twizerimana et al., 202015 that for more grain yield, it is very important to use different kinds of sowing methods so that the high yield values can be identified. the drilling method is advantageous, although it has low population but it produces high production. therefore, özberk et al., 200916, and tapley et al., 201317 suggested that low plant population is only created by less seed rate, which reduced the competition between plant-to-plant for survival resources. spike length (cm) awoke et al., 201718 and kiliç et al., 201018, 19 reported that crop growth and development are considerably affected by the planting method due to the seed’s placement in their depth. the results of different wheat varieties indicated that td-1 had the longest spikes (14cm), followed by imdad2005 (11cm), with sindhu having the shortest spikes (10cm). in sowing methods, the maximum and minimum spike length (13cm and 10cm) were detected in the drilling and broadcasting sowing method. nevertheless, the collaboration revealed that the longest and shortest spikes (16cm and 9cm) were measured for td-1 x drilling sowing and sindhu x broadcasting sowing method (table 3). our findings matched those of attaullah et al., 200720, who conducted an experiment with wheat to determine the effect of different sowing methods on wheat vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 37 effects of various planting approaches on various agronomic characteristics. our findings differed from those of meleha et al., 202021, as the broadcasting approach for seed planting performed better than the other methods. belete et al., 201822 applied different planting methods, which gave good results for most of the traits, but broadcast methods became valuable in their study. for this support, according to zohaib et al., 201923, and muhammad et al., 200924, sowing methods impact various varieties of the same species in different ways, with some types responding better to drilling and others to broadcasting. spikelets spike-1 because of the cultivation of wheat in diversified conditions, spikelets spike-1 reacts variably with the practices of crop management. the findings of several wheat genotypes revealed that td-1 and sindhu had the highest and lowest number of spikelets spike-1 (21 and 12), respectively (table 4). in case of sowing methods, the maximum spikelets spike-1 (18) was expressed in the drilling-sowing method and minimum spikelets spike-1 (15) was observed with broadcasting-sowing method. nonetheless, the highest and the lowest values for this character (23 and 11) were articulated by variety x sowing methods in td-1 x drilling sowing method and sindhu x broadcasting sowing method (table 4). as it has been discussed that drilling methods require low seed rate, which decreases plant population and brings about more spikelets on a single spike and high seed index, because plants show less competition between themselves for available resources. comparable outcomes were likewise found by chaudhary et al., 201525. according poudel et al., 202026, wheat yield and water productivity can be increased by sowing on beds with a managed seed rate. as per meleha et al., 202027, there are a variety of causes for low wheat yield, including planting approaches. so, it is very important to apply a suitable sowing method which must be beneficial for the increase of yield. inappropriate planting method owing to seed rate and crop management always gives low yield28. table 4. spikelets spike-1 of wheat genotypes under different sowing methods. varieties sowing methods means of varieties drilling broadcasting td-1 23 19 21 a imdad-2005 16 15 15 b sindhu 14 11 12 c mean for methods 18 a 15 b varieties sowing method varieties x sowing method s.e ± 0.4240 0.3462 0.5996 l.s.d 0.05% 0.9447 0.7714 1.3361 table 5. grains spike-1 of wheat genotypes under different sowing methods. varieties sowing methods means of varieties drilling broadcasting td-1 62 55 58 a imdad-2005 47 43 45 b sindhu 39 35 37 c mean for methods 48 a 45 b varieties sowing method varieties x sowing method s.e ± 1.0971 0.8958 1.5515 l.s.d 0.05% 2.4444 1.9959 3.4569 effect of different sowing methods on wheat vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 38 grains spike-1 the application of drilling sowing method provides many advantages like double cropping, controlled soil moisture, and narrow row spacing. the findings of wheat varieties revealed that td-1 seemed to have the highest number of grains spike-1 (58), followed by imdad-2005 (45), and sindhu could have the lowest number of grains spike-1 (37) (table 5). for sowing methods, the maximum grains spike-1 (48) was observed with the drilling-sowing method and minimum grains spike-1 (45) was counted with the broadcasting sowing method. td-1 x drilling sowing method and sindhu x broadcasting sowing method, however, produced the highest and lowest values for this trait (62 and 35) (table 5). naresh et al., 201329 succeeded in obtaining good results from their experiment under different sowing methods. resource availability including sunlight capture, moisture and nutrient availability are enhanced by a proper seed sowing method, which leads to ultimately good root system for its initial growth and development from a seedling to a mature plant. for increasing the number of grains in a single spike, 30. debebe et al., 201630 recommended the drilling method and suggested that wheat production can also be increased by different sowing methods such as ridge sowing method, which included more seed rate. this method was very helpful to increase the grain yield and to improve the productivity of wheat bread. seed index (1000 grain weight-g) unsuitable sowing methods lead to unproductive crops due to the lodging, disease and pest attack resulting in lower yield. the results of different wheat varieties indicated that the heaviest seeds (77g) were recorded in td-1, followed by imdad-2005 (60g) (table 6). whereas the lightest seeds (45g) were weighed for sindh. in case of sowing methods, the maximum seed index (63g) was observed with drilling sowing method and minimum seed index (59g) was weighed with the broadcasting sowing method (table 6). though, the interaction between varieties x sowing methods confirmed that heaviest and lightest seeds (81g and 43g) were obtained by td-1 x drilling sowing method and sindhu x broadcasting sowing method, respectively. the drilling method led to more growth and grain yield in the experiment of singh et al., 200931. ehsanullah et al., 201732 investigated crop performance using several seed sowing methods and came up with some impressive end results in favor of the drilling approach. it was suggested by different scientists to use the most appropriate seed sowing methods which play a vital role for the improvement of crop management and crop productivity. table 6. seed index (g) of wheat genotypes under different sowing methods. varieties sowing methods mean for varieties drilling broadcasting td-1 81 73 77 a imdad-2005 65 56 60 b sindhu 47 43 45 c mean for methods 63 a 59 b varieties sowing method varieties x sowing method s.e ± 1.2622 1.0306 1.7850 l.s.d 0.05% 2.8124 2.2963 3.9773 effect of different sowing methods on wheat vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 39 table 7. biological yield (kg.ha-1) of wheat genotypes under different sowing methods. varieties sowing method means of varieties drilling broadcasting td-1 12477 11166 11822 a imdad-2005 9392 8996 9194 b sindhu 8527 7767 8147 c mean for methods 10132 a 9310 b varieties sowing method varieties x sowing method s.e ± 215.19 175.70 304.33 l.s.d 0.05% 479.48 391.49 678.08 table 8. grain yield (kg.ha-1) of wheat genotypes under different sowing methods. varieties sowing methods means of varieties drilling broadcasting td-1 8283 7219 7751 a imdad-2005 6363 6303 6333 b sindhu 5426 4482 4954 c mean for methods 6691 a 6001 b varieties sowing method varieties x sowing method s.e ± 98.810 121.02 171.14 l.s.d 0.05% 220.16 269.64 381.33 biological yield (kg.ha-1) according to the findings of some wheat varieties, td-1 have had the highest biological yield (11822kg.ha-1), followed by imdad-2005 (9194kg.ha-1). sindhu, on the other hand, had the lowest biological yield (8147kg.ha-1). under sowing methods, the maximum and minimum biological yield (10132kg.ha-1 and 9310kg.ha-1) was observed with drilling method and broadcasting method. yet, the highest and the lowest values for these characters (12477kg.ha-1 and 7767kg.ha-1) were demonstrated by td1 x drilling sowing method and sindhu x broadcasting sowing method (table 7). a suitable sowing method not only improves utilization of solar energy through the space between plant to plant, but also sets up a proper position of the crop in the soil33, 34. these results were in conformity with krezel et al., 201335 in favor of the drilling method. in the sowing method experiment, ali et al., 201436 discovered the same findings. umed et al., 200937 additionally announced the exhibition of bread wheat assortments under various line separations including drilling technique. low yield due to broadcast method was also presented by hayat et al., 201738. grain yield (kg.ha-1) the td-1 and imdad-2005 genotypes generated the best grain production in terms of wheat genotype efficiency (7751 and 6333). sindhu had the lowest grain yield (4954kg.ha-1). in case of sowing methods, the drilling method (6691kg.ha-1) produced more grain yield than the broadcast planting method (6001kg.ha-1). however, the interaction between varieties x sowing methods articulated that maximum and minimum grain yield (8283kg.ha-1 and 4482kg.ha-1) were observed under the variety td-1 x drilling sowing method and sindhu x broadcasting sowing method (table 8). hossain et al., 201139 also found some good outcomes from their research work and concluded while experimenting with the genotypes of wheat. increase in the yield was also supported by harishankar et al., 201740 and iqbal et al., 202041, informing about the parameters contributing to the rise in yield. the use of various crop sowing methods also aids in the crop yield and in conserving water by optimizing tillering capability, soil moisture content, and nutrient availability42. drilling effect of different sowing methods on wheat vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 40 methods were shown to be beneficial for cultural practices in crops and agricultural development by kabesh et al., 200943. c o n c l u s i o n according to the data, wheat growth and yield were positively influenced by cultivars and planting methods. variety td-1 and drilling sowing method were likewise shown to have performed best in both the growth and yield metrics. the current data articulated that the variety of td1 x drilling sowing method is a good combination for generating the most wheat yield from other varieties and sowing methods. finally, it was recommended that the drilling method was very good in showing better results and it would be very beneficial for farmers in the upcoming years. c o n f l i c t s o f i n t e r e s t all authors have no conflict of interest. f u n d i n g s o u r c e they study was funded by agronomy section of agriculture research institute, tandojam, sindh. a c k n o w l e d g e m e n t s we are very grateful to agronomy section of agriculture research institute, tandojam for supporting us in this research study. l i s t o f a b b r e v i a t i o n s by biological yield dap diammonium phosphate gy grain yield npk nitrogen, phosphorus, potassium sop sulphate of potash td 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2016; 15(2):154-162. 31. singh y, humphreys e, kukal ss, kaur bsa, thaman s, prashar a, timsina syj, dhillon s, kaur n, smith dj, gajri pr. crop performance in permanent raised bed rice-wheat cropping system in punjab, india. field crops res. 2009; 110:1-20. 32. ehsanullah, shahzad ma, anjum sa, zohaib a, ishfaq m, warraich ea. effect of different sowing methods and planting densities on growth, yield, fiber quality and economic efficacy of cotton. pak j agric res. 2017; 30(1):212-19. 33. rasool t, zohaib a, ahmad r, abbas t, tabassum t, nadeem ma. forage yield and quality in pearl milletsesbania intercropping system under various geometrical patterns. pak j agric res. 2017; 30(1):75-84. 34. ali ma, ali m, sattar m, ali l. sowing date effect on yield of different wheat varieties. j agri res. 2010; 48(2):157-62. effect of different sowing methods on wheat vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 42 35. krezel r, sobkowicz p. the effect of sowing rates and methods on winter triticale grown on light soil. j sci. 2013; 11(3):69-78. 36. ali ma, ali m, din qm. determination of grain yield of different wheat varieties as influenced by planting dates in agroecological conditions of vehar. pak j life soc sc. 2014; 2(1):5-8. 37. umed as, rahman mu, odhano ea, gul s, tareen aq. effects of sowing method and seed rate on growth and yield of wheat. world j agri sci. 2009; 5(2):15962. 38. hayat s, usman k, aminmt. response of zero tillage wheat to row spacing and nitrogen application methods. sarhad j. agri. 2017; 33(4):680-7. 39. hossain m, islam mk, sufian ma, meisner ca, islam ms. effect of planting method and nitrogen levels on the yield and yield attributes of wheat. j biol sci. 2011; 14:127-30. 40. harishankar gpp, tomar gs. growth and yield of wheat (triticum aestivum l.) as influenced by methods of sowing and seed rates. prog res int j. 2017; 12:401-3. 41. iqbal j, zohaib a, hussain m, bashir a, hamza m, muzaffer w, latif mt, faisal n. effect of seed rate on yield components and grain yield of ridge sown wheat varieties. pak j agric res. 2020; 33(3):508-15. 42. poudel pb, poudel mr. heat stress effects and tolerance in wheat: a review. j biol today’s world. 2020; 9(4):217-23. 43. kabesh mo, el-kramany mf, el-naggar gashm, gehan, shb. effects of sowing methods and some bioorganic fertilizations treatments on yield and yield components of wheat. res j agri bio sci. 2009; 5:97102. healthcare waste management in hyderabad sindh vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 175 op e n ac c e s s f u l l l e n g t h a r t i c l e healthcare waste management (hwm) in hyderabad (sindh): a comparison of government, semi-/nongovernment, and private hospitals sehreen moorat*, hiba pervaiz, sasuee rajpar liaquat university of medical and health sciences, university in jamshoro, sindh, pakistan. a b s t r a c t background: population expansion and greater accessibility to healthcare have increased the production of medical waste. along, it is a crucial concern of the government and the healthcare industry for the environment and public safety. hospitals, dentist offices, clinics, clinical laboratories, and other facilities providing healthcare are among the facilities that produce medical waste. when it comes to handling hospital wastes, developing countries are resource-constrained due to which chances of the spread of infectious diseases increase. objective: the main purpose of this study was to determine the percentage of hospitals that follow healthcare waste management practices. along with how well-versed healthcare providers were in terms of awareness, knowledge, and practices. methodology: a survey-based study was performed to obtain healthcare waste disposal data from government, semi-government and private hospitals in the region of sindh. the obtained survey data was fed to the spss in the form of statistics to observe the meaningful results related to healthcare waste management. results: the average waste generation maximum at private hospitals is found to be 71.2%, in government hospitals it is 25% and in semi-govt/ngo hospitals it is 3.8%. whereas, it is also observed that more waste management techniques were followed at private hospitals than at government or semi-government. conclusion: it is necessary to spread awareness among healthcare practitioners and medical staff on healthcare waste management. also, the guidelines and policies should be strictly followed to protect the environment and public health. keywords healthcare waste management, medical waste, survey, spss, waste management techniques. *address of correspondence reen_gopang@yahoo.com article info. received: december 06, 2021 accepted: october 21, 2022 cite this article: moorat s, pervaiz h, rajpar s. healthcare waste management (hwm) in hyderabad (sindh): a comparison of government, semi-/non-government, and private hospitals. 2022; 13(2):175-182. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n nowadays, the healthcare industry is one of the fastestgrowing industries in the world1. the expenditure on health is increased globally and remains rising every year. according to the who report worldwide around 7.8 trillion dollars were spent on health which is 10% of the world's gross domestic product (gdp)2. globally the population is estimated to increase by around 8.6 billion by 2020 and 9.8 billion by 20503. an increasing population and diseases day by day can also increase the expenses of health. therefore, this increasing population would need more healthcare involvements which ultimately increases the generation of healthcare waste. o r i g i n a l a r t i c l e healthcare waste management in hyderabad sindh vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 176 as the diseases are continued to grow, they affect the population aged above 50 years4. the vulnerability increases if the person has already any chronic condition. an increase in the disease increases the cost of multiple morbid conditions (mmcs)5. mccs not only increase the use of specialist care, primary care, and elevated use of medications and hospital admissions. these frequent visits to the hospital increase the use of healthcare disposal items which are extensively used in the healthcare industry. contingent characterization of waste has no importance due to waste heterogeneity in nature. therefore, the focus is on infectious waste and its proper disposal and ways to treat that waste. infectious waste and toxic waste require special handling to avoid the spread of diseases in the environment6. there are numerous types of waste reported in the literature, among which a few are listed below: • the general category includes paper, plastics, and disposable food. • pathological waste includes body tissues, organs, limbs, and body fluids. • chemical waste includes; disinfectants, swabs, and heavy elements. • infectious wastes include waste from patients with infections, natural body excretions, and waste contaminated with blood. • genotoxic wastes include medications used for cancer. • sharps include blades, capillary tubes, reusable pipettes, and shattered glass intravenous needles. it can be observed from figure 1 that 80.30% of the waste produced by the healthcare industry is an infectious waste. whereas, the remaining 19.70% of waste is divided among pathological, pharmaceutical, cytotoxic, chemical wastes, and sharps. the techniques followed to discard the healthcare waste include; • shredding is the process of cutting or de-shaping waste into little bits. it minimizes the volume necessary for efficient disposal with autoclave and sterilizing procedures. it also prevents infectious sharps and waste from being reused also it is an alternative to combustion8. • incineration is a high-temperature, dry oxidation method of converting organic and flammable waste to synthetic, non-flammable material, leading to a reduction in waste weight and volume, at temperatures ranging from 600°c to even more than 1000°c under appropriate conditions, through burning, thermal decomposition, or gasification9. • steam sterilization is the use of non-incineration systems such as autoclaves and hydroclaves to treat pathogenic microorganisms in hazardous hospital waste has been advocated. even though steam sterilization methods kill or stop germs from growing, microbial regeneration and the danger of lingering chemical hazards are the main drawbacks to employing these systems10. figure 1. percentage of waste produced by the healthcare industry7. healthcare waste management in hyderabad sindh vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 177 figure 2. hcw generation worldwide11. figure 2 depicts the production of healthcare waste in 4 major continents of the world. among these africa has the lowest percentage which is 6%. whereas, the highest waste is produced by asia followed by america and europe with 44%, 26%, and 24% respectively. the percentage of waste also has a relation with the population in the continent which relatively affects the number of people hospitalized each day. as can be observed from figure 2 asia has the highest rate of production of waste due to its densely populated countries with high resource constraints12. developing countries have less of facilities and awareness about hygiene practices. pakistan being the 4th most populated country in asia follows fewer practices of healthcare waste management. therefore, this research aimed to determine hospital waste management practices in the region of sindh, the level of knowledge about those practices in hospital staff, incarnation methods followed by the hospital, and to understand the current waste management situation in sindh from multiple standpoints. the obtained information is fed into the spss in the form of statistical data to observe the meaningful results related to healthcare waste management. m e t h o d o l o g y this research is based on the primary data to survey different hospitals. to obtain the primary information qualitative methods were used such as questionnaires (open and close-ended), interviews, and observations. these questionnaires were filled by meeting the hospital personnel like medical superintendents (ms) in government hospitals and hospital representatives in private hospitals (figure 3). figure 3. flow chart of the methodology the above flow chart represents the steps followed in this research. in this study, the targeted area selected was hyderabad.it has been conducted from 52 hospitals among which 45% were government hospitals, 5% were ngo hospitals located in small areas of hyderabad, and 50% were private hospitals. a survey strategy made is built study area selection data collection result interviews data preparation spss analysis survey strateg questionnaire observations healthcare waste management in hyderabad sindh vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 178 on questionnaires, interviews, and direct observations. the data is composed of government, private and semigovernment hospitals of different cities in sindh. the data composed through questionnaires, interviews, and observations are arranged in a proper structure in an ms excel sheet. to get the results, different statistical tests are applied. questionnaire the questionnaire was developed by taking help from secondary data to find the most convenient and relevant questions related to hcw. this questionnaire was divided into sections which cover a total of forty questions. section 1 covered the basic information about the hospital and its staff. the second section contains the waste generated by hospitals per day and the color-coded system of bins they followed. a third section was about techniques they used to dump the waste. these questions help in this research regarding generating meaningful information related to healthcare waste management in hyderabad. sample size it is a difficult task to gather data about hcw management from all hospitals of hyderabad, sindh region. therefore, to accumulate the information in regards to health center waste control practices in the hospitals. it was important to select the sample size. to determine sample size the following cochran equation is used13: 𝑛 = 𝑍2𝑆𝐷2 𝑒⁄ 2 ……….. (1) this equation is only for a simple random sample. • zis the significance level. • sd is the standard deviation. • e is the desired level of precision (i.e. the margin of error). in this study, the value of 'z' is 1.96, the value of 'p' is 0.20, 'q' is obtained by subtracting the value of 'p' from '1', the value of sd is obtained by multiplying the value of 'p' & 'q' and the value of 'e' is 0.109. by putting these values in equation1, we obtained the number sample size which is 52 hospitals. r e s u l t s the analysis of data from the survey questionnaire was fed into spss. in spss, the chi-square test was performed which is used when the relationship between two categorical variables is required. after collecting a random sample of hospitals, and administering a survey to this sample, the frequency distribution of hospital waste management is manually observed within the sample. then the chi-square test is performed in spss to validate the observed frequencies. table 1 contains the results of the waste generated by each bad each day. this data is obtained from government, private, and ngo-based hospitals. the horizontal title shows the frequency of waste in kgs generated in percentage, which is randomly divided into 0.5%, 1%, 1.5%, 2%, 2.5%, and 3%.while it is observed that in 13 government hospitals, 3.8% hospitals generate 0.5 kgs of waste per day, 5.8% generate 1 kg, 7.7 % generate 1.5kgs, 3.8% generate 2 kgs and 3.8% of hospitals generate 3 kgs of waste per day per bed.in total he waste generated is 25% in government hospitals and in private hospitals, the waste generated is 71%. the chi-square test suggest that in 52 hospitals the result is significant (p<0.01). table 2,3, and 4 show the chi-square obtained results and its likelihood ratio for waste generated per day/bed, techniques used to discard waste, and waste storage timing, respectively. in table 2, the value of test statistic is 9.385 with the degree of freedom 15. the corresponding pvalue of the statistic is p=0.857. table 3 shows the result of techniques used to discard waste. the value of test statistic is 10.173 with the degree of freedom 15. the corresponding p-value of the statistic is p=0.809. the footnote for this chi-square test pertains to the expected cell count assumption (i.e., expected cell counts are all greater than 5): no cells had an expected count less than 5, so this assumption was met. whereas, table 4 shows the waste storage timing. the value of test statistic is 8.818 with the degree of freedom 6. the corresponding p-value of the statistic is p=0.184. the footnote for this chi-square test pertains to the expected cell count assumption no cells had an expected count less than 5 and the minimum expected count is 0.02. healthcare waste management in hyderabad sindh vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 179 table 1. waste generated per day/bed. hospital type frequency of waste generated in each hospital per bed/day in %. total .50 kg 1.00 kg 1.50 kg 2.00 kg 2.50 kg 3.00 kg govt no of hospitals 2 3 4 2 0 2 13 % of waste generated in each hospital per bed per day 3.8% 5.8% 7.7% 3.8% 0.0% 3.8% 25.0% ngo no of hospitals 0 1 1 0 0 0 2 % of waste generated in each hospital per bed per day 0.0% 1.9% 1.9% 0.0% 0.0% 0.0% 3.8% private no of hospitals 6 6 9 11 2 2 37 % of waste generated in each hospital per bed per day 11.5% 13.4% 17.3% 21.2% 3.8% 3.8% 71.2% sub total 8 11 14 13 2 4 52 15.4% 23.2% 26.9% 25.0% 3.8% 7.7% 100.0% table. 2. chi-square waste generated per day/bed. variables value df asymp. sig. (2-sided) pearson chi-square 9.385a 15 .857 likelihood ratio 9.836 15 .830 n of valid cases 52 table 3. chi-square test of techniques used to discard waste. value df asymp. sig. (2-sided) pearson chi-square 10.173a 15 .809 likelihood ratio 10.931 15 .757 n of valid cases 52 a. 22 cells (91.7%) have an expected count of less than 5. the minimum expected count is .02. table 4. chi-square tests of waste storage timing. value df asymp. sig. (2-sided) pearson chi-square 8.818a 6 .184 likelihood ratio 9.058 6 .170 n of valid cases 52 a. 9 cells (75.0%) have an expected count of less than 5. the minimum expected count is .02. healthcare waste management in hyderabad sindh vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 180 figure 4. the technique used to discard waste. figure 4 shows the techniques used to discard waste by government, private, semi-private, and ngo-based hospitals. it is observed from the figure that private hospitals follow the incineration and open-air burning around 11.5%. whereas, the government follows incineration at 6 % and open-air burning at 5%. however, private hospitals follow the techniques in a much better way as compared to government hospitals. table 3 shows the chi-square test of waste dumping techniques. figure 5. time of waste storage. healthcare waste management in hyderabad sindh vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 181 figure 5 shows the time of storage of waste followed by hospitals before dumping waste. around 27% of private hospitals due to more facilities and availability ofresources can store waste for 24 hours. whereas, 10% store for 48 hours. table 4 shows the chi-square test performed for storage time. d i s c u s s i o n pakistan environmental protection agency (pepa) has the set of rules for hospital waste management. unfortunately, due to lack of awareness and avoidance by many hospitals the rules are not fully followed. as a result to that violence of hwm rules, pepa has right to take strict action against those hospitals.it is essential to manage the hospital waste since improper disposal results in the subsequent repurposing of hazardously contaminated disposal materials for the production of various plastic goods for residential use.to protect human health and the environment, it is necessary to have safe management of hospital waste. c o n c l u s i o n medical waste management and disposal receive minimal attention, even though they are deemed dangerous due to their contagious and/or poisonous qualities. the results revealed that government hospitals require proper wastehandling techniques. the main cause of concern was a lack of effective employee training on healthcare waste management issues and the dangers that could result from improper treatment. toxicity was found in all wastewater samples from the hospital, especially those from the laboratories. for the safety and health of waste handlers, insufficient steps were taken. the guidelines and recommendations were not implemented at most hospitals. moreover, the training of hospital staff, hcwm awareness, and handling of healthcare waste through using protective gear is more improved in private hospitals than that in government hospitals. therefore, by considering the private hospital's management system there should be a separate healthcare department that handles the waste of the hospital without affecting the staff, and other public in government hospitals. however, the hospital waste management policy should state the proper handling, storage, transportation, and disposal of waste. the policy should be stated through appropriate legislation to protect the environment and public health. the action plan and strategy should be designed with decision-making authorities for proper hospital waste management. a task force should which includes representatives from private and government hospitals should be assigned tasks to begin the process of adaption of policy in hospitals. c o n f l i c t s o f i n t e r s t none. f u n d i n g s o u r c e none. a c k n o w l e d g e m e n t s none. l i s t o f a b b r e v i a t i o n s who world health organization ngo non-governmental organization hcw health care waste sd standard deviation spss statistical package for social sciences df degrees of freedom r e f e r e n c e s 1. kenny c, priyadarshini a. review of current healthcare waste management methods and their effect on global health. healthcare [internet]. 2021 mar 1 [cited 2022 oct 11];9(3). available from: /pmc/articles/pmc7999172/ 2. global spending on health: a world in transition [internet]. 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1/journal.pone.0225595 11. minoglou m, gerassimidou s, komilis d. healthcare waste generation worldwide and its dependence on socio-economic and environmental factors. sustain 2017, vol 9, page 220 [internet]. 2017 feb 6 [cited 2022 oct 12];9(2):220. available from: https://www.mdpi.com/2071-1050/9/2/220/htm 12. khan ba, cheng l, khan aa, ahmed h. healthcare waste management in asian developing countries: a mini-review. https://doi.org/101177/0734242x19857470 [internet]. 2019 jul 3 [cited 2022 oct 12];37(9):863–75. available from: https://journals.sagepub.com/doi/full/10.1177/073 4242x19857470 13. naing nn. determination of sample size. malaysian j med sci. 2003;10(2):84–6. emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 24 op e n ac c e s s f u l l l e n g t h a r t i c l e emergence of antifungal azole resistance in the fungal strains of tinea corporis, tinea capitis, tinea cruris and tinea pedis from the locality of southern punjab, pakistan fatima ismail*, abdul ghani, saba akbar department of biochemistry and biotechnology, the islamia university of bahawalpur, pakistan. a b s t r a c t background: dermatophytes are the most common group of fungi causing fungal infections all over the world. they are classified into three main groups trichophyton, microsporum and epidermophyton. among these, trichophyton has the highest prevalence rate (7090%) as compared to the others. the global emergence of fungal infections is varied due to the socio-economic conditions throughout the world. developing countries, like pakistan, are facing an increase in the number of dermatophytoses, including frequent relapses and treatment failures. objectives: the study have been conducted to identify the emerging fungal species, the role of commonly available antifungals such as azoles including voriconazole, ketoconazole, fluconazole and amphotericin b were used to determine the drug resistance among these species. methodology: nine groups of dermatophytes and non-dermatophyte fungi isolated from the patients of tinea corporis, tinea cruris, tinea capitis and tinea pedis infections were analyzed for phenotypic diversity, antifungal susceptibility and strains identification, was performed by cultural characteristics and microscopy. results: nine groups of isolated fungal strains were identified as trichophyton interdigitale, trichophyton mentagrophyte, trichophyton rubrum amongst dermatophytes class and aspergillus terreus, aspergillus verisocolor, aspergillus niger, acretonium sordidulum and acremonium sclerotigenum of non-dermatophytes class. conclusion: the study revealed trichophytone interdigitale group as more frequent dermatophytes. whereas, among the antifungal drugs, fluconazole that targets the erg 1 gene of ergosterol biosynthesis in fungi is less effective most common antifungal drugs available locally. keywords antifungal drug resistance, azoles, dermatomycosis, filamentous fungi, sensitive phenotype, sterol biosynthesis. *address of correspondence fatima.ismail@iub.edu.pk article info. received: november 05, 2020 accepted: january 05, 2021 cite this article: ismail fatima, ghani a, akbar s. emergence of antifungal azole resistance in the fungal strains of tinea corporis, tinea capitis, tinea cruris and tinea pedis from the locality of southern punjab, pakistan. rads j biol res appl sci. 2021; 12(1):24-38. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n fungal infections have a wide impact on global health. fungi threats nearly a billion people suffer from superficial infections of skin hair and nail, 100 million people suffer from mucosal candidiasis,10 million people developed severe allergic reactions result in million deaths each year reported are all linked with fungal infections1,2. the worldwide death rate due to fungal infections is higher than malaria and breast cancer and is comparable to o r i g n a l a r t i c l e emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 25 tuberculosis (tb) and hiv3. the infections caused by fungi are divided into three main groups: cutaneous, superficial and systematic mycosis4. dermatomycosis is the most common in superficial mycosis reported worldwide. it is a well defined infection of the skin, hair and nails5. based on location and mode of transmission, dermatophytes are further classified into three groups i.e. zoophilic, geophilic and anthropophilic associated with the animal, soil and human beings, respectively. the geophilic group including nannizzia gypsea, epidermatophyton floccosum and alternaria is worldwide recognized as a common plant pathogen and airborne allergen. it is the typical aeroallergen species of the genus and in a majority of cases, the most frequent species associated with human and animal health problems. similarly, the zoophilic group include microsporum canis, trichophyton equinum, trichophyton verrucosum, trichophyton erinacei and microsporum manum whereas, anthropophilic group include trichophyton interdigitale, trichophyton mentagrophyte, trichophyton tonsurans, trichophyton soudanense, trichophyton megninii and trichophyton violaceum6. these species invade the skin due to their ability to digest keratin used as a substrate7. dermatophytosis is a disease of overall significance and a general medical issue in numerous parts of the world, especially in developing countries8, 9. it is an increasing threat in immune-compromised individuals10. increasing number of population, low financial status, and insufficient health condition and trading of inappropriately cleaned or used foot-wears, garments, and barbershop supplies etc. among individuals have been perceived as potential hazardous factors for the multiplication of the disease11. the types of fungi epidermophyton, microsporum, trichophyton, non-dermatophyte molds and yeast have been considered as significant cause of the mycosis12. the worldwide weight of cutaneous contamination was evaluated to be ~1,001,000,00013. these contaminations are more typical among rural than urban population, and the disease, tinea capitis is reported to be more prevalent in males14. literature has indicated that the worldwide burden of dermatophyte disease explicitly was evaluated to be 20-25%14. there are fewer studies conducted on antifungal drug resistance and on fungal stress response. therefore, to this end, investigating human dermatophytosis causing superficial mycosis appears to be one of the priorities in health-related studies. the aim of the study is to investigate the more invasive and frequent types of fungal infections and the fungal species involved, and to find their possible resistance against current azoles and amphotericin b antifungals. m a t e r i a l & m e t h o d s sample collection initially, 74 samples were collected from suspected patients of dermatophytosis at the civil hospital bahawalpur. before the collection of skin scrapings and scalp samples, infection site was cleaned with 70% ethanol. the hair were removed from the scalp in case of infection of tinea capitis and nails were scraped and clipped from tinea unguium patients as previously described15. lactophenol cotton blue staining was used for the microscopic observation of dermatophyte. culture media the samples were cultured on sterile sabouraud dextrose agar (sda), fungal specific medium containing chloramphenicol (0.5g/l) which inhibits most of the bacterial growth, and cycloheximide (0.4g/l) that inhibits saprophytic fungi, and plates were incubated at 30°c for 7 days. spores were collected from the sda slant and spore suspension was prepared in 2ml sterilized water. spores collection was done after oscillating at vortex and transferred into the 2ml sterilized tubes. they were then centrifuged at 4000rpm for 2min. supernatant was discarded and 200ml sterilized water was added into conidial suspension. conidial suspension (2µl) was transferred into 2ml tube adding sterilized h2o to reach up to 2×106 conidial suspension16, 17. identification of agents causing dermatophytosis using microscopy all the isolates were undertaken for the phenotypic examinations. the conidial suspension was prepared as previously describes by samuel et al18. conidia were examined under the light microscope at 100x magnification. emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 26 identification of agents causing dermatophytosis by its region the dna of these isolates was extracted using the method described19. species identification was done using fungal universal primers for intra transcribed spacer regions (its1), the forward and reverse primers used (forward primer: its1 (5’tccgtaggtgaacctgcgg3’) and the (reverse primer: its4 (5’tcctccgcttattgatatgc3’) was used for pcr (mygene l series peltier thermal cycler by uniequip. the pcr cycling conditions were 35 cycles of 95ºc for 1 min, 55ºc for 1 min, and 72ºc for 2 min, followed by an extension step of 72ºc for 10 min20. intra transcribed spacer regions are called the fungal bar code they are non-coding regions which are considered the most standardized nucleotide regions to identify the fungal taxonomy at species levels. amplified product was confirmed by running on 2% agarose gel electrophoresis. amplified pcr products were sent for sanger sequencing to beijing institute of genomics (big). the sequences obtained were analyzed by nucleotide ncbi blast and aligned by multiple sequence alignment tool (clustal w and mega6). antifungal drug susceptibility sabouraud dextrose agar (sda) plates were prepared using antifungal drugs stock solutions: 2mg/ml ketoconazole, 10mg/ml fluconazole 10mg/ml amphotericin b, and 5mg/ml voriconazole. drugs were dissolved in dimethyl sulfoxide (dmso) and added to autoclaved media before pouring onto the petri plates. the concentrations of drugs such as ketoconazole, fluconazole, amphotericin b and voriconazole selected according to their minimum inhibitory concentration (mic) points. the drug sensitivity was conducted on petri plates and inoculated with 2µl spores (2 x 106/ml) conidial dilution and incubated at 30ºc for 7 days. the control was inoculated with no drug. each sensitivity test has been done three times, independently. oxidative stress the investigation was conducted to identify the effect of stress generated by different chemical compounds individually. firstly, the effect of hydrogen peroxide (h2o2) was tested by preparing 2, 4 and 6mm concentrations of hydrogen peroxide and added in sda media for identification of the effect of h2o2 on the growth of the dermatophytes. the media (20ml) was poured in each petri plate and 2µl spore suspension was inoculated and incubated at 30ºc for 7 days. the growth after 4 days on different concentration hydrogen peroxide in media and control was compared. similarly, different concentrations of benzoic acid (2, 4 and 6mm) were used in sda media for identification of the phenotypic effect on the growth of the dermatophytes. the stress responses with growth differences was considered at 6mm benzoic acid (dissolved in dmso). twenty milliliter (20ml) media was poured in each petri plate, and 2µl conidial suspension was added and incubated at 30ºc for 7 days. fungal phenotype on different concentration of benzoic acid media and control was compared. each test has been done three times independently. lastly, stress effect of sodium chloride (nacl) was examined using 4% nacl which was adjusted in the media. conidial suspension (2µl) was inoculated on the media plates and the growth of dermatophytes and nondermatophytes on sda medium was observed. the plates were incubated at 30ºc for 7 days and the effect of sodium chloride on fungal species was observed by comparing with the controls. each test has been done three times independently. enzymatic activity the protease activity was performed as described previously21. for this purpose, a medium was prepared that contain contained dextrose 2%, potassium dihydrogen phosphate 0.1%, magnesium phosphate 0.05% and agar 2%. the media was sterilized and cooled up to 50ºc and 1% bovine serum albumin (bsa) was added. the enriched medium was mixed thoroughly and poured in a sterile petri plates. the spore suspension (2µl) was inoculated and incubated at 30ºc for 7 days. the control was inoculated with no bsa in the media. each test has been done three times independently. candida albicans was used as a positive control. a clear zone around the colony indicated protease production. r e s u l t s a total of 74 clinically diagnosed cases of dermatophytosis were studied from bahawalpur. the age group in this study ranged from 1-50 years. the most common age group affected was 21-30 years (table 1). the highest emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 27 percentage of patients were found to have the infection of tinea corporis 39% followed by tinea capitis 20%, tinea cruris 18%, tinea ungium 11%, tinea faciei 5%, tinea pedis 3%, tinea manum 3%, tinea versicolor 1% as mentioned in (fig. 1). the tinea corporis (39%) was the most common of all tinea patients included in this study. all 74 samples of skin, scalp scrapings and hairs were grown on sda media containing cycloheximide and chloramphenicol. figure 1. pie-chart of diseases caused by dermatophyte. infection due to tinea corporis has the highest percentage of 39% followed by tinea capitis 20%, tinea cruris 18%, tinea ungium 11%, tinea faciei 5%, tinea pedis 3%, tinea manum 3%, tinea versicolor 1%. table 1. age and sex-wise distribution of dermatophytosis in clinical samples. s. no. clinical types age group (in years) sex total %age 0-10 11-20 21-30 31-40 41-50 male female 1. tinea corporis 3 3 11 9 3 12 17 29 39% 2. tinea unguium 2 5 1 2 6 8 11% 3. tinea capitis 14 1 5 10 15 20.2% 4. tinea cruris 1 5 6 1 3 10 13 17.5% 5. tinea faciei 2 1 1 1 3 4 5.4% 6. tinea pedis 1 1 1 1 2 2.7% 7. tinea versicolor 1 1 1 1.3% 8. tinea. manum 1 1 2 2 2.7% total 22 7 26 18 7 24 50 74 100% the results showed the highest percentage of tinea corporis infection (39.3%) as compared to others. females are affected more than males and 21-30 years is a more common age group found. emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 28 macroscopic and microscopic identification of dermatophytes fungal isolates were identified on the basis of macromorphology (forward and reverse color) and micromorphology (microconidia and macroconidia). dermatophytes and non-dermatophyte isolates were separated on the basis of color, shape and number of micro and macroconidia such as trichophyton mentagrophytes (yellow brown to reddish brown colony with numerous microconidia and hyaline macroconidia), trichophyton interdigitale (white to brown colour become reddish brown with age from reverse pigment with pyriform microconidia); non-dermatophyte causing dermatophytosis like acremonium sclerotigenum (white center with large hypha) and aspergillus versicolor showed various color from orange yellow to tan green and penicilli-like conidia. among all the collected fungal samples, nine different species (trichophyton mentagrophyte, trichophyton interdigitale, trichophyton rubrum, aspergillus terreus, alternaria alternata, aerentonium sordidulum, alternaria mean versicolor and acremonium sclerotigenum were identified (table 2). identification from pcr and dna sequencing dermatophytes and non-dermatophytes isolates were identified by using pcr for amplification of its1 and its4 region on the dna gel electrophoresis (fig. 2). seventeen isolates including one from each fungal group and eight unidentified fungal species were selected for further verification. identified fungal sub groups were selected and sequenced on sanger sequencing. dna sequencing was run on blast. all the strains are mentioned with percentage (%) identification by blast and with accession number provided in (table 2). the sequence of nine species identified from blast were aligned by clustal w. phylogenetic tree was prepared based on nine different species of fungal strains (fig. 3). figure 2. its gene pcr amplifications on gel electrophoresis. figure 3. phylogenetic tree of 9 representative dermatophytes and non-dermatophyte species based on analysis of its1 region sequences. the evolutionary history was inferred using the neighbor–joining (nj) method based on the tamura– nei model. emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 29 table 2. dna sequence of eight dermatophytes and non-dermatophytes aligned on ncbi gene bank. s. no. strain no. clinical types identification according to its sequence percentage % identification by blast accession 1 tm(25) tinea corporis trichophyton mentagrophyte 98.21% mn661259.1 2 at(30) aspergillus terreus 99.48% ky053121.1 3 tr(55) trichophyton rubrum 99.69% mn691068.1 4 tm(43) trichophyton mentagrophyte 99.53% mn661259.1 5 ti(22) trichophyton interdigitale 99.70% mh517559.1 6 ti(3) trichophyton interdigitale 99.56% mh517559.1 7 tm(26) tinea cruris trichophyton mentagrophyte 98.22% mn661259.1 8 an(54) aspergillus niger 99.65% mg654699.1 9 av(53) alternaria versicolor 100% mh712290.1 10 ti(42) trichophyton interdigitale 99.44% mt497400.1 11 ti(60) trichophyton interdigitale 100.00% mn178659.1 12 at(39) tinea capitis aspergillus terreus 99.34% mn099077.1 13 aa(47) alternaria alternata 88.89% gu004283.1 14 at(9) aspergillus terreus 100.00% kf971363.1 15 tr(59) trichophyton rubrum 100.00% mn176601.1 16 ar(14) aerentonium sordulum 87.67% mk513818.1 17 as(15) tinea pedis acremonium sclerotigenum 99.65% mk732096.1 result showed nine different species of fungi causing dermatophytosis. four group of tinea infection has been focused. its sequence analysis has been used for the identification of four genera of fungi belong to species of aspergillus, trichophytone group, alternaria alternate, acrentonium sordulum and acremonium sclerotigenum. antifungal drug susceptibility in this study, all the isolates form tinea corporis, tinea cruris, tinea capitis and tinea pedis were tested for drug sensitivity of fluconazole (10µg/ml), ketoconazole (2µg/ml), voriconazole (3µg/ml) and amphotericin b (2µg/ml). only one isolate (ti3) tinea corporis showed resistance against fluconazole at 10µg/ml while others were sensitive against ketoconazole, voriconazole and amphotericin b. one isolate (av53) from tinea cruris showed resistance against fluconazole and amphotericin b at 10µg/ml and 2µg/ml, respectively. tinea capitis (tr59) showed resistance against fluconazole and amphotericin b. the isolate of tinea pedis showed inhibition against all the drugs used in this study (table 3.1, 3.2 and 3.3). the minimum inhibitory concentrations of nine different species of dermatophyte and non-dermatophyte isolates of tinea corporis, tinea cruris, tinea capitis and tinea pedis were carried out. results showed that fluconazole is the least effective antifungal drug in contrast to ketoconazole which is highly effective drug against dermatophyte and nondermatophyte (table 4). fungal adaptations to oxidative stresses the effect of hydrogen peroxide on the growth of dermatophyte and non-dermatophyte species isolated from tinea corporis, tinea cruris, tinea capitis and tinea pedis infections have been studied. the growth of all isolates were inhibited at 6mm h2o2 except aspergillus terreus (at30) and trichophyton interdigitale (ti60) belongs to tinea corporis and tinea cruris, which showed no inhibition in growth and adapted hydrogen peroxide stress. in order to conduct the fungal oxidative stress response, benzoic acid sensitivity test was also conducted. benzoic acid stress inhibited the growth of all isolates except acremonium sclerotigenum (as15), aspergillus terreus emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 30 (at30) and acrentonium sordidulum (ar14) from tinea pedis, tinea corporis and tinea capitis, respectively at 6mm. however, the effect of salt stress on 4% sodium chloride inhibited the growth. however, the effect of salt stress on 4% sodium chloride inhibited the growth of all isolates except aspergillus terreus (at30) isolated form tinea corporis and trichophyton interdigitale (ti60) isolated from tinea cruris. oxidative stress affect the cellular ph that lead to the alterations in metabolic regulations, which arrest the cell growth in the fungal isolates. however, the isolates of aspergillus terreus (at30) form tinea corporis and trichophyton interdigitale (ti60) from tinea cruris tolerated the salt stress due the possible adaptations as (table 5). table 3.1. antifungal drug susceptibility against different dermatophytes and non dermatophytes s. no. strain no. clinical types species vor ktc flu amp 1 tm26 trichophyton mentagrophytes sensitive sensitive similar growth resistance 2 an54 aspergillus niger sensitive sensitive sensitive similar growth 3 ti60 trichophyton interdigitale sensitive sensitive similar growth similar growth 4 ti35 trichophyton interdigtale sensitive sensitive similar growth similar growth 5 tm42 trichophyton mentagrophyte sensitive sensitive similar growth similar growth 6 av53 tinea cruris aspergillus versicolor sensitive sensitive similar growth resistance 7 ti33 trichophyton interdigitale sensitive sensitive sensitive sensitive 8 28 no growth 9 49 no growth 10 52 no growth 11 65 sensitive similar growth sensitive sensitive 12 56 no growth 13 57 no growth 1 69 trichophyton rubrum sensitive sensitive sensitive sensitive 2 ti73 trichophyton interdigitale sensitive sensitive resistant sensitive 3 ti74 tinea faciei trichophyton interdigitale sensitive sensitive sensitive sensitive 4 76 microsporum canis resistant sensitive sensitive resistant 1 tm11 trichophyton mentagrophytes similar growth sensitive similar growth sensitive 2 ca20 candida albicans sensitive sensitive similar growth sensitive 3 ca23 tinea unguium candida albicans similar growth sensitive sensitive sensitive contd… emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 31 4 13 no growth 5 37 no growth 6 38 no growth 7 45 no growth 8 51 no growth drug susceptibility against different species of dermatophyte has been presented on basis of phenotypic growth as comparison with control. table 3.2. antifungal drug susceptibility against trichophyton, alternaria and aspergillus species isolated from tinea corporis infection. s. no. strain no. clinical types species vor ktc flu amp 1 ti3 trichophyton interdigitale sensitive sensitive sensitive sensitive 2 tm19 trichophyton mentagrophytes similar growth sensitive similar growth sensitive 3 ti30 trichophyton interdigitale sensitive sensitive sensitive sensitive 4 ti21 trichophyton interdigitale sensitive sensitive similar growth sensitive 5 ti22 tinea corporis trichophyton interdigitale sensitive sensitive sensitive sensitive 6 ti46 trichophyton interdigitale similar growth sensitive sensitive sensitive 7 tm25 trichophyton mentagrophytes sensitive sensitive sensitive sensitive 8 aa41 alternaria alternata sensitive sensitive sensitive sensitive 9 70 trichopyton rubrum sensitive sensitive similar growth sensitive 10 av43 aspergillus versicolor sensitive sensitive sensitive sensitive 11 ti58 trichophyton interdigitale similar growth sensitive similar growth sensitive 12 ti34 trichophyton interdigitale sensitive sensitive sensitive sensitive 13 ti59 trichophyton interdigitale similar growth sensitive sensitive sensitive 14 8 no growth 15 10 no growth 16 18 no growth contd… emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 32 17 27 no growth 18 77 trichophyton. rubrum sensitive sensitive similar growth resistant 19 29 no growth 20 32 no growth 21 33 no growth 22 36 no growth 23 40 no growth 24 44 no growth 25 50 no growth 26 55 no growth 27 61 no growth 28 64 trichophyton interdigitale similar growth similar growth sensitive sensitive 29 67 trichophyton interdigitale similar growth similar growth resistant sensitive results has been presented on bases of phenotypic growth in comparison with control. table 3.3. antifungal drug susceptibility against different species of dermatophytes and non dermatophytes. s. no. strain no. clinical types species vor ktc flu amp 1 at39 aspergillus terreus sensitive sensitive sensitive sensitive 2 an48 aspergillus niger sensitive sensitive similar growth resistant 3 aa47 tinea capitis aspergillus alternata sensitive sensitive sensitive similar growth 4 at9 aspergillus terreus sensitive sensitive sensitive sensitive 5 aa1 aspergillus alternata sensitive sensitive sensitive sensitive 6 aa2 aspergillus alternata sensitive sensitive sensitive sensitive 7 tr55 trichophyton rubrum sensitive sensitive similar growth resistant 8 as14 aspergillus sordidulum sensitive sensitive sensitive sensitive 9 17 no growth 10 24 no growth 11 62 trichophyton mentagrophyte sensitive sensitive sensitive sensitive 12 63 aspergillus terreus resistant resistant sensitive 13 71 trichophyton interdigitale sensitive sensitive sensitive sensitive 14 72 trichophyton mentagrophyte sensitive similar growth sensitive sensitive 15 78 candida albicans sensitive similar growth sensitive 1 as15 tinea pedis aspergillus sclerotigenum sensitive sensitive sensitive sensitive contd… emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 33 2 68 trichophyton rubrum sensitive similar growth similar growth resistant 1 an4 tinea versicolor aspergillus niger sensitive sensitive similar growth similar growth 2 66 tinea manum trichophyton mentagrophyte sensitive similar growth similar growth sensitive 3 75 trichophyton rubrum similar growth resistant sensitive drug susceptibility against nine different species of dermatophyte and non-dermatophyte has been presented on the basis of phenotypic growth as comparison with control. table 4. the mic pattern of nine different species of dermatophytes and non-dermatophytes. s. no. strains no. mic (minimum inhibitory concentration) flu ktc amb vor 1 trichophyton interdigitale 4 >10 1.4 >2 1.8 2 trichophyton mentagrophyte 3 >10 2 >2 1.4 3 trichophyton rubrum 2 >10 1.8 >2 1.4 4 aspergillus terreus 3 >10 1 >2 2 5 acretonium sclerotigenum 1 >10 1.4 >2 1 6 aspergillus versicolor 1 8 1 2 2 7 acrentonium sordidulum 1 >10 2 2 3 8 alterneria alternata 1 >10 >2 >2 3 9 aspergillus niger 1 >10 2 >2 2 the mic (minimum inhibitory concentration) of nine different species of dermatophytes and non-dermatophytes obtained using flu (fluconazole), ktc (ketoconazole), amb (amphotericin b), vor (voriconazole). mic has been conducted using clsi, 2020 guideline. table 5. comparison of effect of oxidative stress (h2o2, benzoic acid and nacl stress) in dermatophytes with controls. s. no. strains clinical types identified species effect of stress on growth compared to control benzoic acid 6mm h2o2 6mm nacl 4% 1 ti(3) tinea corporis trichophyton interdigitale sensitive sensitive sensitive 2 tm(25) trichophyton mentagrophyte sensitive sensitive no effect 3 at(30) aspergillus terreus no effect no effect no effect 4 tr(55) trichophyton rubrum sensitive sensitive sensitive 5 tm(43) trichophyton mentagrophyte sensitive sensitive sensitive 6 ti(22) trichophyton interdigitale sensitive sensitive sensitive contd… emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 34 7 tm(26) tinea cruris trichophyton mentagrophyte sensitive sensitive sensitive 8 an(54) aspergillus niger sensitive sensitive sensitive 9 ti(60) trichophyton interdigitale no effect no effect no effect 10 ti(42) trichophyton interdigitale sensitive sensitive sensitive 11 av(53) aspergillus versicolor sensitive sensitive sensitive 12 at(39) tinea capitis aspergillus terreus sensitive sensitive sensitive 13 aa(47) alternaria alternate sensitive sensitive sensitive 14 at(9) aspergillus terreus sensitive sensitive sensitive 15 tr(59) trichophyton rubrum sensitive sensitive sensitive 16 ar(14) acretonium sordidulum no effect sensitive sensitive 17 as(15) tinea pedis acremonium sclerotigenum no effect sensitive sensitive oxidative stress of h2o2, benzoic acid and nacl on fungal isolates isolated from tinea corporis, tinea cruris, tinea capitis and tinea pedis has been conducted in comparison to controls. sda protease sda protease medium medium medium medium figure 4. protease activity on bovine serum albumin (bsa) medium gave a clear halo zone around the colony and indicated protease production {p (protease production), n (no protease production)} ca+ct (candida albicans) as + ve control. emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 35 enzymatic activity in order to know whether fungal pathogenicity depend on enzymatic secretions (particularly protease enzyme during dermatophytosis), the enzyme activity such as protease was analyzed for all isolates on protease media, the result showed that dermatophyte and non-dermatophyte species such as trichophyton mentagrophyte (25), aspergillus terreus (30), trichophyton interdigitale (22), trichophyton interdigitale (3) the non-dermatophytes like alternaria alternate (47) and aspergillus terreus (9) isolated from tinea capitis produced protease enzyme (fig. 4). d i s c u s s i o n dermatophytosis annually affects millions of people all over the world. the prevalence of dermatophytosis has been enhanced in immuno-compromised patients suffering from diseases like diabetes mellitus, aids, morbid obesity diseases, and transplant patients also reported by pierard et.al22. antifungal drug-resistance has become a major hurdle in the treatment of fungal infection. this study was conducted in bahawalpur, pakistan. clinically diagnosed fungal isolates were collected. the fungal infections found to be common in the age group of 21-30 years and more frequent in female as compared to male. tinea corporis was found as the most common type of infection (35%), followed by tinea cruris (29%), tinea capitis (30%), and tinea pedis (6%). initially, phenotypic identification was done on the basis of color of colony and shape of microconidia and macroconidia such as trichophyton mentagrophytes (yellow brown to reddish brown colony with numerous microconidia and hyline macroconidia), trichophyton interdigitale, (white to brown colour that become reddish brown with age) with pyriform microconidia) and non-dermatophytes causing dermatophytosis like acremonium sclerotigenum (white center and thin hyphae) and aspergillus versicolor (color varies from orange yellow to tan green)23. in addition, nine different species including trichophyton interdigitale, trichophyton mentagrophyte, aspergillus versicolor, trichophyton rubrum, aspergillus terreus, aspergillus niger, alternaria alternata, acrenotinum sordidulum and acremonium sclerotigenum were identified by sequencing its region24. the results shown similarity with the previous studies in which dermatophyte species (trichophyton interdigitale, trichophyton mentagrophyte and trichophyton rubrum) non-dermatophyte species (aspergillus versicolor, aspergillus terreus, aspergillus niger, alternaria alternata, acrenotinum sordidulum and acremonium sclerotigenum) caused dermatophytosis in human beings25. the sequence of nine different species among 17 of dermatophyte and non-dermatophyte causing dermatophytosis were aligned by clustal w. for the phylogenetic tree the isolates of common fungal group were omitted. phylogenetic tree relationship revealed the homology between identified strains of dermatophytes and non-dermatophytes26. antifungal drug susceptibility tests showed only two fungal groups (aspergillus versicolor (av53) and trichophyton rubrum (tr59) non-responsive against amphotericin b, three isolates (trichophyton interdigitale) ti3, (trichophyton rubrum) tr59 and aspergillus versicolor) av53 were resistant against fluconazole probably due to the possible mutation in the fluconazole drug target gene squalene epoxidase. all other isolates were sensitive against voriconazole and ketoconazole due to their potential broad spectrum of the molecular target. such as the azoles target cytochrome p-450 dependent enzymes in fungi. inhibition of the membrane bound enzymes of p450 family accumulate the toxic intermediate such as lanosterol, eburicol and the toxic 14α-methyl-3,6-diol, which reduces the permeability of cell membrane and inhibit the growth of fungi27, 28. in order to examine the adaptability of the oxidative stress (hydrogen peroxide, benzoic acid and nacl stress), 17 various fungal groups were analyzed at a concentration of 6mm. the results showed that all the isolates except aspergillus terreus (at30) and trichophyton interdigitale (ti60) were inhibited against hydrogen peroxide under oxidative stress. the mechanism of hydrogen peroxide involved in the cytotoxicity in oxygen reduction which generates more reactive and cytotoxic oxygen species such as the hydroxyl radical (•oh) which is a powerful oxidant and can initiate oxidation and cause damage to nucleic acids, proteins, and lipids. these results were similar to the previous study28 in which most of the isolates of dermatophytes shown inhibition against hydrogen peroxide. however the oxidative stress of benzoic acid inhibited at 6mm benzoic acid except acremonium sclerotigenum (as15), trichophyton interdigitale (ti60) and aspergillus terreus (at30), which does not show any effect emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 36 on growth. the mechanism of benzoic acid stress induce the accumulation of benzoic acid in cells, which decrease the cytoplasmic ph of the cell and lead to cell death29. the reason for their resistance against benzoic acid should be their adoption to low ph30. a high concentration of sodium chloride in the environment may cause an intracellular imbalance in the na+/k+ ratio resulting in a loss of potassium and a metabolic disturbance, which reduces the growth of dermatophytes31. this study showed the salt stress at high concentration diminished the fungal growth. study identified that ph 5.6 is a suitable ph for the growth of dermatophyte (data not shown). the mechanism of ph alterations in bioprocess of cytoplasmic enzymes in microorganisms’ effect on the ion uptake from the nutrient medium32. protease enzyme essay was also examined and it was identified that dermatophyte and non-dermatophyte species such as trichophyton mentagrophyte (25), aspergillus terreus (30), trichophyton interdigitale (22), trichophyton interdigitale (3), alternaria alternate (47) and aspergillus terreus (9) produce protease enzyme, which helps in pathogenesis by breaking down the protein on a surface layer of the host and form the colony on the stratum corneum of the skin. these results were identical to the study of elavarashi et al33. c o n c l u s i o n study identified tinea corporis as major cause of fungal infection, more common in the age group of 21-30 years, and the high prevalence rates were found in female as compared to male in the region of southern punjab pakistan. selected isolates from nine fungal groups were further identified by the identification of its gene sequencing. the resistance to azole and polyene group was determined. study revealed amphotericin b and fluconazole found to be the least effective against aspergillus terreus, trichophyton mentagrophyte, aspergillus niger, aspergillus versicolor, trichophyton rubrum and trichophyton interdigitale. the role of various oxidative stresses on fungal morphology was determined and found out that hydrogen peroxide and benzoic acid produce less stress on the growth and morphology of aspergillus terreus and trichophyton metagrophyte. whereas, the 4% nacl stress had not been tolerated by fungal isolates except aspergillus terrus and trichophyton interdigitale which reduced the cellular nutritional uptake and did not ponder the growth retardation. thus, among the four groups of tinea infections, we concluded that in tinea corporis, aspergillus terreus is more resistant to most of the stress responses and do not induce growth arrest in fluconazole and amphotericin b exposure and adapt to the oxidative stresses. the trichophyton interdigitale isolated from tinea cruris infection have stress adaptations and do not induce growth retardation in physiological stress responses. in tinea capitis and tinea pedis acremonium sordidulum and acremonium sclerotigenum were nonresponsive to benzoic acid stress, respectively. fungal protease production concluded that aspergillus terrues, trichophyton mentagrophyte, trichophyton interdigitale and alternaria alternate excrete protease production for the possible adaptations to the oxidative stresses in tinea infections invading. hence, this can be concluded that in dermatophytosis, fungi adapt to the physiological stress and excrete protease enzyme to invade on the human epidermal layer. whereas, the adaptations of the antifungal azole in dermatophytes and non-dermatophytes may be due to the abnormal production of the sterol derivatives in fungal cell. thus, it is suggested to analyze the possible abnormal production of fungal sterols particularly in aspergillus terreus and trichophyton interdigitale. e t h i c a l a p p r o v a l the ethical approval was taken from institutional bioethical research committee (ibc), the islamia university of bhalwalpur, pakistan. c o n f l i c t s o f i n t e r e s t none. f u n d i n g s o u r c e was hec grant no. srgb-1911. a c k n o w l e d g m e n t s author is thankful to dr jameel shaheen from civil hospital, bahawalpur in facilitating the work. l i s t o f a b b r e v i a t i o n s amb amphotericin b bsa bovine serum albumin clsi clinical laboratory standard institute emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 37 dmso dimethyl sulfoxide flc fluconazole hiv human immunodeficiency virus itc itraconazole its internal transcribed spacer ktc ketoconazole mic minimum inhibitory concentration sda sabouraud dextrose agar tb tuberculosis vor voriconazole r e f e r e n c e s 1. bongomin f, gago s, oladele ro, denning dw. global and multi-national prevalence of fungal diseases-estimate precision. j fungi. 2017; 3(4):5763. 2. casadevall a, pirofski l. the damage-response framework of microbial pathogenesis. nat rev microb. 2003; 1(1):17-25. 3. drouhet e, dupont b. chronic mucocutaneous candidosis and other superficial and systemic mycoses successfully treated with ketoconazole. rev infec dis.1980; 2(4):606-19. 4. borgers m, degreef h, cauwenbergh g. fungal infections of the skin: infection process and antimycotic therapy. curr drug tar. 2005; 6(8):849-62. 5. dvorak j, otcenášek m. geophilic, zoophilic and anthropophilic dermatophytes. a review. mycopathol mycol appl. 1964; 23(4):294-6. 6. raheem ademola r, omolade olabowale a, folorunso jamiu b, oluwadun a, onilude a. comparative study of keratinolytic activities of dermatophytes in various keratin substrates. vir mycol. 2013; 2(117):2161-517 7. nweze e, eke i. dermatophytosis in northern africa. mycoses. 2016; 59(3):137-44. 8. nweze e. dermatophytosis in western africa: a review. pak j biol sci. 2010; 13(13):649-56. 9. evans egv, gentles jc. essentials of medical mycology: churchill livingstone; 1985. 10. moto jn, maingi jm, nyamache ak. prevalence of tinea capitis in school going children from mathare, informal settlement in nairobi, kenya. bmc res not. 2015; 8(1):274-85. 11. moammar h, cheriyan g, mathew r, al-sannaa n. incidence and patterns of inborn errors of metabolism in the eastern province of saudi arabia, 1983-2008. ann saudi med. 2010; 30(4):271-7. 12. asadi ma, dehghani r, sharif mr. epidemiologic study of onychomycosis and tinea pedis in kashan, iran. jundishapur. j micro. 2009; 2(2):61-8. 13. sharma v, kumawat tk, sharma a, seth r, chandra s. dermatophytes: diagnosis of dermatophytosis and its treatment. african j micro res. 2015; 9(19):128693. 14. neji s, makni f, cheikhrouhou f, sellami a, sellami h, marreckchi s, et al. epidemiology of dermatophytoses in sfax, tunisia. mycoses. 2009; 52(6):534-8. 15. fuller l, barton r, mohd m, proudfoot l, punjabi s, higgins e. british association of dermatologists' guidelines for the management of tinea capitis 2014. brit j derma. 2014; 171(3):454-63. 16. santos dda, barros meds, hamdan js. establishing a method of inoculum preparation for susceptibility testing of trichophyton rubrum and trichophyton mentagrophytes. j clin micro. 2006; 44(1):98-101. 17. leck a. preparation of lactophenol cotton blue slide mounts. comm eye heal. 1999; 12(30):24-32. 18. samuel t, ebabhi a, adekunle a. identification of some human pathogenic fungi using four dna extraction methods. j app sci envir manag. 2017; 21(6):1079-83. 19. shehata as, mukherjee pk, aboulatta hn, el akhras ai, abbadi sh, ghannoum ma. single-step pcr using (gaca) 4 primer: utility for rapid identification of dermatophyte species and strains. j clin micro. 2008; 46(8):2641-5. 20. taha m, elfangary m, essa s, younes a. species identification of dermatophytes isolated from human superficial fungal infections by conventional and molecular methods. j egyptian wom derm soc. 2017; 14(2):76-84. 21. weinstein mp, lewis js. the clinical and laboratory standards institute subcommittee on antimicrobial susceptibility testing: background, organization, functions, and processes. j clin micro. 2020; 58(3):19. 22. piérard g, piérard-franchimont c, hermanns-lê t, hermanns j, delvenne p. dermatophyte growth in glabrous skin dermatophytoses at immunocompromised hosts. j med diag meth. 2015; 4:1000-186. 23. taha m, elfangary m, essa s, younes a. species identification of dermatophytes isolated from human emergence of antifungal azole resistance in fungal strains vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 38 superficial fungal infections by conventional and molecular methods. j egyptian wom derm soc. 2017; 14(2):76-84. 24. kalita jm, sharma a, bhardwaj a, nag vl. dermatophytoses and spectrum of dermatophytes in patients attending a teaching hospital in western rajasthan, ind j fam med prim car. 2019; 8(4):141826. 25. makimura k, tamura y, mochizuki t, hasegawa a, tajiri y, hanazawa r, et al. phylogenetic classification and species identification of dermatophyte strains based on dna sequences of nuclear ribosomal internal transcribed spacer 1 regions. j clin micro. 1999; 37(4):920-4. 26. martinez-rossi nm, bitencourt ta, peres nt, lang ea, gomes ev, quaresemin nr, et al. dermatophyte resistance to antifungal drugs: mechanisms and prospectus. front micro. 2018; 9:1108-17. 27. balkan c, ercan i, isik e, akdeniz es, balcioglu o, kodedová m, et al. genome-wide elucidation of drug resistance mechanisms for systemically used antifungal drugs amphotericin b, caspofungin and voriconazole in the budding yeast. aac. 2019: 4(2):6876. 28. wirsching s, michel s, morschhäuser j. targeted gene disruption in candida albicans wild‐type strains: the role of the mdr1 gene in fluconazole resistance of clinical candida albicans isolates. mol micro. 2000; 36(4):856-65. 29. maccarthy kg, dahl mv. inhibition of growth of trichophyton rubrum by the myeloperoxidasehydrogen peroxide-chloride system. j invest dermatol. 1989; 92(4):1-7. 30. warth ad. mechanism of action of benzoic acid on zygosaccharomyces bailii: effects on glycolytic metabolite levels, energy production, and intracellular ph. appl envir micro. 1991; 57(12):3410-4. 31. warth ad. resistance of yeast species to benzoic and sorbic acids and to sulfur dioxide. j food prot. 1985; 48(7):564-9. 32. kane j, fischer j. the effect of sodium chloride on the growth and morphology of dermatophytes and some other keratolytic fungi. canad j microb. 1975; 21(6):742-9. 33. elavarashi e, kindo aj, rangarajan s. enzymatic and non-enzymatic virulence activities of dermatophytes on solid media. j clin diag res. 2017; 11(2):23-9. potential of leaf extract of (m. oleifera) as seed priming agent and foliar fertilization of cotton (g.hirsutum) vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 148 op e n ac c e s s f u l l l e n g t h a r t i c l e potential of leaf extract of miracle plant (moringa oleifera l.) as seed priming agent and foliar fertilization of cotton (gossypium hirsutum l.) bushra urooj panhwar1,*, abdullah keerio1, aasia akbar panhwar2, rabia akbar panhwar3, faiz hussain panhwar4, abdul razzaque channa4 1plant physiology / chemistry section, central cotton research institute, sakrand, sindh, pakistan. 2department of food science and technology, s. a. u., tandojam, pakistan. 3northwest a & f university, yangling, shaanxi 712100, china. 4plant breeding and genetics, central cotton research institute, sakrand, sindh, pakistan. a b s t r a c t background: a key desire of a farmer is to produce a higher yield with low inputs. through proper nutrient management, potential yield can be picked but mostly cotton is not grown with balanced nutrition. in this scenario, seed priming and foliar fertilizers have the advantage of quick plant responses. therefore, a cost-effective, farmers-friendly, and full of nutrients supplement should be considered. moringa is known as a miracle plant and its role as a seed priming agent and foliar spray has been observed in many other crops. objective: in this regard, present study was conducted to evaluate the effect of the leaf extract of a miracle plant (moringa oleifera l.) on the growth and development of cotton. methodology: two cotton varieties cris-585 and cris-342 were seeded with an experimental design of split-plot repeated thrice. the experiment included i) control ii) foliar spray of tap water iii) seed priming with leaf extract of miracle plant (mle), iv) seed priming and foliar spray with mle v) foliar spray with mle. the extract was 3% solution. cotton seeds were soaked to complete seed priming for 3 hours. after emergence 30, 60, and 90 days were selected to apply the foliar spray. results: it is proved from the application of leaf extract that seed priming, as well as a foliar spray on cotton crops, had significant (p=0.05) effects on its yield and related parameters. the highest boll weight (3.8 g) and seed cotton yield (3844 kg ha-1) were recorded when a combined application of seed priming and foliar spray was done followed by the sole application of the foliar spray. while the minimum values of boll weight and seed cotton yield were found from the control where no seed priming or foliar spray was done. conclusion: it is concluded that the application of leaf extract of miracle plants as seed priming and foliar spray in cotton crops significantly affect seed cotton yield and its traits. keywords cotton, miracle plant (moringa oleifera l.), seed priming, foliar spray, phytohormone *address of correspondence uroojpanhwar@gmail.com article info. received: august 26, 2021 accepted: october 07, 2022 cite this article: panhwar bu, keerio a, panhwar ak, panhwar ra, panhwar fh, channa ar. potential of leaf extract of miracle plant (moringa oleifera l.) as seed priming agent and foliar fertilization of cotton (gossypium hirsutum l.). 2022; 13(2):148-156. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. o r i g i n a l a r t i c l e potential of leaf extract of (m. oleifera) as seed priming agent and foliar fertilization of cotton (g.hirsutum) vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 149 i n t r o d u c t i o n the outstanding growth and development of the cotton (gossypium hirsutum l.) plant are likely in the balanced presence of water and dissolved minerals (salts) in their rhizosphere, likewise, the role of phytohormones cannot be denied. through proper nutrient management, potential yield can be picked.1,2 in most cases, cotton is not grown with balanced nutrition and needs some supplements. in this scenario, seed priming3 and foliar fertilizers4 have the advantages of quick plant responses, and it is predominantly important when the rhizosphere is unable to fully furnish the plant and the plant itself has inadequate root growth. a major wish of a cotton producer is to get a higher yield with low inputs. here an economical, easy-to-handle, farmers-friendly, and full of nutrients supplement should be considered for foliar spray as well as seed priming. to fulfill these needs, the leaf extract of a miracle plant (moringa oleifera l.) was selected as its role for seed priming and foliar spray fertilization has been observed in numerous studies.5,6mle (leaf extract of miracle plant) is a good source of a derivative (purine adenine) called zeatin of a known plant hormone cytokinin, phenols, vitamins, minerals, antioxidants and a group of companionable solutes that safeguard cells under stresses.7,8 cytokinins are known to stimulate or inhibit numerous plant growth processes and developmental attributes including seed germination. these physiological processes are regulated by cytokinins endogenously as well as when applied exogenously on the intact plant.9 in addition to zeatin, mle is rich in antioxidants, therefore, the defense system of a cotton plant can likely to be improved in response to natural as well as man-made stresses.10therefore, it can be assumed that transmission of zeatin occurs during the paused stage of germination and promotes germination eventually,11when these compounds can be absorbed through a foliar spray.9 seed is the first step that affects and reflects the final yield. seed emergence is a strategic factor in this regard. therefore, it is important to ensure something better regarding its management. it is important to adopt a suitable (economical and low-risk) approach to grow more for the increasing population of pakistan with fewer available resources (land and water) in an exceedingly hostile environment. therefore, seed priming is considered an easy, inexpensive and eco-friendly approach, which was proposed by harris12 to enhance the seed performance during seed germination. it improves germination and establishment, during the phases of flowering and maturation, plant population, seedling length, plant height, and the yield of many crop species13,14 and it also boost tolerance against abiotic stress3, insect pests15,16 and disease.6 successful seed priming is accomplished through water uptake that process occurs in three steps (uptake of moisture, origination of physiological processes, and end of germination and start of seedling) respectively.17 abundant literature is available showing the importance of foliar fertilization. it is a good addition to soil application. spraying through leaf extract of miracle plants has proved some prominent effects such as a longer and more vigorous lifetime and more resistance to pests and diseases6. this validates its prospective to be used as a foliar spray to hasten plant growth and development of cotton. as synthetic sources of growth-promoting hormones are costly, there is a need to explore natural sources which are reasonable, environmentally defensive farmer friendly, and realistic under natural conditions.10 therefore, it was decided to study the response of mle as a priming agent and foliar supplement. m a t e r i a l a n d m e t h o d s experimental site and conditions: an experiment was established at central cotton research institute (ccri), sakrand, sindh, pakistan (latitude 26.0996930, longitude 68.2996450). the experimental trial was arranged in a split-plot design replicated thrice. sowing was done on 15th may 2020 of two approved cotton varieties viz. cris585 and cris-342. recommended doses i.e., 145 kg n ha-1, 56 kg p ha-1, and 62 kg k ha-1 were applied to the soil. potassium and phosphorus were applied at the seedbed preparation for crop husbandry and nitrogen was in the first, middle, and last splits followed by sowing, budding, and peak flowering stages. weather condition is presented in fig 1, 2, and 3 and soil analysis is presented in table 1 as well. all other recommended agronomic practices were done according to the requirements. potential of leaf extract of (m. oleifera) as seed priming agent and foliar fertilization of cotton (g.hirsutum) vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 150 table 1. soil characteristics of the experimental plot before sowing of cotton crop. parameters results from various soil depths cm soil depth cm 0-15 15-30 30-60 ph 8.1 7.9 8.0 conductivity, dsm-1 1.47 1.46 1.48 available nutrients, mg kg-1 nitrate nitrogen (no3-n) 8.6 7.9 6.6 phosphorus (p) 4.1 2.2 1.7 potassium (k) 120 101 110 figure 1. average maximum and minimum temperature c0 figure 2. mean relative humidity%. 2 9 .8 3 9 .9 4 1 .6 4 1 .7 4 0 .7 3 6 .5 3 8 .2 3 4 .9 1 4 .3 2 2 .3 2 4 .7 2 7 .5 2 7 .8 2 5 .3 2 4 .9 2 0 .8 m a r c h a p r i l m a y j u n e j u l y a u g u s t s e p t e m b e r o c t o b e r month average maximum temperature average minimum temperature 5 1 .2 3 8 .8 3 7 .6 4 6 .2 4 8 .9 5 9 .5 5 5 5 4 .1 m a r c h a p r i l m a y j u n e j u l y a u g u s t s e p t e m b e r o c t o b e r month mean relative humidity % potential of leaf extract of (m. oleifera) as seed priming agent and foliar fertilization of cotton (g.hirsutum) vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 151 figure 3. average rainfall mm figures 1, 2, and 3 followed the average maximum and minimum temperature c0, mean relative humidity %, and average rainfall mm, respectively, at ccri, sakrand in 2019. the details of the experimental units were set as: i) control (without priming and foliar spray), ii) foliar spray of simple water used for extract preparation (fsw), iii) seed priming with mle 3% solution (spm), iv) seed priming and foliar spray mle 3% solution (spfs), v) foliar spray with mle 3% solution (fs). preparation of leaf extract: zeatin is rich in young leaves and tender branches18, therefore, young leaves were detached from the tree standing at ccri, sakrand. leaves were rinsed thoroughly and after rinsing 30g of leaves were grounded using a pestle mortar. the ground material was then added to 1l tap water and stirred for some movement thoroughly. staining was done through a fine cotton cloth to prepare approximately 3% solution.19,20 usually, 6-8 tanks (20 l) capacity is used per acre.6 cottonseed priming: cotton seeds were soaked in a 3% solution of mle for 3 h at a 1:5 seed-to-solution ratio.21 after priming, cottonseeds were sown directly. foliar spray: because of the peak nutrient requirement stages of the cotton crop, mle was sprinkled at 30 (one month after sowing), 60 (flowering), and 90 (boll opening) days after sowing (figure.5). electrical conductivity of seed leachates: for determination of electrolyte leakage, one gram of cotton seeds was soaked in 50 ml of deionized water. before soaking, seeds were cleaned and air-dried. electrical conductivity was measured in µs cm-1 g-1 starting from 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, and finally at 24 hours after soaking. statistical analysis: the data was analyzed using the software statistic. the usa. ver. 8.1. mean separations were done by sed, lsd, and tukey's pair-wise test. the analysis of variance (anova) was done by following a two-factor factorial completely randomized design. r e s u l t s a n d d i s c u s s i o n this study observes the potential of leaf extract of miracle plant (mle) as a natural plant growth enhancer for cotton crops. its analysis shows that they have substantial quantities of mineral essential nutrients. furthermore, it is enriched with antioxidants (enzymatic as well as nonenzymatic) that promotes it as a natural growth enhancer.10 mle was evaluated for its potential to act as a seed priming medium and foliar spray solution to improve growth and yield in cotton. the hypothesis was developed that the growth, development, and seed cotton yield constraint can be overcome by seed priming and foliar spray thus exploring the easily adoptable best application for cotton crops. it has been reported to hasten the growth and yield of wheat crops3 and improve resistance to pests and diseases.6 its method of application (seed priming and foliar spray) is also important to affect growthenhancing characteristics. in former studies, it has been 4 0 2 4 0 9 2 4 9 4 7 1 3 m a r c h a p r i l m a y j u n e j u l y a u g u s ts e p t e m b e ro c t o b e r month average rainfall mm potential of leaf extract of (m. oleifera) as seed priming agent and foliar fertilization of cotton (g.hirsutum) vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 152 witnessed that seed priming has increased the seed germination and seedling vigor along with final yield in wheat crops3 while working with cotton crops its positive effects have been observed on growth and development and contending cotton leaf curl virus disease using foliar spray on cotton crop.2 in the present study the results regarding plant height (cm) at 75, 90, and 135 days after sowing (das) are statistically non-significant (p=0.05) but visually good growth was observed from all the treatments either through seed priming or foliar spray on both varieties as compared to control (table 2). the results are accorded with the findings of panhwar et al.,3 where both vegetative and reproductive growth was increased with the use of mle on wheat crops. this also ratifies that mle has the potential to show long-term effects at later growth stages after germination. as mentioned earlier it is gifted by nature in having a substantial source of zeatin a plant hormone and other bio-stimulating compounds along with essential nutrients, antioxidants, and a complete family of amino acids in ready-to-take form7,10therefore exogenously applied mle through the foliar spray and seed priming improved the plant defense system, secondary metabolites and antioxidative system that is why it may be assumed that these compounds have transferred to the growing seed during the germination and boosted amylase movement that increased starch breakdown11, eventually improving successive growth and development of a cotton plant. as it has already been processed by grinding hence it may release a higher amount of k+, ca2+, mg2+, and po43and other nutrients as mentioned earlier when dissolved in water that might have transferred to seed during imbibition. on average, the maximum value of plant height (160.8 cm) at 135 das was noted from seed priming + foliar spray of mle 3% solution (t4) and the minimum value (147.8 cm) was observed from control (t1). improvement in height of cotton plants that emerged from primed seeds was possibly due to the excellent source of all essential nutrients which are known to promote the growth and development of a plant. a similar trend was observed in the number of bolls counted in plant 1 (table 3). on average, the highest number of bolls counted plant-1 (65.2) was noted from t4 and the minimum (35.8) was from control (t1) recorded at 135 das. in addition, the cris-585 produced the highest plant height and the number of bolls counted plant-1 as compared to cris-342 but statistically non-significant (p=0.05). as stated by foidl et al.,10both vegetative as well as reproductive growth were probably increased due to the presence of essential nutrients, ascorbates, zeatine, etc in mle. table2. plant height (cm) recorded at various growth stages. treatments variety plant height (cm) das 75 90 135 t1(control) cris-585 112.90b 17.73c 153.47ab cris-342 111.03b 17.26c 142.20b t2 (fs w) cris-585 115.77b 19.633bc 158.20ab cris-342 114.50b 21.933bc 155.67ab t3 (sp) cris-585 130.40ab 33.067abc 154.87ab cris-342 131.13ab 31.867abc 151.20ab t4 (sp+fs mle) cris-585 137.67a 39.400a 164.60a cris-342 128.13ab 34.200ab 157.00ab t5 (fs mle) cris-585 137.23a 38.033a 156.40ab cris-342 125.80ab 33.900ab 152.07ab lsd values (5%) treatment (t) 12.84 9.01 ns variety (v) 4.64 ns 3.45 t×v ns ns ns sp = seed priming, w = water, fs = foliar spray, mle = leaves extract of miracle plant. the means sharing the same letters did not differ significantly at p= 0.05. potential of leaf extract of (m. oleifera) as seed priming agent and foliar fertilization of cotton (g.hirsutum) vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 153 table3. number of bolls counted plant-1 at various growth stages. treatments variety number of bolls counted plant-1 das 75 90 135 t1(control) cris-585 1.93c 17.73c 36.27a cris-342 3.00bc 17.26c 35.40a t2 (fs w) cris-585 4.20bc 19.63bc 37.13a cris-342 1.70c 21.93bc 63.93a t3 (sp) cris-585 9.50ab 33.06abc 53.80a cris-342 6.20bc 31.86abc 72.00a t4 (sp+fs mle) cris-585 8.60abc 39.40a 97.40a cris-342 4.20bc 34.20ab 102.93a t5 (fs mle) cris-585 15.70a 38.03a 48.27a cris-342 6.30bc 33.90ab 52.87a lsd values (5%) treatment (t) 2.10 ns 8.34 variety (v) 3.41 10.59 61.2 t×v ns ns ns sp = seed priming, w = water, fs = foliar spray, mle = leaves extract of miracle plant. the means sharing the same letters did not differ significantly at p= 0.05. table 4. boll weight (g) and seed cotton yield (kg ha-1). treatments variety boll weight (g) seed cotton yield (kg ha-1) t1 (control) cris-585 3.52ab 2330b cris-342 3.11b 2563ab t2 (fs w) cris-585 3.58ab 2873ab cris-342 3.13b 3106ab t3 (sp mle) cris-585 3.89ab 2796ab cris-342 3.20b 3339ab t4 (sp+fs mle) cris-585 4.10a 4194a cris-342 3.64ab 3495ab t5 (fs mle) cris-585 3.82ab 3029ab cris-342 3.63ab 3339ab lsd values (5%) treatment (t) 0.34 ns variety (v) 0.25 ns t×v ns 813.26 sp = seed priming, w = water, fs = foliar spray, mle = leaves extract of miracle plant. the means sharing the same letters did not differ significantly at p= 0.05. potential of leaf extract of (m. oleifera) as seed priming agent and foliar fertilization of cotton (g.hirsutum) vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 154 figure 4. seed membrane permeability test. t1= no priming, t2= mle priming, v1= cris-342 and v2= cris-585 figure 5. pictorial view of foliar spray of mle on cotton crop taken at the experimental field of ccri sakrand during the experiment (used, photographs taken by author during, 2020). application of mle as seed priming or foliar spray in cotton crops significantly (p=0.05) affected seed cotton yield and boll weight g (table 4). on average the highest boll weight (3.8 g) and seed cotton yield (3844 kg ha-1) were recorded from t4, followed by t5 (3.7 g and 3184 kg ha-1) and t3 (3.5 g and 3068 kg ha-1), respectively. while the minimum values of boll weight (3.3 g) and seed cotton yield (2446 kg ha-1) were found in the control plot (t1). the increased seed cotton yield of primed seed was possibly the result of the initialization of metabolic potential of leaf extract of (m. oleifera) as seed priming agent and foliar fertilization of cotton (g.hirsutum) vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 155 reactions that enhanced embryo growth14, which eventually hastened cell division. also as mentioned earlier ascorbate-enriched mle increases ascorbic acid contents which promotes growth and development and ultimately final yield when applied exogenously. to assess the vigor and viability of seeds a possible test can be done which is the electrical conductivity (ec) of seed leachates (water that has infiltrated through a seed with some of its constituents). from ec ds/m values we can conclude that lower ec shows high vigor and vice versa. also, it is negatively correlated to germination and positively correlated to damaged seeds as it shows the integrity of the cell membrane.22,23 it has been observed from the present study that seed soaking treatment remains effective in decreasing the electrolyte conductivity of cotton seed leachates, which shows plasma membrane strength and stability, therefore, treated seeds has the lowest ec than the control. (figure 4). c o n c l u s i o n the results indicated that the application of leaf extract of miracle plants as seed priming or foliar spray in cotton crops significantly affected seed cotton yield and its traits. it is observed from the present study that seed soaking treatment is effective in decreasing the electrolyte conductivity of cotton seed leachates, which shows plasma membrane strength and stability. e t h i c a l a p r o v a l none. c o n f l i c t o f i n t e r e s t none. f u n d i n g s o u r c e none. a c k n o w l e d g e m e n t s none. l i s t o f a b b r e v i a t i o n s das days after sowing ec electrical conductivity fs foliar spray fsw foliar spray of simple water mle leaf extract of miracle plant spfs seed priming and spray mle 3% spm seed priming with mle r e f e r e n c e s 1. panhwar rb, akbar a, panhwar bu, panhwar ga, bai-li f. effects of plant spacing and nitrogen fertilizer levels on cotton yield and growth. int j sci environ technol. 2018;7(1):313–24. 2. urooj panhwar b. reducing the incidence of cotton leaf curl virus disease through potassium nutrition of cotton (gossypium hirsutum l.). acad lett [internet]. 2022 apr 12; available from: https://www.academia.edu/76414465/reducing_t he_incidence_of_cotton_leaf_curl_viru s_disease_through_potassium_nutrition _of_cotton_gossypium_hirsutum_l_ 3. panhwar bu. seed priming… a cohort to boost wheat harvest in a hostile environment. acad lett [internet]. 2021 oct 28; available from: https://www.academia.edu/60636731/seed_priming_ a_cohort_to_boost_wheat_harvest_in_a_hostile_envi ronment 4. oosterhuis d. foliar fertilization: mechanisms and magnitude of nutrient uptake. proc fluid forum. 2009;15–7. 5. farooq m, usman m, nadeem f, rehman h ur, wahid a, basra sma, et al. seed priming in field crops: potential benefits, adoption, and challenges. crop pasture sci [internet]. 2019;70(9):731. available from: http://www.publish.csiro.au/?paper=cp18604 6. panhwar bu, keerio a, shah n, panhwar aa, panhwar rb, magsi wa, et al. considering leaf extract of miracle tree (moringa oleifera l.) and potassium nutrition for contending cotton leaf curl virus (clcuv) disease of cotton (gossypium hirsutum l.). j appl res plant sci [internet]. 2022 jul 1;3(02):229–35. available from: https://joarps.org/index.php/ojs/article/view/67 7. fuglie lj. the miracle tree: moringa oleifera: natural nutrition for the tropics. church world serv dakar. 2001;172. 8. arif m, kareem s, ahmad n, hussain n, yasmeen a, anwar a, et al. exogenously applied bio-stimulant and synthetic fertilizers to improve the growth, yield potential of leaf extract of (m. oleifera) as seed priming agent and foliar fertilization of cotton (g.hirsutum) vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 156 and fiber quality of cotton. sustainability [internet]. 2019 apr 11;11(7):2171. available from: https://www.mdpi.com/2071-1050/11/7/2171 9. fageria nk, filho mpb, moreira a, guimarães cm. foliar fertilization of crop plants. j plant nutr [internet]. 2009 may 14;32(6):1044–64. available from: http://www.tandfonline.com/doi/abs/10.1080/0190416 0902872826 10. foidl n, makkar hps, becker k, foild n, km s. the potential of moringa oleifera for agricultural and industrial uses. what dev potential moringa prod. 2001;1–20. 11. farooq m, basra sm, wahid a, ahmad n. changes in nutrient-homeostasis and reserves metabolism during rice seed priming: consequences for seedling emergence and growth. agric sci china [internet]. 2010 feb;9(2):191–8. available from: https://linkinghub.elsevier.com/retrieve/pii/s16712927 09600833 12. harris d, tripathi r, research a d. on-farm seed priming to improve crop establishment and yield in dry direct-seeded rice. direct seeding: research strategies and opportunities. booksgooglecom [internet]. 2002;231–40. available from: https://books.google.com/books?hl=fr&lr=&id=ifl_oxh xb5oc&oi=fnd&pg=pa231&dq=harris+d.,+tripathi+r .s.,+joshi+a.+(2002)+onfarm+seed+priming+to+improve+crop+establishment +and+yield+in+dry+directseeded+rice,+in:+pandey+s.,+mortimer+m.,+wade+ l.,+tuong+t.p.,+ 13. basra sma, farooq m, tabassum r, ahmad n. physiological and biochemical aspects of pre-sowing seed treatments in fine rice (oryza sativa l.). seed sci technol [internet]. 2005 oct 1;33(3):623–8. available from: http://www.ingentaconnect.com/content/ista/sst/2005/ 00000033/00000003/art00009 14. nawaz j, hussain m, jabbar a, nadeem ga, sajid m, subtain m, et al. seed priming a technique. int j agric crop sci. 2013;6(20):1373–81. 15. urooj panhwar b, keerio a. suppressing the sucking insect pests of cotton through repulsive moringa (miracle) tree leaf extract – a review. acad lett [internet]. 2022 jun 4; available from: https://www.academia.edu/81125201/suppressing_th e_sucking_insect_pests_of_cotton_through_repulsive _moringa_miracle_tree_leaf_extract_a_review 16. panhwar bu. repelling insidious and burgeoning pests of cotton. acad lett [internet]. 2021 jul 7; available from: https://www.academia.edu/49919985/repelling_in sidious_and_burgeoning_pests_of_cott on 17. bewley jd. seed germination and dormancy. plant cell [internet]. 1997 jul 1;1055–66. available from: https://academic.oup.com/plcell/article/9/7/10551066/5986415 18. iqbal m, ashraf m. presowing seed treatment with cytokinins and its effect on growth, photosynthetic rate, ionic levels and yield of two wheat cultivars differing in salt tolerance. j integr plant biol [internet]. 2005 nov;47(11):1315–25. available from: https://onlinelibrary.wiley.com/doi/10.1111/j.17447909.2005.00163.x 19. batool s, khan s, basra sma. foliar application of moringa leaf extract improves the growth of moringa seedlings in winter. south african j bot [internet]. 2020 mar;129:347–53. available from: https://linkinghub.elsevier.com/retrieve/pii/s02546299 19300043 20. khan s, ibrar d, bashir s, rashid n, hasnain z, nawaz m, et al. application of moringa leaf extract as a seed priming agent enhances growth and physiological attributes of rice seedlings cultivated under water deficit regime. plants [internet]. 2022 jan 19;11(3):261. available from: https://www.mdpi.com/2223-7747/11/3/261 21. nouman w, siddiqui mt, basra sma, afzal i, rehman hu. enhancement of emergence potential and stand establishment of moringa oleifera lam. by seed priming. turkish j agric for [internet]. 2012 jan 1; available from: https://journals.tubitak.gov.tr/agriculture/vol36/iss2/9 22. full issue. international rules for seed testing. int rules seed test [internet]. 2016;2016(1):1–384. available from: https://doi.org/10.15258/istarules.2016.f 23. marin m, laverack g, powell aa, matthews s. potential of the electrical conductivity of seed soak water and early counts of radicle emergence to assess seed quality in some native species. seed sci technol [internet]. 2018 apr 1;46(1):71–86. available from: http://www.ingentaconnect.com/content/10.15258/sst. 2018.46.1.07 chemical characterization of treated & rejected wastewater of ro plants vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 60 op e n ac c e s s f u l l l e n g t h a r t i c l e chemical characterization of treated and rejected wastewater of reverse osmosis treatment plants in the area of allama iqbal town, lahore naveed aslam dogar1,2,*, haroon iftikhar2, noor-ul-ain khalid3, muhammad shahid4, sajjad ahmad2 1department of chemistry, government college university, lahore, pakistan. 2department of chemistry, government college of science, wahdat road lahore, pakistan. 3department of chemistry, forman christian college university, lahore, pakistan. 4department of chemistry, lahore garrison university, lahore, pakistan. a b s t r a c t background: water is one of the most essential requirements of life. life is not possible without water. polluted water on the other hand can affect the health badly. reverse osmosis (ro) plants are used to remove dissolved solids including harmful and toxic materials from wastewater. objectives: to investigate the quality of 6 treated and rejected wastewater samples of ro plants water being consumed in the areas of college block, hunza block of allama iqbal town, and gulberg iii lahore, pakistan. methodology: both qualitative and quantitative analyses were done by using the parameters like chemical oxygen demand (cod), biological oxygen demand (bod), total dissolved solids (tds), cl-, ph, nitrate, nitrite, so42-, na+, k+ and heavy metals like cr3+, fe3+, cu2+, zn2+ and mn2+ through flame emission spectroscopy, uv-vis spectroscopy, atomic absorption spectroscopy, and volumetric analysis. results: the sample analysis indicated that these parameters lie within the permissible limits with reference to national environmental quality standards (neqs) values with some exceptions. the ph of treated water of college block (sample a) was 9.219, which is slightly higher than normal ph value, which is between 6.5-8.5. the value of cr3+ ion in rejected water of gulberg iii was 0.06ppm, which is also higher than the normal limit. conclusion: all the parameters of treated and rejected wastewater indicated the suitability of water samples for population of respective areas, but the values of rejected wastewater are towards an increase, and should therefore be treated before dumping. keywords biochemical oxygen demand, chemical oxygen demand, heavy metals, ph, ro plant, rejected wastewater. *address of correspondence naveedaslamdogar@gmail.com article info. received: june 11, 2021 accepted: april 19, 2022 cite this article dogar na, iftikhar h, khalid na, shahid m, ahmad s. chemical characterisation of treated and rejected wastewater of reverse osmosis treatment plants in the area of allama iqbal town, lahore. 2022; 13(1):60-69. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n when two moles of hydrogen and one mole of oxygen combine together, we get a colorless and odorless compound. this product is known as water. it is an essential requirement of every living cell without which life is impossible. water is a universal solvent or solvent of life. a cell contains more than 70% of water. the human body contains almost 60% of water in which the brain and heart almost contain 73%, lungs contain 83%, skin has 64%, o r i g i n a l a r t i c l e chemical characterization of treated & rejected wastewater of ro plants vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 61 muscles and kidneys contain 79% and the bones contain 31% water1. an average human body requires almost 2000ml to 2500ml of water but this may vary according to the temperature, pressure, and by other environmental conditions. water is very essential for normal life, but polluted water can badly effect the human health and can cause deadly water borne diseases like vomiting, diarrhea, e. coli infections, typhoid, dysentery, hepatitis a, b, c and e, etc2. both natural and human activities are responsible for water pollution. volcanic eruptions, earthquakes, tsunamis, etc. are natural sources but they are not as harmful as anthropogenic activities related to the generation of industrial domestic and commercial waste3. the government of punjab has taken some serious actions in 2013 and installed many ro plants for the purification of drinking water in different areas of lahore, pakistan. this study was planned for the chemical characterization of treated and rejected wastewater of reverse osmosis treatment plants in the area of allama iqbal town and gulberg iii, lahore4. this research has extreme benefit in gaining a better understanding of the water quality in these areas. m a t e r i a l s a n d m e t h o d s all the treated and rejected water samples were collected from reverse osmosis plants of college block and hunza block of allama iqbal town and gulberg iii, lahore, pakistan. tests for chloride (cl-) determination apparatus and chemicals required for the determination included 20ml to 25ml burette graduated in 0.1ml, burette support, 100ml graduated cylinder, titrating flask, beakers, pipette, silver nitrate solution and potassium chromate as an indicator. agno3 (0.16m) solution was prepared by adding 2.73g of agno3 in 100ml of distilled water in a burette. water (10ml) was pipetted out, which was to be tested in the titrating flask. two to 3 drops of potassium chromate were added to the flask as an indicator. it was titrated against the standard solution till agno3 turned red. the volume of agno3 used was recorded till the end point5. biological oxygen demand (bod) analysis the apparatus required for bod were 20ml to 25ml burette, burette support, 100ml graduated cylinder, titrating flask, beakers, pipette, reagents, silver nitrate solution, potassium chromate as an indicator, 500ml conical flask, pipette bulb, pipette with elongated tips and 250ml graduated cylinders and washed bottles. chemicals required were calcium chloride, magnesium sulphate, ferric chloride, di-sodium hydrogen phosphate, potassium di-hydrogen phosphate di-sodium hydrogen phosphate, ammonium chloride, manganese sulphate, potassium hydroxide, potassium iodide, sodium azide, conc. sulphuric acid, starch indicator, sodium thiosulphate and distilled water. four (300ml) bod bottles were taken and 10ml of sample was added in two of them while the remaining two were filled with diluted water alone for blank. glass stoppers were placed to preserve one blank and one sample in the bod incubator at 20°c. the other two bottles containing one sample and one blank were investigated immediately. then, 2ml of alkali iodide azide reagent was added. sufficient time was required for complete reaction with oxygen. next, 2ml of conc. h2so4 was added and 10ml of the solution was pipetted out from the bottle and transferred to the erlenmeyer flask, which was then standardized with sodium thiosulphate solution. when the solution became pale yellow, starch indicator was added to it which turned the solution blue. titration was continued till blue color turned to colorless (endpoint). the titration process was repeated for concordant readings. after 5 days, the incubated sample and blank bottles were titrated to find dissolved oxygen (do) value in mg/l. the titration process was repeated for concordant readings6, 7. chemical oxygen demand (cod) determination the apparatus included cod digester, burette and burette stand, cod vials with stand, 250ml erlenmeyer flask, pipette & pipette bulb, tissue paper, and wash bottles. the chemicals required were potassium dichromate, conc. sulfuric acid, ferrous ammonium sulphate (mohar salt), silver sulphate, ferroin indicator, and organic free distilled water. reagents required included 0.25n solution of potassium dichromate, 0.1n ferrous ammonium sulphate solution, chemical characterization of treated & rejected wastewater of ro plants vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 62 and ferroin indicator. concentrated sulphuric acid was also required. h2so4 and the sample were taken in a reflux flask and 10ml of 0.25n k2cr2o7, h2so4 and agno3 were added reagent in it, mixed and refluxed for two hours. then, 150ml distilled water was added to dilute it. the indicator was added till the color changed from green to wine red, which is the end point. the experiment was performed against blank8. total alkalinity determination alkalinity is due to the presence of co2, hco31-, co32, and ohetc. it may come in water form acid rain and earths’ natural buffering system etc. it can be determined easily by using methyl-orange as an indicator and n/50 sulphuric acid solution titration9. ph determination ph meter is used for the determination of ph. standard buffer solution was used for the calibration of glass electrode and then ph of the sample was measured10. determination of total hardness buffer solution of ph 10 was used along with erichrome black-t indicator and edta as a standard solution (0.01m). value of caco3 in ppm expressed the total hardness of water11. determination of calcium ions standard edta solution of 0.01m was used along with ebt and buffer of ph 10. the sample was taken and boiled to remove bicarbonates. then, it was titrated against edta solution using ebt as an indicator12. determination of nitrate and nitrite ions in water nitrate ion can be measured spectroscopically. salicylic acid under basic conditions forms a stable complex with nitrate ion, which can be estimated by a spectrophotometer at 410nm. chromophore absorption is directly proportional to amount of nitrate present. blank is prepared using distilled water with simple normal reagent. for the determination of nitrite ion, spectrometer, pipette, glass stopper flask, beaker and distilled water were required. colored reagents were produced by adding 100ml of 85% phosphoric acid and 10ml sulphanilamide mixed in 800ml of water. n-1-naphthylethylene diamine dihydrochloride (1g) was added and diluted up to 1000ml by distilled water. this coloring agent was then stored in the dark. further, 0.05n sodium oxalate was prepared, followed by the preparation of a stock nitrite solution of 0.018n. this solution required 1ml of chcl3 for its preservation. standard 0.05n kmno4 was also used in this analysis. further, 50ml of sample was taken and 2ml of coloring reagent was added to it as a chromophoric reagent. the absorbance was measured at 543nm, followed by a waiting period of 10min to 120min after addition of the coloring agent. standard curve was used to estimate the sample nitrite concentration13. measurement of total dissolved solids (tds) first, 100ml of filtered water was taken using the whatmann filter paper. the water was evaporated in an electric oven at 110°c. the amount of solid residue in the sample was then weighed14. formula used was: tds (mg/l) = [(a-b)*1000*1000] / sample volume (ml) where, a = weight of dried residue + dish (g) b = weight of dish (g) determination of heavy metals atomic absorption spectroscopy (aas) is used for the determination of heavy metals. standard solution (5ppm, 10ppm, 15ppm and 20ppm) of fe, cr, pb, cd and mn were prepared and tested by aas. comparison was used between the standard and unknown sample to determine heavy metals in water15. estimation of na+ and k+ ion by the help of flame photometric method after calibration of the instrument with the help of standard and adjusting the reading between 0-10mg/l and 0-100mg/l, distilled water was aspirated to bring zero mark reading and the sample was applied to the flamephotometer. the readings were accordingly noted16. determination of sulphate concentration in water sample for this determination, magnetic stirrer, physical balance, measuring cylinder along with spectrophotometer etc. were used. then, 50ml of the sample was taken along with buffer and 2ml conditioning agent. a pinch of bacl2 was added and stirred for 1min at a defined speed. its absorbance was measured at 420nm. afterwards, 5mg to 40mg of standard curve was used for the determination of sulphate ions17. chemical characterization of treated & rejected wastewater of ro plants vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 63 iron determination porcelain dish, measuring cylinder, glass rod, wash bottles, iron wire and spectrometer etc. were the apparatus used for iron determination. glacial acetic acid, hcl, ammonium acetate buffer, hydroxyl amine hydrochloride solution 1,10-phenenthroline solution, ammonium ferrous sulphate solution and 0.1n kmno4 were the reagents required for this estimation. water sample (50ml) was taken and 2ml hcl was added in it. the solution was heated till its volume reached to 20ml. 10ml of ammonium acetate buffer and 4ml of 1,10phenenthroline were added in it. the solution was then incubated in the dark for 20min and then absorbance was measured at 510nm. the sample value was compared with the calibration curve obtained from known concentration18. r e s u l t s samples a and a* represent the treated and rejected water of college block, respectively. samples b and b* represent the treated and rejected water of hunza block, respectively. similarly, samples c and c* represent the treated and rejected water of gulberg iii block, respectively. the parameters like ph, tds, total hardness, ca2+ ions, cl1ions, total alkalinity, bod, cod19 values are given in table 1. all these values were compared with national environmental quality standards. table 1 shows that the treated sample a (collected from the college block ro plant) has a ph value greater than neqs, therefore it is not much suitable for drinking. all other parameters of all the samples lie within the permissible limits. table 1. sample parameters measured for treated sample a (collected from the college block ro plant). parameters a a* b b* c c* neqs ph 9.239 7.965 8.262 7.940 7.715 7.619 6.5-8.5 tds 120 180 100 110 200 230 <1000 total hardness 25 93 26 37 33 86 <500 ca2+ 40 65 18 88 40 93 <500 total alkalinity 80 130 94 128 102 288 <300 cl113 20 15 25 30 85 <250 cod 56 74 33 55 10 69 150mg/l bod 26 58 12 18 28 60 80mg/l figure 1. ph values of all the samples. figure 2. tds values of all the samples. chemical characterization of treated & rejected wastewater of ro plants vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 64 figure 3. total hardness of all the samples. figure 4. total ca+2 ions in all the samples figure 5. total alkalinity of all the samples. figure 6. total cl-1 ions in all the samples. figure 7. cod values of all the samples. figure 8. bod values of all the samples. 0 200 400 600 a a* b b* c c* neqs ca2+ chemical characterization of treated & rejected wastewater of ro plants vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 65 table 2. measurement of ions in all the ro plant samples. metal a a* b b* c c* neqs (ppm) fe3+ 0.00 0.00 0.00 0.00 0.00 0.00 2.0 zn2+ 0.00 0.00 0.00 0.00 0.00 0.00 0.01 cu2+ 0.00 0.00 0.00 0.00 0.00 0.00 <0.05 mn2+ 0.00 0.00 0.00 0.00 0.00 0.00 <0.05 k+ 1.4567 3.5674 1.05 2.7768 1.4612 3.4013 12 na+ 0.00 0.00 0.00 0.00 0.00 0.00 250 table 3. measurement of na+ / k+ levels in all the ro plant samples. parameters (ppm) a a* b b* c c* neqs (mg/l) na+ 30 65 40 88 21 22 250mg/l k+ 2 5 4 7 5 6 12mg/l figure 1 represented that the ph value of treated water from the college block is not within the neqs permissible limit, while fig. 2 to fig. 8 showed that all other parameters such as total alkalinity, total hardness, tds, ca+2 and cl-1 ions concentration, bod and cod values are within the permissible limits, and the treated water sample can be used and is safe for drinking purposes. by using atomic absorption spectroscopy, the metals like fe3+, zn2+, cu2+, mn2+, k+ and na+ were estimated and their values are given in table 2. it shows that only k+ ions are present in all the ro plant samples. the concentration of k+ ions lies within the safety limit of neqs values. na+ and k+ ions were estimated by flame photometric method (table 3). the values lie within the safety limits. uv-visible spectrophotometer studies were conducted to find the ppm percentage of no21-, no31-, fe3+, cr3+, and so41-. from the data in table 4, it can be concluded that the rejected water of gulberg iii contained 0.06ppm of cr3+, which exceeds the safety limits of neqs. the sample of rejected water (sample b*) of hunza block had 42.20ppm of no21which is very high as compared to neqs, which is 12ppm only. variations of different parameters like ph, tds, total hardness, ca2+, total alkalinity, and clion for the treated and rejected water of hunza block, college block and gulberg iii were investigated for seven days as shown in tables 5-10. table 4. measurement of specific cations and anions level in all the ro plant samples. parameters (ppm) a a* b b* c c* neqs (ppm) no310.000 0.085 0.004 0.000 0.000 0.000 12 no220.00 0.002 5.747 42.20 0.044 0.110 12 so421.321 92.62 23.34 27.46 29.62 27.26 1000 fe3+ 0.151 0.108 0.744 0.02 0.173 0.220 2.0 cr3+ 0.00 0.001 0.010 0.00 0.003 0.06 <0.05 chemical characterization of treated & rejected wastewater of ro plants vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 66 table 5. measurement of different parameters in hunza block (treated water) sample. sample hunzablock parameters (ppm) 1stday (ppm) 2ndday (ppm) 3rdday (ppm) 4thday (ppm) 5thday (ppm) 6thday (ppm) 7thday (ppm) 01 ph 6.9 7.2 7.95 7.75 7.8 7.95 7.81 02 tds 1.5 2.0 8.0 4.01 3.04 1.80 2.00 03 total hardness 50 56 33 76 31 53 61 04 ca2+ 85 88 88 90 85 89 86 05 total alkalinity 92 101 79 83 105 36 45 06 cl125 28 21 18 17 24 19 table 6. measurement of different parameters in hunza block (rejected water) sample. sample hunzablock parameters (ppm) 1stday (ppm) 2ndday (ppm) 3rdday (ppm) 4thday (ppm) 5thday (ppm) 6thday (ppm) 7thday (ppm) 01 ph 6.95 7.26 7.05 7.65 7.88 7.95 8.01 02 tds 257 230 150 311 324 180 200 03 total hardness 101 177 303 116 201 153 101 04 ca2+ 115 168 88 260 115 275 111 05 total alkalinity 92 110 119 88 155 163 155 06 cl1115 98 141 37 111 204 19 table 7. measurement of different parameters in college block (treated waste water) sample. sample college block parameters (ppm) 1stday (ppm) 2ndday (ppm) 3rdday (ppm) 4thday (ppm) 5thday (ppm) 6thday (ppm) 7thday (ppm) 01 ph 7.5 7.35 7.45 7.25 7.86 7.68 7.50 02 tds 125 126 110 120 118 125 127 03 total hardness 98 102 92 97 90 97 91 04 ca2+ 60 65 70 65 70 68 72 05 total alkalinity 88 81 94 87 80 85 88 06 cl114 22 29 36 31 20 13 table 8. measurement of different parameters in college block (rejected waste water) sample. sample college block parameters (ppm) 1stday (ppm) 2ndday (ppm) 3rdday (ppm) 4thday (ppm) 5thday (ppm) 6thday (ppm) 7thday (ppm) 01 ph 6.5 7.15 7.45 6.25 8.86 7.08 7.04 02 tds 448 156 350 430 128 445 527 03 total hardness 228 132 302 207 210 307 411 04 ca2+ 104 225 110 215 118 182 372 05 total alkalinity 266 319 401 213 120 115 88 06 cl1133 221 194 200 103 119 103 chemical characterization of treated & rejected wastewater of ro plants vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 67 table 9. measurement of different parameters in gulberg iii (rejected water) sample. sample gulberg iii parameters (ppm) 1stday (ppm) 2ndday (ppm) 3rdday (ppm) 4thday (ppm) 5thday (ppm) 6thday (ppm) 7thday (ppm) 01 ph 7.65 6.28 7.05 7.51 7.11 8.00 7.65 02 tds 100 103 110 204 107 309 102 03 total hardness 304 302 166 117 136 66 133 04 ca2+ 270 282 168 76 117 101 109 05 total alkalinity 119 104 137 198 165 274 196 06 cl1111 135 121 119 112 106 105 table 10. measurement of different parameters in gulberg iii (treated water) sample. sample gulberg iii parameters (ppm) 1stday (ppm) 2ndday (ppm) 3rdday (ppm) 4thday (ppm) 5thday (ppm) 6thday (ppm) 7thday (ppm) 01 ph 7.1 6.13 6.0 7.11 7.9 6.95 8.0 02 tds 1.00 1.03 1.10 2.04 1.07 3.09 1.02 03 total hardness 30 32 66 47 36 49 34 04 ca2+ 70 82 68 76 67 60 86 05 total alkalinity 99 104 107 98 96.5 97.4 96 06 cl115 11 18 19 12 16 15 d i s c u s s i o n this work was planned to evaluate the quality of treated and rejected wastewater used in the vicinity of allama iqbal town and gulberg iii in lahore, pakistan. table 1 shows the parameters like ph, tds, hardness, chloride ion, alkalinity, cod, and bod are within the permissible limits of treated wastewater samples. as reported by elorm and sudesh, the parameters are very important for understanding how to make it re-useable, as the improvement in waste can make it re-useable for this growing population20. tables 2 and 3 revealed that the heavy metals like zn2+, cu2+ and mn2+ are not detected in the treated or rejected wastewater of all the samples, and the concentration of na+ and k+ was within acceptable limits in all samples. if these metals are present, then removal of these heavy metals is very important because heavy metals are carcinogenic and even in rejected water, they must be removed before disposal21. the presence of sulphate, phosphate and other nitrate ions does not make the water safe for drinking because they can precipitate the calcium and magnesium present in the human body, resulting in the weakening of bones and loss of minerals in the human body. the kidney stones are composed of oxides and phosphates of calcium, and their presence can be a dangerous threat to humans as well as animals22. these acidic radicals can also create boiler scales in industry and can be very harmful to machine life23. table 4 showed that the concentrations of nitrite, nitrate, sulphate, ferric, and chromium lie within safe limits, which are considered to be safe for domestic life. the ph of all the treated wastewater samples increased because the concentrations of sulphates, nitrates and other acidic radicals decreased in these, while they increased in rejected wastewater samples. chromium is very harmful and has many side effects, such as irregular heartbeats, sleep disturbances, headaches, mood changes, and allergic reactions. chromium may also increase the risk of kidney or liver damage24. the value of chromium in rejected wastewater from gulberg iii (sample c*) exceeded the safety limit and should be treated properly before dumping. tables 5-10 showed the collection of treated and rejected wastewater for consecutive seven days, and study of their parameters reveals that the water chemical characterization of treated & rejected wastewater of ro plants vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 68 meets standard drinking values with some variations among each other during consecutive 7-day studies. c o n c l u s i o n conclusively, the ro plants seem to be efficient in treating water and enabling it suitable for the population of these areas. this treated and rejected wastewater is within permissible limits but in rejected wastewater, values of some parameters increased, which can be threatening to human health due to the accumulation of the elements in the environment. c o n f l i c t o f i n t e r e s t no conflicts of interest have been declared by the authors. f u n d i n g s o u r c e there was no funding source and all research was done by self-support. a c k n o w l e d g e m e n t s by the grace of almighty, i, naveed aslam dogar, acknowledge the motivation and moral support provided by my respected colleagues and seniors. i am really thankful to dr. nawaz chaudhary, dr. dildar ahmed, dr. muhammad pervaiz and dr. mushtaq for their encouragement and valuable suggestions during the entire work. l i s t o f a b b r e v i a t i o n s aas atomic absorption spectroscopy bod biochemical oxygen demand cod chemical oxygen demand ebt erichrome black-t edta ethylenediaminetetraacetic acid m molar solution n normal solution neqs national environmental quality standards ph potential of hydrogen ro reverse osmosis tds total dissolved solids r e f e r e n c e s 1. westall f, brack a. the importance of water for life. space sci rev. 2018; 214(2):1-23. 2. jackson s. how much water does a culture need? environmental water management’s cultural challenge and indigenous responses. water environ res. 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md, rijkenberg mj, statham pj, stinchcombe mc, achterberg ep, mowlem mjttiac. determination chemical characterization of treated & rejected wastewater of ro plants vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 69 of nitrate and phosphate in seawater at nanomolar concentrations. 2008; 27(2):169-82. 15. willis j. determination of lead and other heavy metals in urine by atomic absorption spectroscopy. anal chem. 1962; 34(6):614-7. 16. chen m-j, hsieh y-t, weng y-m, chiou ry-y. flame photometric determination of salinity in processed foods. food chem. 2005; 91(4):765-70. 17. esterby sr. review of methods for the detection and estimation of trends with emphasis on water quality applications. hydrol process. 1996; 10(2):127-49. 18. harvey jr ae, smart ja, amis e. simultaneous spectrophotometric determination of iron (ii) and total iron with 1, 10-phenanthroline. anal chem. 1955; 27(1):26-9. 19. adams sa, matthews ce, ebbeling cb, moore cg, cunningham je, fulton j, et al: the effect of social desirability and social approval on self-reports of physical activity. am j epidemiol. 2005; 161(4):38998. 20. obotey ezugbe e, rathilal s. membrane technologies in wastewater treatment: a review. membranes. 2020; 10(5):89-97. 21. klaassen cd. heavy metals and heavy-metal antagonists. pharmacol. 1996; 12:1851-75. 22. ali i. new generation adsorbents for water treatment. chem rev. 2012; 112(10):5073-91. 23. hall re. a system of boiler water treatment based on chemical equilibrium. ind eng chem res. 1925; 17(3):283-90. 24. levina a, lay pa. chemical properties and toxicity of chromium (iii) nutritional supplements. chem res toxicol. 2008; 21(3):563-71. prevalence of cestode parasites of gallus domesticus in karachi, sindh, pakistan vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 157 op e n ac c e s s f u l l l e n g t h a r t i c l e prevalence of cestode parasites of gallus domesticus in karachi, sindh, pakistan rubab malik*, nasira khatoon department of zoology, university of karachi, karachi-75270, pakistan. a b s t r a c t background: poultry production in pakistan is growing remarkably and contributing significantly to the economy. however, disease outbreaks are the most serious threat to poultry productivity. among other poultry diseases which cause huge damage to poultry, parasitic diseases are also included, but no significant work has been done on this area of concern in pakistan. objective: this study is carried out to evaluate the prevalence rate of one of the major helminth parasite groups, cestodes, and their effects on the health of chickens in the districts of korangi, karachi, sindh. method: between february 2018 and january 2019, 76 chickens were examined for cestodes infection. cestodes were extracted from the gut and fixed in f.a.a. fixative for 24 hours with a little cover slip pressure. the cestodes were then thoroughly cleaned with 70% ethanol, stained with mayer's carmalum, dehydrated in progressively stronger alcohol, clarified with clove oil, rinsed with xylene, and permanently mounted in canada balsam. results: the overall prevalence was 52.6%. parasite burden is high in males (63.8%) compared to females (45.6%). six species of cestodes parasites were identified from the intestine of the infected chickens. raillietina tetragona, r. cesticillus, r. echinobothridia, r. ransomi, choanotaenia infundibulum, and raillietina sp. are the cestode species that have been found. their prevalence was 42.1%, 34.2%, 26.3%, 22.2%, 15.7%, and 7.8%, respectively. the prevalence rate also varies from season to season. spring had the highest rate of infection at 60%, summer was next at 58%, and autumn and winter both had a rate of 40%. conclusion: the study concludes that heavy infestation has a negative impact on the physical health of chickens and points to the need to control cestodes parasite infection in order to reduce mortality and increase poultry yield. keywords poultry, prevalence, cestodes parasites, chickens, karachi. *address of correspondence rubabmalik943@gmail.com article info. received: december 12, 2021 accepted: november 01, 2022 cite this article: malik r, khatoon n. prevalence of cestode parasites of gallus domesticus in karachi, sindh, pakistan. 2022; 13(2):157-162. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n in pakistan, the poultry industry is a big part of the economy because it makes eggs and meat, which are the cheapest sources of protein. it contributes 29% of the total meat production of the country, 5.8% of the agriculture sector, and 1.3% of the overall gdp. it has shown excellent growth and become a source of 1.6 million people’s employment. among other poultry diseases which cause huge damage to poultry, parasitic diseases are also included, but no significant work has been done on this area of concern in pakistan. the common gastrointestinal helminth parasites cause negative effects on the health of chickens, which then leads to considerable damage and great economic losses to the poultry industry due to malnutrition, decreased food conversion ratio, weight loss, lower egg production, and death in young birds1. parasites also make the flocks less resistant to diseases and make diseases worse2,3. chicken cestodiasis not only causes loss of body weight but also may cause several problems, o r i g i n a l a r t i c l e prevalence of cestode parasites of gallus domesticus in karachi, sindh, pakistan vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 158 such as enteritis, loss of blood, loss of production, nervous manifestations, and death. parasitic infection causes villous atrophy, catarrhal enteritis, granuloma formation in the duodenum, desquamation of villi and submucosal gland congestion, an inflammatory response, and vacuolation of epithelial cells4. however, poultry production in pakistan is growing remarkably and contributing significantly to its economy. however, disease outbreaks pose the greatest threat to productivity. among other diseases that cause huge damage to poultry, parasitic diseases are also included, but no significant work has been done on this area of concern in pakistan. it is necessary to investigate the rate of infection of various cestode parasites in chickens. the current research aims to study the prevalence and intensity of infection of cestode parasites of gallus domesticus and to study the difference in rate of parasitic infection in different seasons and months to analyse the effect of seasonal variation on parasitic infection. m a t e r i a l s a n d m e t h o d s the viscera of 76 freshly slaughtered chickens have been collected from common shops located in korangi, karachi, from february 2017 to january 2018. samples were subjected to necroscopic and parasitological examination. cestodes were collected from the gastrointestinal tract and preserved in acetic acid formalin alcohol solution (afa) for 8 to 12 hours. then it is kept in 70% alcohol overnight and stained with alum carmine for 3-5 minutes and is continuously checked to prevent overstrain. then it is dehydrated through 70%, 90% and 100% alcohol for 20-30 minutes for each grade. it is then bathed in clove oil for 12 minutes and washed in xylol for 2-3 minutes. finally, permanent slides were prepared by using canada balsam. cestodes were identified under a light microscope with 10x magnification according to their morphological characteristics5. however, pearson's coefficient of correlation 'r' was used to examine the relationship among seasons and infection prevalence. at the 5% threshold of significance (p ≤ 0.05), statistical significance was recognised. spss version 20 was used to analyse the data. r e s u l t s a n d d i s c u s s i o n the author intends to investigate the prevalence of cestode parasite infection in the korangi creek area. the sampling area is selected due to the author's residence in the area, which provides easy access to get samples. the study found that, out of 76 chickens, 40 had different species of cestodes (table 1). six species belonging to three genera were found in the digestive tract of chickens. the overall prevalence of cestodes was 52.6%. parasite burden is high in males (63.8%) as compared to females (45.6%) (table 2). six species of cestodes parasites were found in the intestines of infected chickens. raillietina tetragona, r. cesticillus, r. echinobothridia, r. ransomi, choanotaenia infundibulum, and raillietina species are the species of cestodes that have been found. their prevalence was 42.1%, 34.2%, 26.3%, 22.2%, 15.7%, and 7.8%, respectively (table 3). table 1. overall prevalence of cestodes infection. helminthes group host examined host infected prevalence % cestodes 76 40 52.6 table 2. prevalence of cestodes in relation to host’s sex. sex of host host examined host infected prevalence (%) male 30 19 63.8 female 46 21 45.6 prevalence of cestode parasites of gallus domesticus in karachi, sindh, pakistan vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 159 table 3. prevalence of cestodes parasites. genus host examined host infected prevalence (%) raillietina tetragona 76 32 42.1 raillietina cesticillus 76 26 34.2 raillietina echinobothridia 76 20 26.3 raillietina ransomi 76 17 22.2 choanotaenia infundibulum 76 12 15.7 raillietina sp. 76 6 7.8 table 4. seasonal incidence of recorded cestodes’ species. season host examined host infected prevalence % spring 15 9 60.0 summer 36 22 58.3 autumn 15 6 40.0 winter 10 3 40.0 raillietina tetragona was reported in chicken 48.3%6 and 51.66%7 from pakistan; 14.5%8 from western cameron; 21.3%9 from tanzania; and 2.0%10 from nigeria; 7.4%11 from egypt; 51.42%12; and 9.16%13 from india; 57.5%14 from thailand; 68.75%15 from algeria; 19.68%16 from bangladesh; and 65.94%17 from ethiopia r. echinobothrida reported from the chicken with prevalence of 19.2% 6 from pakistan, 10.4%18 from iran, 48.3%14 from northern thailand, 25.3%19 from nigeria, from brazil20, 91.9%21 and 6.6%11 from egypt, 39.86%17 from ethiopia, 83.3%15 from algeria, 37.83%22 from phitsanulok province, 2.3% 23 from trinidad, 50% 24 from bangladesh and 46.3%9 from tanzania while the same specie has been reported in pigeon 61.76%25 from greece and 32%26 from libya. r. cesticillus was reported from birds with prevalence of 56.6%24 from bangladesh, 55.07%17 from ethiopia, 83.5%27 from pakistan, 23.22%28 from india, 29.1%15 from algeria and 21.3%19 from nigeria, 0.9%23 from trinidad, 5.83%7 and 0.8%6 from pakistan, 12.5%14 from thailand and, also reported from brazil20. choanotaenia infundibulum was reported from hyderabad, pakistan, with a prevalence of 89.5%27, while choanotaenia sp. with prevalence of 20% reported from the same host in quetta, pakistan6. outside of pakistan, the same species has been reported from zimbabwe29, from trinidad23, from tanzania9, from algeria15, from japan30 and from brazil31. the difference in prevalence might be due to the variation in the environmental conditions of the area32, including high temperature, humidity or dryness, annual rainfall, and the availability of intermediate hosts33, it could also be due to sharing the same habitat with migratory birds, which increases the chance of harbouring parasites34. similar attribution is also given that environmental alterations, especially increasing temperature, may have affected the occurrence of helminth infection 14, and another author stated that more than one million birds migrate from central asia to pakistan during winters. during migration, they also carry parasites, which result in new host and locality records35. moreover, the study found that the prevalence rate also varies from season to season. the highest infection has been observed in spring at 60%, followed by summer at 58.3%, and in autumn and winter, the same infection has been observed at 40% (table 4). this suggested that there was not enough variation in the prevalence and intensity of infection in different seasons. the results suggest that high temperatures increase the susceptibility of the hosts to parasitism6. although parasites were discovered to be prevalent throughout the year, with a higher prevalence and parasitic load, they were found to be most prevalent in the summer season. furthermore, multiple infections of helminth parasites have also been found in many chickens. similarly, a high intensity of parasites has also been observed. the intestine prevalence of cestode parasites of gallus domesticus in karachi, sindh, pakistan vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 160 was fully blocked with the worm burden. grossly, the intestine showed a heavy infestation of cestodes, due to which the mucosal lining of the intestine was damaged. chronic catarrhal enteritis, hemorrhagic enteritis, and nodular enteritis have been observed. the intestinal wall was thickened and inflamed with hemorrhagic necrotic spots. table 5. seasonal incidence of each specie of recorded cestodes. season r.tetragona r.cesticillus r.echinobothrida r.ransomi choanotaenia infundibulum raillietina sp. p-value no. % no. % no. % no. % no. % no. % spring 8 88.8 6 66.6 4 44.4 2 22.2 2 22.2 0 0 0.028 summer 17 77.2 15 68.1 13 59 11 50 8 36.3 4 18.1 0.002 autumn 5 83.3 4 66.6 3 50 2 33.3 2 33.3 1 16.6 0.005 winter 2 66.6 1 0 0 0 2 66.6 0 0 1 33.3 0.041 table 6. monthly incidence of different types of recorded cestodes’ species. month r.tetragona r.cesticillus r.echinobothridia r.ransomi choanotaenia infundibulum raillietina sp. p-value no % no % no % no % no % no % feb 3 100 2 66.6 2 66.6 1 33.3 1 33.3 0 0 0.017 mar 2 100 2 100 1 50 0 0 0 0 0 0 0.093 apr 3 75 2 50 1 25 1 25 1 25 0 0 0.025 may 7 77.7 7 77.7 6 66.6 5 55.5 3 33.3 2 22.2 0.002 jun 3 75 3 75 2 50 2 50 2 50 1 25 0.001 jul 7 77.7 5 55.5 5 55.5 4 44.4 3 33.3 1 11.1 0.004 aug 1 100 0 0 1 100 1 100 1 100 1 100 0.004 sep 2 100 2 100 1 50 1 50 1 50 0 0 0.013 oct 2 66.6 2 66.6 1 33.3 0 0 0 0 0 0 0.093 nov 1 50 1 50 0 0 2 100 0 0 0 0 0.102 dec 0 0 0 0 0 0 0 0 0 0 0 0 0 jan 1 100 0 0 0 0 0 0 0 0 1 100 0.175 prevalence of cestode parasites of gallus domesticus in karachi, sindh, pakistan vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 161 c o n c l u s i o n the present study concludes that the prevalence of parasitic infection by cestodes is very high, resulting in the mortality of chickens. the high prevalence of infection leads to invisible production and economic losses. raring area, season, availability, and contact with the intermediate host also affect the prevalence and intensity of infection. based on the result, the following recommendations are forwarded: as the cestodes parasites have a sub-clinical occurrence, studies that focus on these ideas should be conducted. the public, especially those who are related to poultry farming, should be aware of the occurrence and economic significance of gastrointestinal parasites. elimination of intermediate hosts in the rearing area and extensive early-season larval control have been recommended. e t h i c a l a p p r o v a l not required. c o n f l i c t s o f i n t e r s t no conflict of interest. f u n d i n g s o u r c e none. a c k n o w l e d g e m e n t s i am thankful to my supervisor for her guidance and support. l i s t o f a b b r e v i a t i o n s afa acetic acid 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in indigenous chicken (gallus gallus domesticus) in the temperate himalayan region of kashmir-short communication. vet arh. 2010;80(2):323-8. 29. percy j, pias m, enetia bd, lucia t. seasonality of parasitism in free range chickens from a selected ward of a rural district in zimbabwe. afr j agric res. 2012;7(25):3626-31. 30. gamra ow, antia re, falohun oo. intestinal cestodes of poultry raillietina echinobothrida and choanotaenia infundibulum infection in a commercial japanese quail (coturnix coturnix japonica) farm in apomu, osun state, nigeria. sci j zool. 2015;4(4):20-5. 31. monte gl, cavalcante dg, oliveira j. parasitic profiling of japanese quails (coturnix japonica) on two farms with conventional production system in the amazon region. pesqui vet bras. 2018;38:847-51. 32. bennema sc, vercruysse j, morgan e, stafford k, höglund j, demeler j, von samson-himmelstjerna g, charlier j. epidemiology and risk factors for exposure to gastrointestinal nematodes in dairy herds in northwestern europe. veterinary parasitology. 2010;173(3-4):247-54. 33. charlier j, thamsborg sm, bartley dj, skuce pj, kenyon f, geurden t, hoste h, williams ar, sotiraki s, höglund j, chartier c. mind the gaps in research on the control of gastrointestinal nematodes of farmed ruminants and pigs.transbound emerg dis. 2018;65:217-34. 34. besier rb, kahn lp, sargison nd, van wyk ja. the pathophysiology, ecology and epidemiology of haemonchus contortus infection in small ruminants. adv parasitol. 2016;93:95-143. 35. birmani na, dharejo am, khan mm. catatropis sp.(trematoda: notocotylidae) from the black coot, fulica atra linnaeus, 1758 (gruiformes: rallidae) in sindh province of pakistan. japs, j of animal and plant sci. 2011;21(4):872-3. plant parasitic nematodes associated with maple (acer caesium) and poplar (populous nigra) in district swabi vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 183 op e n ac c e s s f u l l l e n g t h a r t i c l e plant parasitic nematodes associated with maple (acer caesium) and poplar (populous nigra) in district swabi bilal muhammad*, mian sayed khan, saira department of zoology, university of swabi, anbar, khyber pakhtunkhwa, 23561, pakistan a b s t r a c t background: parasitic nematodes are very destructive to crops, ornamental plants and woody plants as well which cause most severe diseases in plants and feed on the roots of these plants. objective: main aim of this study is to identify different species of nematodes associated with maple (acer caesium) and poplar (populous nigra) trees at district swabi, khyber pakhtunkhwa, pakistan. methodology: some maple and poplar growing areas of district swabi were surveyed during the year 2019, for the occurrence and incidence of parasitic nematodes. soil samples collected from different localities were analyzed for the nematodes. plant-parasitic nematodes were extracted from soil by cobb methods, and identified the species after fixation. result: maximum analyzed simples were infected and among all localities seven different species of nematodes has been recorded. maximum infection was recorded in zaida, manki and anbar. following nematodes were found named paratylenchus larvae, aphelenchus avenae, helicotylenchus dihystera, tylenchus, trichodorous and meloidogyne incognita. ratio of occurrence among all different varieties of nematodes meloidogyne incognita species shows most abundance in two localities lahor and zaida. conclusion: the results clearly indicate the fact that studied area is mostly infected with different types of parasitic nematodes species which highly affect the maple (acer caesium) and poplar (populous nigra) plants. keywords nematodes, plant parasitic nematodes, maple, poplar, infection, meloidogyne incognita, paratylenchus, tylenchus. *address of correspondence bilaluoswabi2021@gmail.com article info. received: october 26, 2021 accepted: december 20, 2022 cite this article: muhammad b, khan ms, saira. plant parasitic nematodes associated with maple (acer caesium) and poplar (populous nigra) in district swabi. 2022; 13(2):183-192. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n nematodes commonly known as round worms the word nematoda is a greek word “nemos,” means “thread”. nematodes are worm-like, generally minute, colorless and unsegmented animals. nematodes are aquatic animals. plant parasitic nematodes colonize in the rhizosphere. and accomplished in the rhizosphere and exert beneficial effects on the plants1.the most numerous, multicellular animals are nematodes on earth. the size of nematodes is 0.2mm-6mm dwelling in many hosts like plants and animals2. mostly plant and animals are infected at least single species of parasitic nematode. nematodes make a wild range of relationship with other species. at large numbers of nematodes species are parasitic in nature which defined on host plant and animals3. mostly nematodes are live in deep water soil. nematodes are found almost every imaginable habitat including in the rhizosphere of plant, salt water, and fresh water as well as with other organism as a parasite. but mostly nematodes are abounded in the upper layer of soil where plant roots, other organic matters are abounded. according to survey there are 1–10 million nematodes/m are present in soil4,5. nematodes are classified on the base of their feeding o r i g i n a l a r t i c l e plant parasitic nematodes associated with maple (acer caesium) and poplar (populous nigra) in district swabi vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 184 habits and structure of their mouth parts. we enlist the feeding habits of some most common nematodes groups which are fungal-feeder, predators, omnivores, bacterialfeeder, and plant parasites. the predators nematodes feed on protozoa and some other soil nematodes as well. omnivore feeding habits depends on food availability and environmental conditions; for example during lack of their primary food source omnivores act as predators and also feed on bacteria and fungi. a small amount of soil contains numerous numbers of nematodes. phylum nematoda consists of 20,000 described species. members of phylum nematoda have existed from one billion year which gives them the credibility of the ancient organism on earth6.the phylum nematode diverged into two classes chromadorea and enoplea so long ago (figure.1). according to this classification ecologist easily understand the position of nematodes in soil food web chain. plant parasitic nematodes almost feed on all parts of plants like seeds, roots, stem, flower, and leaves. parasitic nematodes use specialize organ called stylet through which they puncture the plant cells during feeding and suck the nutrients from plant and having special esophageal glands7.the 1st plant parasitic nematode was reported by needham (1743) for the 1st time he observed the symptom of galling in wheat plant8 .one of the important species in agriculture of parasitic nematodes are identified by berkeley, he first time observed that in roots of cucumber are root-knot nematodes1. plant parasitic nematodes are different from each other in life style. some nematodes are take nutrition externally known as ectoparasitic nematodes. ectoparasitic nematodes feed from the outside the host plant. while other endoparasitic nematodes enter to the host root and take nutrition from it. xiphinema (california dagger nematode) is the example of ectoparasitic nematodes it cause viral infection in grapevine and cause on large scale of economics loses world widely9. endoparasitic nematodes are further divided into two groups. figure 1. classification of nematodes5 orders subclass classes phylum nematoda chromadorea chromadoria order 1 rhabditida order 2 plectida order 3 araeolaimida order 4 monhysterida order 5 desmodorida order 6 desmoscolecida order 7 chromadorida enoplea enoplia order 1: dorylaimida and longidoridae order 2: mermithida order 3: monorchida order 4: dioctophymatida order 5: trichinellida order 6: isolaimida order 7: muspiceida order 8: marimermithida plant parasitic nematodes associated with maple (acer caesium) and poplar (populous nigra) in district swabi vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 185 migratory nematodes. these types of nematodes feed briefly on a particular site and then move to another site e.g. xiphinema, longidorus and trichodorus. sedentary nematodes. these typed of nematodes are feed on one site of the host cell for long time and remain attached with it e.g. pratylenchus and hirschmanniella10. root-knot nematodes. root-knot nematodes contain over one hundred species which are most threaten species to the agriculture field including meloidogyne javanica, meloidogyne arenaria, meloidogyne incognita and meloidogyne hapla. meloidogyn spp. globally distributed, having a large number of hosts to be attach11. cyst nematodes. second in list is cyst nematodes (heterodera and globodera spp.) are ranked to economic and agricultural more damaging nematodes. cyst nematodes enter to the host plant root tips and secret the esophageal gland from the stylet. esophageal gland promotes degradation of protoplast and cell wall fusion of other adjacent cells and form syncytium12. lesion nematodes. lesion nematodes (pratylenchus spp.) are ranked on 3rd position in the list of more damaging nematodes to agriculture. lesion nematodes get nutrition from vascular tissues for which they damage cortex of roots and then enter to vascular tissues, as a result plants show some above ground symptoms of chlorosis, necrosis on root and reduction of leaf size and number13. burrowing nematode. burrowing nematode (radopholus similis) is migratory plant parasitic nematodes. burrowing nematodes are listed quarantine plant pest in worldwide14. maple and poplar trees: maple (acer caesium) and poplar (populus nigra) trees are mostly planted or self-grown trees in pakistan. the plantations of populus nigra are the 1st choice of farmer because of his rapid growth and domesticated importance. the woods of populus nigra are commonly used for the manufacturing of matchsticks. the poplar trees are disseminated throughout pakistan. there are 20 species of poplar tree found in pakistan but mostly poplar (populus nigra) specie is found in district swabi which are cultivated or self-growing in this area. maple (acer caesium) is slow growing plant approximately 0.2 to 0.5 cm growth/year. 20 to 25m tall and the shape of leaves are simple having 3 to 5 lobes and 8 to 20 cm wide. it can reproduce from both sources seed and from vegetative means. it distributed throughout pakistan, nepal and india. in pakistan mostly found in swat, muree hills, chitral, dir, swabi and azad kashmir.the wood of acer caesium use in bobbins, carving, furniture and ornamental15. r e v i e w l i t e r a t u r e according to malakhov (1994) the parasitic nematodes having negatively impact on agriculture field, human health and other many wildlife animals. there are parasitic nematodes present everywhere having special host to be affected16. gregory and egnin, (2017) were conducted that from his research that parasitic nematodes cause lot of destruction in agriculture field in past. cyst nematodes, lesion nematodes, and root-knot nematodes listed on top of the scientifically most economically important species17. another study was conducted by gnamkoulamba et al., (2018) which proposed that rice is the second most important cereal in togo. among parasites which affect rice production, nematodes as parasites are considered to be contributing too much regarding detrimental effect over all as about 71% nematodes parasitic genera like hirschmanniella spp., xiphinema spp. ,meloidogyne spp., helicotylenchus spp. ,tylenchid spp., and pratylenchus spp., were been collected during study which established the evidence that nematodes could be important constraints for rice production. study of nematodes declared that the species of root-knot nematode is commonly most pathogen to tomato crop including ethiopia which is seen as been spread worldwide attacking a vast variety of crop plants and destroying economy of different countries18. bridge et al., (2005) concluded from his survey that rice production is affected by over 100 species of nematodes. meloidogyne spp. is on the top of this list that is been distributed through world, but the hirschmanniella oryzae is most effective regarding rice production which also plant parasitic nematodes associated with maple (acer caesium) and poplar (populous nigra) in district swabi vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 186 known as rice root nematode which has been reported in following asian countries such as pakistan, india, nepal, bangladesh, thailand, vietnam, sri lanka, china, japan, and korea19. al-mohithef et al., (2018) conducted a survey on some genera of parasitic nematodes associated with vegetable crops in riyadh, (saudi arabia). survey showed that among all the identified genera m. javanica is more common among all vegetables crops. from tennessee nurseries in 1981, 27 red maple, 48 dog woods, and 17 peach were sampled for nematodes identification 57 species of nematodes in 24 genera were found. the most detected species of nematodes were xiphinema americanum and paratylenchus projectus, which were found 78% and 88% respectively found in sites. and others species of nematodes were found in 10% in sites or above than 10%. but the effect of paratylenchus projectus, were more than others is mean xiphinema americanum and paratylenchus projectus, have been large effect on maple, dogwood, and peach plants in tennessee20. hussein et al. (2016) surveyed the montpellier maple from the different regions boyer-ahmad, sepidar and kohgiluyeh of iran. he studied the rhizosphere of montpellier maple during his study he found a new species which belong to genus glenchus. the new species have some different characters from other species of genus glenchus like they have long stylet approximately 7-8 µm in length21. the first report on nematodes associated with poplar tree was published from pakistan in 1993 by dr. aly khan and his colleagues who mentioned in their book “nematodes associated with some trees of khyber pakhtunkhwa” that they collected 120 soil samples from the rhizosphere of populous nigra. they collected those samples from six different locations of district mardan. ten different species and nine genera were isolated from the rhizosphere of populous nigra. tylenchorhus annulatus showed large population with 180-205 nematodes per 100 ml of soil residue, helicotylenchus dihystera 95-130 nematodes per 100 ml of soil residue and hoplolaimus seinhorst and pratylenchus penetrans showed the lowest percentage of occurrence with 2-8 nematodes per 100 ml of soil residue22. m a t e r i a l s a n d m e t h o d study area: the area of interest for research on plant parasitic nematodes on maple tree and poplar (populus nigra) was district swabi. swabi lies between indus river and kabul river 100 kilometer from country capital islamabad and 100 kilometer from provincial capital peshawar. district swabi is situated at 34º 7', 48º n and 72º 28', 11" e of kpk, pakistan. there are five tehsil in district swabi. lahor is a tehsil of swabi which is the largest tehsil in district swabi which consist of 10 villages (figure. 2). figure 2. map of district swabi showing different villages (online, accessed december, 2019) plant parasitic nematodes associated with maple (acer caesium) and poplar (populous nigra) in district swabi vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 187 from which we have collected the samples of maple and poplar trees initially. the climate of district swabi is subtropical warm humid in summers and mild in winters. average temperature is 22.2 c and annually average precipitation is 639 mm of district swabi. average rainfall in november is 12 mm therefore this month is a driest month of year. while the precipitation in august (wettest month of year) is 137 mm. hottest month is june average temperature of this month 41.2  c and the coldest month of year is january average temperature of this month is 10.2 c. the climate of tehsil lahor is suitable for cultivation of maple and poplar trees. generally the cultivation starts between mid-february and mid-march. poplar tree is one of the poplar wood producing trees. poplar tree can grow above 85 feet in height and 36 inches diameter in 5 to 6 years. mostly maple and poplar trees can survey in any environment hot and cool both condition can’t effect the cultivation of maple and poplar. m e t h o d o l o g y collection of samples: in this study the soil were taken for samples collection and some roots with it from approximately 1 to 2 feet deep from the base (rhizosphere) of maple and poplar trees and dump it in plastic bag. at least 2 samples were collected from each locality (maple and poplar) from one area, and brought these collections to the lab and analyzed them for the nematodes. extraction of nematodes: in this study cobbs sieving method (1918)23 were used and baermann funnel technique (schindler, 1961)24 for the extracting of nematodes from the collected soil samples. in lab mixed the soil with water in tub and stirred it. then that liquid passed from 100µ mesh sieves in tub. discord the residue which present in 100 meshes and passed the liquid which passed previously from 100µ mesh sieves now passed them through 200µ mesh sieves, and then passed the liquid from 300µ mesh sieves discord the liquid after passing it from 300µ mesh sieves. washed the residue of 200µ and 300µ mesh sieves and kept it in beaker. take the baermann funnel, rubber tube, and circular wire mesh and filter paper. attached the rubber to the mouth of baermann funnel and clamped it shut. put the wire mesh on baermann funnel and placed the filter paper on them. and then added the residue on filter paper mixed with water. filled the baermann funnel with water and left it for 24 hours that all nematodes sinked down in bottom. after 24 hours took 50 ml water from the rubber tube the nematodes were sinked there. isolation of nematodes: first take the 50 ml of water from bearrman funnel in beaker then poured some of water from the respective beaker in petry dish to observe through stereoscopic microscope in order to find out nematodes which were picked up with the help of dropper after seeing in the respective arrangement and placed them into the cavity block. killing of nematodes: then after isolation kept the cavity block on heater at very low temperature because high temperature is destructive to the cell morphology of the respective entity (nematode) for the purpose of killing respective nematode through proper scientific protocol to observe accurately. following heat killing then water was removed from the cavity block. dehydration: after removing water from cavity block taf were added to cavity block and kept it for 48 hours in room temperature to get it dehydrated. after removing water from cavity block taf were added to cavity block and kept it for 48 hours in room temperature to get it dehydrated. staining: after 48 hours taf were removed from cavity block while glycerin were added instead to cavity block for staining and kept it in oven for 72 hours. preparation of slides: after the staining the next step was to prepare permanent slides. this started with cleaning of the slide and cover slip with xylene with the help of tissue paper gently so to avoid rupturing and scratches of slides. then glycerin was dropped over the central part of cleaned slide and then through eyelash brush nematodes picked up from cavity block and placed into the glycerin drop on respective slide. maximum number of nematodes which are allowed to be placed on a single slide range 7-9 for certain and accurate result which was kept in mind during the process for having maximum accuracy, after keeping nematodes over slide plant parasitic nematodes associated with maple (acer caesium) and poplar (populous nigra) in district swabi vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 188 four pieces of paraffin wax were placed on four spots around the glycerin drop so that to fully surround the drop along with our study object over which cover slip was kept to cover the object then heated it up by keeping on hot plate in order to melt paraffin wax which covered all the surface of slide and hold the slip tightly to prevent object movement during observation, after fully melting of wax over slide, slide was removed from hot plate and kept at room temperature till getting it fully dried. after this entire described process finally slid were ready to be observed for nematode identification. r e s u l t s soil samples from six localities were collected and analyze for the identification of parasitic species of nematodes associated with poplar (populus nigra) and maple (acer caesium). the results of study shows that samples were found infected with nematodes showing high infection. maximum samples of poplar (populus nigra) tree were found infected. table. 1 is shown that four different species of parasitic nematodes aphelenchus avenae and tylenchus larvae were identified in anbar and one species of paratylenchus larvae was identified in manki and one species of parasitic nematodes helicotylenchus dihystera was found in tordher district swabi. table 1. plant parasitic nematodes in poplar tree (populus nigra). s.no tree name number of soil samples location nematodes 1 poplar 2 manki paratylenchus larvae 2 poplar 2 anbar aphelenchus avenae 3 poplar 2 tordher helicotlenchus dihytera 4 poplar 2 anbar tylenchus larvae 5 poplar 2 lahor free living 6 poplar 2 beka free living figure 3. frequency of free living and parasitic nematodes in populus nigra. plant parasitic nematodes associated with maple (acer caesium) and poplar (populous nigra) in district swabi vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 189 figure 3 shows that the most infected area is anbar which shows 75% infestation followed by manki with 25% of infestation rate. table. 2 is shown that three species of parasitic nematodes identified in which trichodorous larvae determine in zaida and two species of meloidogyne incognita were identified in lahor and zaida. the most abounded species of nematodes paratylenchus, aphelenchus avenae, helicotylenchus dihystera, tylenchus, trichodorous and meloidogyne incognita were mostly distributed among the different area of district swabi. the most infected area is zaida with 70% of infestation rate followed by lahor which having 30% of infestation rate which has been shown in table. 3 and figure 4. in below figure. 4 we determine that zaida is the most infected site with 70% infestation rate. table 2. plant parasitic nematodes in maple tree (acer caesium). s.no tree name number of soil samples location nematodes 1 maple 2 lahor meloidogyne incognita 2 maple 2 zaida meloidogyne incognita 3 maple 2 zaida trichodorous larvae 4 maple 2 tordhair free living 5 maple 2 manki free living 6 maple 2 anbar free living figure 4. frequency of free living and parasitic nematodes in acer caesium. anbar lahor manki zaida nabi parasitic nematodes 0 30 0 70 0 free living nematodes 50 25 70 15 35 50 25 70 15 35 0 30 0 70 0 plant parasitic nematodes associated with maple (acer caesium) and poplar (populous nigra) in district swabi vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 190 table 3. categorization of identified species on the bases of their abundance. nematodes host localities abundance paratylenchus larvae poplar manki 65-70 aphelenchus avenae poplar anbar 20-30 helicotlenchus dihytera poplar tordher 30-35 tylenchus larvae poplar anbar 70-80 meloidogyne incognita maple lahor 43-60 meloidogyne incognita maple zaida 75-80 trichodorous larvae maple zaida 45-50 d i s c u s s i o n present survey confirms the occurrence of parasitic nematodes in maple and poplar trees in district swabi and we also aim that from discussion our study clear this fact that meloidogyne spp. and pratylenchus spp. is not host specific and cause destruction only in rice and vegetables but also destructive for poplar and maple tree also it prove that the determine species are not host specific they can cause infestation in any maple and poplar too. the damaging of ornamental, woody, crops due to the effect of parasitic nematodes is a universal problem including pakistan. malakhov (1994) stated that every parasitic nematodes have specific host to affect these creatures are very disaster for crop, animal health and livestock16. according to the survey of aly in 1993 of populous nigra of district mardan, ten different species and nine genera were isolated from the rhizosphere of populous nigra. tylenchorhus annulatus showed large population with 180205 nematodes species per 100 ml of soil residue, helicotylenchus dihystera showed 95-130 nematodes. species per 100 ml of soil residue, and hoplolaimus seinhorsti and pratylenchus penetrans showed the lowest percentage of occurrence with 2-8 nematode per 100 ml of soil residue. while during our survey four different species of parasitic nematodes aphelenchus avenae, tylenchus larvae, paratylenchus larvae, and helicotlenchus dihytera were recorded in the samples of popalr trees. and two different species of parasitic nematodes trichodorous larvae and meloidogyne incognita larvae were recorded on samples of maple trees22. al-mohith et al., (2018) survey in riyadh region of saudi arabia for the parasitic nematodes was associated with vegetable crops. the results of this survey showed that the most threaten root nematodes species to crops is m. javanica. which mostly affect the vegetable crops in riyadh region. from tennessee nurseries in 1981, 27 red maple, 48 dogwoods, and 17 were samples for nematodes identification 57 species of nematodes in 24 genera were found. the most detected species of nematodes were xiphinema americanum and paratylenchus projectus, which were found 78 % and 88 % respectively found in sites, and other species of nematodes were found in 10 % in sites or above than 10 %. but the result showed that the paratylenchus projectus effect were more than xiphinema americanum on maple, dogwood, and peach plants in tennessee20. bridge et al., (2005) concluded from his survey that over 100 species of parasitic nematodes affect production of rice world widely, meloidogyne spp is the most widely distributed specie throughout the world, while hirschmanniella oryzae which is mostly associated with ricehas been reported in asian countries such as pakistan, india, nepal, bangladesh, thailand, vietnam, sri lanka, china, japan and korea19. c o n c l u s i o n the results of this study show that samples were found infected with nematodes showing high infection. the most abounded species of nematodes paratylenchus, aphelenchus avenae, helicotylenchus dihystera, tylenchus, trichodorous and meliodogyne incognita were mostly distributed among the all maple and poplar cultivated area of district swabi. there were two different species of parasitic nematodes trichodorous larvae and meloidogyne incognita larvae were identified in zaida and plant parasitic nematodes associated with maple (acer caesium) and poplar (populous nigra) in district swabi vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 191 one species of parasitic nematodes meloidogyne incognita larvae were identified in lahor district swabi, and four different species of parasitic nematodes associated with populus nigra were found. aphelenchus avenae and tylenchus larvae were identified in anbar and one species of paratylenchus larvae was identified in manki and one species of parasitic nematodes helicotlenchus dihytera were found in tordher district swabi. from above results and discussion we conclude that, the various villages of district swabi are suffering from various parasitic nematode infestation which are ultimately become the cause for many diseases in maple and poplar trees. c o n f l i c t s o f i n t e r s t no conflict of interest. f u n d i n g s o u r c e none. a c k n o w l e d g e m e n t s we are thank full to university of swabi for providing research facility. l i s t o f a b b r e v i a t i o n s spp specie kpk khyber pakhtoon khawa taf tri ethylamine formaldehyde n north e east r e f e r e n c e s 1. berkeley m. the botany of the antarctic voyage ii. florae novae-zealandiae, 1855: p. 172-210. 2. mark b, and dee rd.the worm in the world and the world in the worm. bmc biology, 2012. 10(1): p. 57. 3. jones jt, annelies h, etienne gjd, hari sg,johannes h, et al.top 10 plant‐parasitic nematodes in molecular plant pathology. molecular plant pathology, 2013. 14(9): p. 946-961. 4. davisel, macguidwinae.lesion nematode disease. plant health instr, 2000. 5. poinar g. the natural history of nematodes., 1983. 6. wang ms, limin li, rapid screening assay methods and devices. 1999, google patents. 7. maierc, bilas f, weinbauer mg, watremez p, peck ma, et al.respiration of mediterranean cold-water corals is not affected by ocean acidification as projected for the end of the century.biogeoscience, 2013. 8. needham f, john t, robert, henry. new microscopical discoveries: london: smithsonian libraries; 1745. 9. villate l,fievet v, hanse b, delemarre f, plantard o, et al.spatial distribution of the dagger nematode xiphinema index and its associated grapevine fanleaf virus in french vineyard. phytopathology, 2008. 98(8): p. 942-948. 10. williamsonvm, husseyrs.nematode pathogenesis and resistance in plants. the plant cell, 1996. 8(10): p. 1735. 11. poulin r,aseeb sr.evolution of parasitism along convergent lines: from ecology to genomics. parasitology, 2015. 142(s1): p. s6-s15. 12. gheyseng, fenoll c.gene expression in nematode feeding sites. annual review of phytopathology, 2002. 40(1): p. 191-219. 13. jones m, nyarko j.molecular biology of root lesion nematodes (pratylenchus spp.) and their interaction with host plants. annals of applied biology, 2014. 164(2): p. 163-181. 14. sasserj, freckman d.a world perspective on nematology; the role of the society. pp: 7-14. vistas on nematology. society of nematologists, hyatsville, md, 1987. 15. mahmooda, caccamo dv,tomecek fj, malik gm.atypical and malignant meningiomas: a clinicopathological review. neurosurgery, 1993. 33(6): p. 955-963. 16. malakhovv.classification of the pseudocoelomates. nematodes, structure, development, classification and phylogeny. smithsonian institute press, washington, 1994: p. 175-201. 17. gregory b, egnin , bonsi c. the impact of plantparasitic nematodes on agriculture and methods of control. nematology-concepts, diagnosis and control. 2017; 10(1): 121-151. 18. gnamkoulambaa, tounou ak, tchabi a, kolombia ag, agboka k, et al.occurrence, abundance and distribution of plant-parasitic nematodes associated with rice (oryza spp.) in different rice agroecosystems in togo. international journal of biological and chemical sciences, 2018. 12(2): p. 618-635. 19. bridge j, plowright ra, peng d.nematode parasites of rice. plant parasitic nematodes in subtropical and tropical agriculture, 2005. 2: p. 87-130. 20. almohithef, a.h, al-yahyaf.a, al-hazmia.s, dawabah a.a.m, lafi h.a, prevalence of plant-parasitic nematodes associated with certain greenhousevegetable crops in riyadh region, saudi arabia. j. saudi soc. agric. sci.2018. 19 (1),22–25. plant parasitic nematodes associated with maple (acer caesium) and poplar (populous nigra) in district swabi vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 192 21. husseinm,abdollahi m,karegar a.description of aglenchus microstylus n. sp.(nematoda, tylenchidae) from iran with a modified key to the species of the genus. nematropica, 2016. 46(1): p. 38-44. 22. khan, a. and bilqees, f.m. basiria bajorensis n. sp. (nematoda: tylenchida) from pakistan. proceedings of parasitology,1993.15: 14-16. 23. cobb n. estimating the nematode population of the soil. agric. tech. circ. u.s. dept. agriculture. 1918; 48 p. 24. schindler a. f, a simple substitute for a baermann funnel. pl. dis. replr; 1961 p 747-748. green synthesis of agnps and antibacterial activity against mdr pathogens vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 98 op e n ac c e s s f u l l l e n g t h a r t i c l e green synthesis of silver nanoparticles from flowers of helianthus annus and tagetes erecta and their antibacterial activity against mdr pathogens sadaf shaheen1, zelle humma2, iqra arooj1, ⃰, sana batool1 1department of microbiology & molecular genetics, the women university, multan, pakistan. 2department of biochemistry & biotechnology, the women university, multan, pakistan. a b s t r a c t background: multidrug resistant (mdr) organisms are of major concern to healthcare sector as increasing antimicrobial resistance has made it obligatory to focus on alternative options. investigating the medicinal benefits of nanoparticles synthesized from biological sources is relevant in this regard. objectives: main purpose of the current study was to explore antibacterial potential of silver nanoparticles (agnps) synthesized from flower extracts of helianthus annus and tagetes erecta against mdr bacteria. methodology: mdr bacteria were isolated from sewage water of hospitals and characterized by gram staining and biochemical analysis. from aqueous and methanolic extracts of h. annus and t. erecta flowers, silver nanoparticles were prepared and then characterized and confirmed by visual analysis, ftir spectroscopy, and uv-visible spectroscopy. antibacterial activity of crude extracts and their nanoparticles was evaluated by well diffusion and disk diffusion methods, respectively. results: bacterial isolates included mdr escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae, salmonella spp., shigella spp. and proteus spp. as well as methicillin-resistant staphylococcus aureus were identified. instant color change in extract solutions upon addition of silver nitrate confirmed the formation of nanoparticles. ftir and uv-visible spectroscopy showed response of biomolecules present in flower extracts during formation of silver nanoparticles, indicating the presence of different phytochemicals. antibacterial activity of crude extracts increased greatly after synthesis of nanoparticles and they produced zones of inhibition against all of the mdr bacteria included. methanolic extracts and their nanoparticles possessed more antibacterial power compared to aqueous extracts. conclusion: our findings suggest the presence of potent antibacterial components in flowers of h. annus and t. erecta which necessitates further evaluation. keywords antibiotic resistance, helianthus annus, multidrug resistance, nanoparticles, mdr, tagetes erecta. *address of correspondence iqra.6051@wum.edu.pk article info. received: may 09, 2021 accepted: july 06, 2021 cite this article shaheen s, humma z, arooj i, batool s. green synthesis of silver nanoparticles from flowers of helianthus annus and tagetes erecta and their antibacterial activity against mdr pathogens. 2021; 12(2):98-107. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n multidrug resistant (mdr) organisms are the principal cause for the morbidity and mortality worldwide. in terms of contagious diseases, mdr pathogens are the obligatory reason for annual mortality1. due to acquired chromosomal mutations or transposon-mediated transfer, microbes incorporate genes that make them intrinsically resistant to o r i g i n a l a r t i c l e green synthesis of agnps and antibacterial activity against mdr pathogens vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 99 antibiotics2. methicillin-resistant staphylococcus aureus (mrsa) and extensive-spectrum beta-lactamase (esbl) producers have attained the ability to dissolve penicillins and cephalosporins. members of several bacterial genera have acquired esbl genes and produce enzymes that enable them to demonstrate resistance against multiple antimicrobial agents3. the mechanism of resistance in microbes may involve metabolic rearrangements in some pathogens4. bacteria acquire antibiotic resistance owing to the modification of extracellular proteins and dna, as well as formation of thick biofilms5. rapidly increasing resistance against known antibiotics necessitates the exploration of alternative drug sources to combat microbial diseases. phytochemicals present in plants such as vitamins (a, c, e, and k), flavonoids, terpenoids, saponins, enzymes, pigments, and minerals have been reported to possess antimicrobial, antioxidant and anti-cancerous activities6. according to the world health organization (who), approximately 80% of people rely on medicinal plants for treatment of ailments7. recently, nanotechnology has extended biological systems for the production of nanoscale materials by using superficial techniques8. regarding biomedical approaches, nano techniques are being used in tissue engineering, drug delivery, and biosensor production5. in phyto-nanotherapy, the effect of plants is combined with metal nanoparticles which are equivalent to synthetic drugs, with the advantage of being associated with less side effects9. nanoparticles clasp more surface atoms which enhances their functional aptitude. among metals employed for nanoparticle synthesis, silver holds great promise owing towards exhibition of high level of toxicity against microorganisms and representation of the minimal harmful effects on mammals10. silver has been incorporated in many ointments and creams where it acts as an antimicrobial agent to prevent infections from burns and wounds11. silver nanoparticles (agnps) synthesized from flower extracts have exhibited antimicrobial activity against several pathogens12. they were detected as a reservoir of ag+ ions, which lead to deprivation of cell membrane by incorporating themselves into bacterial proteins13. this biosynthesis-based approach is attractive because agnps synthesis could be done extra-cellularly by using cell-free extracts of biomolecules loaded in free amino acids and organic acids. agnps suspension was shown to demonstrate intrinsic antibacterial activity against mdr pathogens as well, and it has been established that the colloidal agnps could eradicate growth of pathogenic bacteria14. with reference to helianthus annus, ozonated vegetable oils like canola, olive, and sunflower are used against bacterial infections15. tagetes erecta flower possesses many pharmacological properties and is also used as food colorant as it possesses many bioactive compounds which are the key determinants for its antioxidant and antibacterial activity16. in view of these observations, the present study aimed at exploring the antibacterial potential of agnps synthesized from helianthus annus and tagetes erecta flower extracts against mdr pathogens. m a t e r i a l s a n d m e t h o d s isolation and characterization of bacteria sewage water was collected from nishtar hospital and family hospital, multan, pakistan, in air-tight containers. serial dilutions of sewage water were plated onto nutrient agar to obtain pure isolates, which were then characterized based on gram staining, growth on mcconkey, emb and mannitol salt agar, as well as a series of biochemical tests including mr-vp, tsi, citrate utilization, starch hydrolysis, gelatin hydrolysis, and indole production. using kirbybauer disk diffusion method, mdr status of isolates was ascertained. for this purpose, isolated organisms were cultured on mueller-hinton agar plates and incubated at 37ºc for 18-24hrs after placement of antibiotic disks. antibiotics used for this purpose included vancomycin (30µg), imidazole (20µg), imipenem (30µg), augmentin (10µg), ceftazidime (30µg) and methicillin (30µg). subsequently, zones of inhibition were measured using clsi guidelines so as to establish status of bacteria as sensitive (s), intermediate (i) or resistant (r) to the antibiotics used. preparation of flower extracts floral part of two plants helianthus annus and tagetes erecta, were collected from field area of multan in sterile plastic bags and transported to the laboratory of department of microbiology and molecular genetics, the women university, multan. the collected floral parts were washed several times with running water followed by distilled water in last step to remove dust particles. for green synthesis of agnps and antibacterial activity against mdr pathogens vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 100 aqueous extraction, 20g of fresh petals of each flower were added to 60ml of distilled water and boiled on slow heat for 1hr with continuous stirring. then the extract was filtered, allowed to cool down at room temperature and stored at 4ºc for future use. for the preparation of methanolic extracts, petals were oven dried at 45ºc for 3-5 days and grinded into fine powder later on. oven-dried powder (20g) of each flower was added separately to 60ml of methanol and mixed with continuous shaking in shaking incubator at 15,000rpm for 72hrs. then, it was evaporated to obtain concentrated solution and stored at 4ºc for further use. synthesis and characterization of silver nanoparticles stock solutions of flower extracts were added to 5mm colorless silver nitrate solution separately in dark room to minimize photoactivation of silver. color change observed after 5min and 10min served as an indication for successful synthesis of nanoparticles. these solutions were centrifuged 2-3 times at 12,000rpm for 15min, supernatant was discarded every time to obtain purified particles and pellet was oven-dried at 45-50°c12. characterization of these silver nanoparticles (agnps) was done by uvvisible spectroscopy and fourier transform infrared (ftir) spectroscopy. ftir spectrum was recorded in the range of 500-4000cm-1. various modes of vibration were observed in transmittance mode. the reduction of ag+ to ag° was monitored by measuring the uv-visible spectrum within the range of 400-800nm at a resolution of 10nm in the uv-vis spectrophotometer including methanol as control. analysis of antibacterial activity of flower extracts and agnps for the assay of antibacterial potential of crude floral extracts as well as synthesized agnps, two alternative approaches were employed: well diffusion and disk diffusion. all the analyses were performed in triplicates so as to minimize chances of error. for well diffusion analysis to check antibacterial activity of crude extracts, holes of 6mm were punched on mueller hinton agar plates after swabbing isolates on them. extract solution (20μl) was added to each well and plates were left at room temperature for 1hour to allow diffusion of extracts followed by incubation at 37ºc for 24hrs. afterwards, zones of inhibition were measured and recorded in mm. for agnps activity analysis, filter paper disks were made by soaking those in agnps solution for 30min and subsequently drying for 5-10min. these discs were placed onto mueller hinton agar plates after swabbing of isolates and incubated at 37ºc for 24hrs, subsequent to which, zones of inhibition were measured in mm. r e s u l t s bacterial isolates and their mdr status isolated organisms included escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae, salmonella spp., shigella spp. and proteus spp. whose identity was ascertained following the scheme provided by bergey’s manual of systematic bacteriology. mdr status of these isolates was established based on resistance to more than one of the antibiotic drugs tested as documented in table 1. with the exception of s. aureus, all pathogens were mdr. s. aureus was however, resistant to methicillin. majority of the isolates demonstrated resistance against vancomycin and augmentin whereas, the most effective drug against majority of the pathogens was ceftazidime. table 1. antibiotic sensitivity profiles of bacteria isolated from sewage water. test organism vancomycin imidazole imipenem ceftazidime augmentin methicillin e. coli s r s r r i s. aureus s i i s s r p. aeruginosa r i r s r i k. pneumoniae r i r s r s salmonella spp. r s s i i r shigella spp. s i r s r i proteus spp. r i s r r i green synthesis of agnps and antibacterial activity against mdr pathogens vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 101 characterization of agnps synthesized from floral extracts silver nanoparticles (agnps) synthesized from h. annus and t. erecta floral extracts were characterized based on visual analysis, ftir spectroscopy and uv-visible spectroscopy. visual analysis on the basis of color change is used as a convenient indication for successful synthesis of agnps and the reason for the observation of this color change is surface plasmon resonance. for h. annus, the color of solution changed from colorless to dark yellow 5min after the addition of silver nitrate solution while that of t. erecta changed to dark green (fig. 1). after 10min, the color became dark as resonance increases with the passage of time. ftir analysis was performed to evaluate the presence of characteristic phytochemicals in floral extracts, which act as reducing and capping agents. ftir bands for h. annus were observed in different regions with the broadest absorbance band observed at 3404.30cm-1 (fig. 2). ftir bands for t. erecta were also observed at different values but the broadest band was observed at 3401cm-1 (fig. 3). uv-visible spectroscopy analysis was performed to establish reduction of ag+ to agº which is characteristic feature of silver demonstrating maximum absorbance in the range of 425nm. absorbance for both flowers was between 1.0 and 1.5, in the range of 400450nm which confirmed the presence of agnps (fig. 4). figure 1. visual analysis of agnps synthesized using (a) h. annus and (b) t. erecta floral extracts. figure 2. ftir analysis of agnps synthesized from h. annus flowers. green synthesis of agnps and antibacterial activity against mdr pathogens vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 102 figure 3. ftir analysis of agnps synthesized from t. erecta flowers. figure 4. uv-visible spectrum of agnps synthesized from (a) h. annus and (b) t. erecta flowers. antibacterial activity of crude floral extracts and their agnps in general, the antibacterial activity of crude floral extracts was enhanced after synthesis of their agnps, although exceptions existed (table 2). this observation is reinforced by the fact that aqueous floral extract of h. annus was completely ineffective against e. coli and salmonella spp. while that of t. erecta was not efficacious against p. aeruginosa. however, these demonstrated antibacterial effect against aforementioned pathogens subsequent to the synthesis of agnps. it was further documented that crude extract as well as agnps of h. annus flowers exhibited stronger bactericidal effect against most of the mdr isolates (fig. 5). however, t. erecta flowers were much more effective against e. coli as compared to h. annus flowers. likewise, in comparison with corresponding extract or agnps of h. annus flowers, crude aqueous extract of t. erecta flowers possessed stronger green synthesis of agnps and antibacterial activity against mdr pathogens vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 103 bactericidal activity against salmonella spp. and agnps synthesized from its methanolic extract displayed more potency against shigella spp. on the whole, methanolic extracts of both flowers as well as agnps synthesized from it were more effective against mdr isolates in comparison with aqueous extract and agnps synthesized from it, and the difference was noteworthy in some cases. the strong antibacterial potential of both flowers was revealed by the observation that agnps synthesized from methanolic extract of h. annus completely cleared the growth of s. aureus, and k. pneumoniae and those of t. erecta cleared growth of shigella spp. table 2. antibacterial activity assay of h. annus and t. erecta floral extracts and their synthesized agnps. test organism / type of extract agnps of t. erecta (zone of inhibition in mm) agnps of h. annus (zone of inhibition in mm) crude extract of t. erecta (zone of inhibition in mm) crude extract of h. annus (zone of inhibition in mm) e. coli aqueous extract 17 12 08 00 methanol extract 13 15 19 06 s. aureus aqueous extract 14 18 12 17 methanol extract 14 clear 16 16 p. aeruginosa aqueous extract 10 16 00 14 methanol extract 11 16 04 14 k. pneumoniae aqueous extract 13 17 06 15 methanol extract 12 clear 12 16 salmonella spp. aqueous extract 12 15 07 00 methanol extract 14 19 06 08 shigella spp. aqueous extract 14 15 09 10 methanol extract clear 19 09 12 proteus spp. aqueous extract 08 10 15 16 methanol extract 12 12 14 19 green synthesis of agnps and antibacterial activity against mdr pathogens vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 104 figure 5. evaluation of antibacterial activity of extracts by well diffusion and disk diffusion methods: a) crude extracts against methicillin-resistant s. aureus, t= t. erecta, h= h. annus; b) crude extracts against proteus spp., t= t. erecta, h= h. annus; c) agnps against e. coli, t= t. erecta, h= h. annus, ag=agno3 solution, c=control; c) agnps against p. aeruginosa, t= t. erecta, h= h. annus, ag=agno3 solution, c=control. d i s c u s s i o n due to its multi-targeted action, silver has already been established as a promising agent in overcoming antibiotic resistance because bacterial cell membranes have been documented to be harmed by agnps owing to structural changes, and this property can be used to enhance susceptibility of bacteria towards antibiotics17. silver nanoparticles (agnps) have specific optical, electrical, and thermal properties, as well as a large surface area to volume ratio, allowing them to associate with bacterial surfaces optimally, resulting in superior antimicrobial activity18. binding of silver ions to bacterial cell macromolecules causes structural alterations and deformations as well as degradation of essential enzymes by interaction with thiol groups, cumulating in the form of oxidative stress19. plant-based compounds possess pharmacologically significant attributes as their secondary metabolites act as anti-oxidant, anti-microbial, anti-cancer, anti-inflammatory agent etc20. furthermore, various parts of plants are non-toxic, economic, eco-friendly and safe for the synthesis of nanoparticles with an added advantage of being environment friendly21. in the present study, nanoparticles were synthesized using t. erecta and h. annus whose flowers are used for ornamental, cosmetic and medicinal purposes. characterization of agnps synthesized from t. erecta and h. annus flower extracts relied upon spectroscopic analysis. visual analysis in terms of color change serves as a simple spectroscopic signature marking the successful synthesis of nanoparticles12. color change observed after addition of silver nitrate solution to crude flower extracts was rapid and instantaneous and the intensity of color increased with the passage of time due to shift in surface plasmon resonance. several previous studies have documented change in the color of solution as an indication for formation of nanoparticles from various plant sources and it occurs due to reduction of silver ions and excitation of surface plasmon vibrations which are characteristic attributes of silver22-24. to identify the functional groups responsible for reduction of silver ions to agnps, ftir spectrum was generated in the range of 500green synthesis of agnps and antibacterial activity against mdr pathogens vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 105 4000cm-1. ftir spectrum for t. erecta produced peaks in the range of 557.45-3862.58cm-1 and h. annus produced the broadest band at 3404.30cm-1. these narrow peaks correspond to functional groups like flavonoids, saponins, terpenoids etc. these observations were in accordance to those reported earlier for some other plant sources25. the presence of narrow peaks implies that nanoparticles synthesized were of small size within the range of 1-100nm. presence of agnps was further confirmed by uv-visible spectroscopy and maximum absorbance was observed at 425nm. this is characteristic attribute of silver which is used to confirm the presence of agnps, and has been reported previously by other researchers as well26. two different types of extracts were prepared from flowers of t. erecta and h. annus i.e. aqueous and methanolic. methanolic extract was more effective against mdr organisms possibly due to the presence of hydrophobic phytochemicals in the solution. effectiveness of methanolic extract compared to other solvents has been reported for aegle marmelos fruit as well27. silver nanoparticles (agnps) of h. annus possessed greater antibacterial activity as compared to t. erecta particularly against grampositive bacteria. this was probably due to difference in cell wall composition as thin wall of gram-negative bacteria leads to poor penetration of silver nanoparticles28, 29. additional factors determining antibacterial activity of agnps include availability of particles in the medium, their electrostatic interactions and bacterial sensitivity to them30, 31. we observed a general increase in the antibacterial potential of both floral extracts subsequent to the synthesis of agnps which is in line with previous reports32. plant-agnps exhibited antibacterial activity against all the pathogens tested but particularly against s. aureus, shigella spp. similar to these findings, zones of inhibition have been shown by synthesized nanoparticles against pathogens like s. aureus, b. cereus, and p. aeruginosa33. additionally, antibacterial potential of plant-agnps has been reported against mdr pathogens including e. coli, k. pneumoniae and a. baumannii34. the exact mechanism underlying antibacterial potential of agnps has not been deciphered yet. theories proposed in this regard implicate altered membrane permeability, generation of reactive oxygen species, release of membrane lipopolysaccharides as well as disintegration of membrane potential35-38. c o n c l u s i o n inappropriate use of antibiotics has become significant and predominant problem as it is leading towards rapid increase in resistance among pathogens. the combined effect of plant and metal nanoparticles is equivalent to synthetic drugs with lower side effects. biosynthesis-based approach is attractive and effective as demonstrated by antibacterial potential of h. annus and t. erecta flowers against mdr organisms in this study which must be explored by further research. c o n f l i c t s o f i n t e r e s t none. f u n d i n g s o u r c e none. a c k n o w l e d g m e n t s none. l i s t o f a b b r e v i a t i o n s agnps silver nano particles clsi clinical and laboratory standards institute ftir fourier transform infrared mdr multi drug resistant r e f e r e n c e s 1. zahlane k, ouafi at, barakate m. the clinical and epidemiological risk factors of infections due to multidrug resistant bacteria in an adult intensive care unit of university hospital center in marrakesh-morocco. j infect public health. 2020; 13(4):637-43. 2. li b, webster tj. bacteria antibiotic resistance: new challenges and opportunities for implant‐associated orthopedic infections. j orthop res. 2018; 36(1):2232. 3. shah as, karunaratne k, shakya g, barreto i, khare s, paveenkittiporn w, et al. strengthening laboratory surveillance of antimicrobial resistance in south east asia. bmj. 2017; 358-63. 4. zampieri m, enke t, chubukov v, ricci v, piddock l, sauer u. metabolic constraints on the evolution of antibiotic resistance. mol syst biol. 2017; 13(3):91725. green synthesis of agnps and antibacterial activity against mdr pathogens vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i 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aureus (mrsa). j clin microbiol antimicrob. 2016; 15(1):1-3. 20. akram m, hamid a, khalil a, ghaffar a, tayyaba n, saeed a, et al. review on medicinal uses, pharmacological, phytochemistry and immunomodulatory activity of plants. int j immunopathol pharmacol. 2014; 27(3):313-9. 21. shahid m, shahzad a, malik a, sahai a. recent trends in biotechnology and therapeutic applications of medicinal plants. dordrecht springer. 2013. 22. alsammarraie fk, wang w, zhou p, mustapha a, lin m. green synthesis of silver nanoparticles using turmeric extracts and investigation of their antibacterial activities. colloids surf b: biointerfaces. 2018; 171:398-405. 23. gavamukulya y, maina en, meroka am, madivoli es, el-shemy ha, wamunyokoli f, et al. green synthesis and characterization of highly stable silver nanoparticles from ethanolic extracts of fruits of annona muricata. j inorg organomet polym mater. 2020; 30(4):1231-42. 24. tamilarasi p, meena p. green synthesis of silver nanoparticles (ag nps) using gomphrena globosa (globe amaranth) leaf extract and their characterization. mat today: proc. 2020; 33:2209-16. 25. yazdi me, khara j, sadeghnia hr, bahabadi se, darroudi m. biosynthesis, characterization, and antibacterial activity of silver nanoparticles using rheum turkestanicum shoots extract. res chem intermed. 2018; 44(2):1325-34. 26. jacob sj, prasad vs, sivasankar s, muralidharan p. biosynthesis of silver nanoparticles using dried fruit extract of ficus carica-screening for its anticancer activity and toxicity in animal models. food chem toxicol. 2017; 109:951-6. green synthesis of agnps and antibacterial activity against mdr pathogens vol. 12 (2), dec 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 107 27. devi m, devi s, sharma v, rana n, bhatia rk, bhatt ak. green synthesis of silver nanoparticles using methanolic fruit extract of aegle marmelos and their antimicrobial potential against human bacterial pathogens. 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nanoparticles biosynthesized using chenopodium murale leaf extract. j saudi chem soc. 2014; 18(4):356-63. 33. zarei z, razmjoue d, karimi j. green synthesis of silver nanoparticles from caralluma tuberculata extract and its antibacterial activity. j inorg organomet polym mater. 2020; 30:4606-14. 34. alharbi fa, alarfaj aa. green synthesis of silver nanoparticles from neurada procumbens and its antibacterial activity against multi-drug resistant microbial pathogens. j king saud univ sci. 2020; 32(2):1346-52. 35. amro na, kotra lp, wadu-mesthrige k, bulychev a, mobashery s, liu gy. high-resolution atomic force microscopy studies of the escherichia coli outer membrane: structural basis for permeability. langmuir. 2000; 16(6):2789-96. 36. lok cn, ho cm, chen r, he qy, yu wy, sun h, et al. proteomic analysis of the mode of antibacterial action of silver nanoparticles. j proteome res. 2006; 5(4):916-24. 37. panáček a, kvitek l, prucek r, kolář m, večeřová r, pizúrová n, et al. silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity. j phys chem b. 2006; 110(33):16248-53. 38. kim js, kuk e, yu kn, kim jh, park sj, lee hj, et al. antimicrobial effects of silver nanoparticles. nanomed: nanotechnol biol med. 2007; 3(1):95-101. . in vitro phytochemical analysis & antioxidant assay of fruit extracts vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 50 op e n ac c e s s f u l l l e n g t h a r t i c l e in vitro phytochemical analysis and antioxidant assay of fruit extracts of sapindus mukorossi gaertn. and acacia concinna dc faiza anum1,*, ayesha alam2, aroosa aftab1, labeeb ali3, arifa tahir4, shahid raza5 1department of botany, lahore college for women university, pakistan. 2department of interactive agriculture, united arab emirates university, uae. 3department of chemical and petroleum engineering, united arab emirates university, uae. 4department of environmental sciences, lahore college for women university, pakistan. 5department of biotechnology, university of central punjab, pakistan. a b s t r a c t background: sapindus mukorossi, commonly known as areetha, and acacia concinna, commonly known as shikakai reported to have potential naturally occurring medicinal properties. the prime medicinal actions included cleaning, therapeutic mode of action, antifungal properties and cited as important ingredients in ayurvedic medicines. the antioxidant potential of these plants could be the best alternative for the commercially synthesized antioxidants added in medicinal and cosmetic products. objectives: to examine the variety of phytochemicals and antioxidant potential of fruit extracts of both plants’ species. methodology: the dry fruits were powdered and extracts of different polar and non-polar solvents were used to examine the presence of secondary metabolites in plants bearing fruits by using phytochemical tests and dpph assays for antioxidant potential. results: the experimental outcomes revealed a positive response in case of cardiac glycosides, flavonoids, coumarins and terpenoids and showed negative results for phlobatannins and anthraquinones, respectively. positive results were obtained in case of total antioxidant assay as compared to that of dpph assay, in comparison to the standard antioxidant agents available commercially that were utilized in the study. conclusion: this study concluded acacia and sapindus fruits to be organic replacement for commercially synthesized antioxidants and a potential source of secondary metabolites. keywords dried fruits powder, medicinal plants, phytochemistry, plant extracts, polar, nonpolar solvents, secondary metabolites. *address of correspondence faizaanum55@gmail.com article info. received: july 26, 2021 accepted: may 31, 2022 cite this article: anum f, alam a, aftab a, ali l, tahir a, raza s. in vitro phytochemical analysis and antioxidant assay of fruit extracts of sapindus mukorossi gaertn. and acacia concinna dc. 2022; 13(1):50-59. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. o r i g i n a l a r t i c l e in vitro phytochemical analysis & antioxidant assay of fruit extracts vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 51 i n t r o d u c t i o n phytochemicals are organic compounds that may act as defense mechanism of plants against different predators1. the beneficial and multipurpose pharmacological properties of medicinal flora are fundamentally reliant on their phytochemical components2. various kinds of plants are used for medicinal purposes owing to their medicinal potential3. at the present time, a major drawback is that out of the 250,000-500,000 known plant species on the planet earth, only a few plants have been examined phytochemically for their potential properties4. therefore, there is a dire need to perform research on the identification of bioactive compounds that are beneficial for treating numerous human ailments caused by bacterial and fungal infections5. sapindus mukorossi (reetha) and acacia concinna (shikakai) are potential medicinal plants mostly used in manufacturing of hair care products and detoxifiers6. sapindus mukorossi is mostly found in the tropical and subtropical territories of asia having tropical weather7. it is a tall deciduous tree which attains a height of 20m. sapindus mukossori has pharmaceutical properties e.g. antidandruff, mucolytic, and spermicidal. it is utilized in the treatment of diseases like psoriasis (skin infections), chlorosis (a sort of weakness), dermatitis, normal tingles, and in ailment caused by helicobacter pylori, a gut pathogen8. acacia concinna, commonly called shikakai is a remedial plant, the fruit of which is utilized, traditionally known as "fruit for hair"9. the plant owns bioactive natural metabolites having anticoagulant (repressing blood coagulation), and antiplatelet properties10. the leaves are used for the treatment of diabetes and furthermore, for skin ailments in india, myanmar, and thailand11. scientists performed a phytochemical investigation of sapindus mukorossi extracts in water and ethanol which showed the presence of various secondary metabolites for example, phytosterols, flavonoids, alkaloids, phenolics, saponins, tannins, and glycosides12. in this research, we have investigated the presence of complex secondary phytometabolites such as triterpenes, saponins, glycosides, flavonoids, alkaloids, tannins, and anthraquinones. leaf extracts were prepared by using various polar and non-polar chemical solvents. the main aim of this research work was to examine the utilization of medicinal plants depending upon their naturally occurring phytochemical nature and finding their potential for targeted secondary usage. m a t e r i a l s a n d m e t h o d s plants selected for examination were phytochemically analyzed and then subjected for an antioxidant assay. analytical grade chemicals were used in the study for precise reaction chemistry. review of experimental approach the plant extracts were stirred on the magnetic stirrer for 5h at room temperature. the spectrophotometric calculations of samples were done by using uv/vis spectrophotometer (uv-1650pc, shimadzu, japan). preparation of reagents first, 0.6m h2so4 (95-97%) was prepared by dissolving 1.66ml h2so4 in 100ml of distilled water in a volumetric flask. potassium dihydrogen phosphate was prepared by dissolving 0.3808g potassium dihydrogen phosphate in 100ml of distilled water. ammonium molybdate (4mm) was prepared by dissolving 0.0784g of ammonium and 0.3g molybdate in 100ml of distilled water, respectively. plant materials fruit samples of s. mukorossi gaertn. and a. concinna (willd.) dc. were sorted, washed and extraction was done by utilizing petroleum ether, dis. water, methanol and chloroform solvents. plant extract preparation to make extracts, samples were sun dried, grounded, and the powder was preserved in amber shaded containers at 4ºc. solvent extraction: maceration for this, 15g powdered sample was added into 30ml of (non-polar and polar) solvents for at least 7 days (approx. 160hrs). samples were filtered, isolated, and preserved in glass vials. the leftover was set for next extraction. ash, moisture content and acid moisture content, total ash, and acid insolubility were determined by using standard methods13. in vitro phytochemical analysis & antioxidant assay of fruit extracts vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 52 phytochemical assay phytochemical assay was conducted as per standard procedures to estimate the following metabolites14: alkaloids approx. 0.25g of powdered plant material was mixed in 4ml of 1% hcl followed by stirring and heating. six drops of mayors reagent were added to the 1ml of plant filtrate displaying creamy orange color compound as an indicator of an alkaloids. frothing test (saponins) distilled water (5ml) was poured into 0.5g powder plant sample and shaken vigorously to observe frothing intensity. coumarins initially, 0.5g of plant sample with 0.1n naoh was added in a secure test tube with filter paper and kept in a water bath for 10min. later, the filter paper was observed under bright (uv) light for the presence of yellow inflorescence. anthraquinones here, 3ml of 1% hcl was added into 0.5g of plant sample and filtered, followed by the addition of 2ml benzene and shaken. benzene on the top was decanted and few drops of 10% ammonium hydroxide were added. the generation of violet, red, or pink color solution demonstrated the indication of anthraquinones. terpenoids (liebermann-burchard test) plant extract (2ml) was dissolved in 2ml of chloroform and filtered. for the estimation, a drop of sulphuric acid and methanol was added to the filtrate. appearance of bluegreen color ring in the tube confirmed the presence of terpenoids. flavonoids plant powder (5g) and petroleum ether were dissolved together and mixed in 20ml of 80% ethanol and filtered. then, 3ml of filtrate was mixed with 4ml of 1% koh to see dim yellow shade substance indicating flavonoids. phlobatannins petroleum ether (5ml) and 1% hcl when mixed into 0.25g plant sample generated red precipitates showing the presence of phlobatannins. tannins iron chloride (1%) and 10ml of distilled water were added to 0.25g sample and filtered. tannins were indicated with the production of dark green to dark blue tinge color. cardiac glycosides (keller-kiliani test) for this, 0.5g of sample was mixed with 2ml of glacial acetic acid and few drops of 1% iron chloride along with 1ml of extracted sulphuric acid to indicate the presence of cardiac glycosides by the formation of green-blue color. assessment of antioxidant activity total antioxidant and dpph (2,2-diphenyl-1-picrylhydrazyl) radical scavenging was conducted by using standard methods15. the transfer of electron is responsible for the reduction of an antioxidant reagent. the sample capacity to scavenge the radicals was observed by spectrophotometer and quantified in bht (butylated hydroxytoluene) with α-tocopherol equivalents as standards16. dpph (2,2-diphenyl-1-picrylhydrazyl) radical scavenging measure dpph assay was used to assess the scavenging of free radicals in solvents. homogenous solutions were prepared by adding 0.5mg/ml solute in dmso (dimethyl sulphoxide). dpph and dmso were mixed and kept for 30min in dry shade. dpph radical scavenging was conducted by using spectrophotometer at 517nm absorbance with three replicates. the antioxidant agent action was derived as % as17: sc % = absorbance of control − absorbance of test absorbance of control × 100 the control group included all experimental ingredients except the sample however, testing of dpph scavenging action of bht and α-tocopherol was done at diverse concentrations, e.g., 0.5, 1, 2.5 and 5mg/ml to observe correlation between them. statistical software used for analysis were spss and minitab 2.0, respectively. total antioxidant assay (taoa) determination all solutions with concentration 0.1ml or 0.5mg/ml were shaken up by adding reagent (1.9ml) carrying (4mm (nh4)6mo7o24, 28mm na2so4 and 0.6m h2so4) and set for in vitro phytochemical analysis & antioxidant assay of fruit extracts vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 53 incubation for 1hr at 95°c temperature. furthermore, the mixture was set for cooling and estimation of wavelength was done at calibrated spectrophotometer set at a wavelength of 695nm along with blank. similar estimation was conducted for bht (0.5mg/ml) antioxidant potential. statistical analysis mean ± s.e and standard deviation of all triplicates of data sets were calculated and means were compared by using the turkeys’ multiple range tests at a significance level of α=0.5 as probability values. data sets were further analyzed using two-way anova, in completely randomized design to estimate significant effects of the factors. r e s u l t s a n d d i s c u s s i o n both test species are known to be utilized for the treatment of different ailments since many decades (table 1) with high antioxidant potential. therefore in vitro assays were analyzed for total phenolics and flavonoids. qualitative phytochemical analysis plant material was examined for various compounds21. most of the compounds were identified as secondary metabolites that give particular shade and fragrance to the plant. in this study, it has been noticed that every test shows a particular shading as the end point (table 2 & fig. 1). secondary metabolites tests in the phytochemical investigation of s. mukorossi gaertn and a. concinna (willd.) dc., lack of cream and orange precipitates demonstrated the absence of alkaloids. formation of froth in the sample analysis indicates the presence of saponins. absence of white froth indicated the absence of anthraquinones. emanation of yellowish inflorescence in uv light the demonstrated the presence of coumarins in the examination of s. mukorossi and a. concinna (willd.). fruits formation of blue green ring demonstrated the indication of terpenoids. a light-yellow shading in the extracts demonstrated that flavonoids were present. sign of earthy green shading demonstrated that tannins are available while the absence of caramel green color indicated that tannins were not present. no deposition of red precipitates demonstrated that phlobotannins are missing in individual plant sample. sign of blue green color demonstrated that glycosides were present (table 2). table 1. medicinal plant species analysed for the study. scientific name common name family growth form moisture content (g/100g) ash content (g/100g) acid insoluble ash/ silicates (g/100g) part analyzed fruit medical importance reference sapindus mukorossi garten. areetha soap berry spaindaceae deciduous tree 4.24± 0.098 16.96± 0.24 10.7± 0.05 fruit berry epidermal cleanser, insecticide, headache, migraine, eczema 18, 19 accacia concinna dc. shikakai fabaceae legume 5.07± 0.032 23.7± 0.15 21.03± 0.23 fruit pod pod shampoo, natural oil, cleaning agent, detergent, jaundice 20, 11 in vitro phytochemical analysis & antioxidant assay of fruit extracts vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 54 table 2. detection of secondary metabolites in plant samples. phyto constituent indication result images s. mukorossi a. concinna alkaloids orange color appear ✔ saponins froth formation occur ✔ anthraquinone white colour frouth × coumarins yellow inflorescence ✔ terpenoids occurrence of ring ✔ flavaniods appearance of yellow color ✔ tannins brownish green coloration not appeared × contd…. in vitro phytochemical analysis & antioxidant assay of fruit extracts vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 55 phlobotanins no red ppt. form × cardiac glycosides violet ring and brown color appear ✔ figure 1. secondary metabolites concentration g/100g. table 2 shows the presence and absence of all secondary metabolites that were tested in the plant samples. results for the presence of alkaloids, saponins, coumarins, terpenoids, flavonoids and cardiac glycosides were positive while, anthraquinones and phlobatannins were absent in s. mukorossi and a. concinna. figure 1 shows the average metabolite concentrations g/100g in the acacia plant samples showed highest flavonoid content up to 9.5± 0.20 and lowest alkaloid content upto 0.51± 0.04. s. mukorossi reported highest concentration of coumarins up to 7.6± 0.15 and lowest values of alkaloids up to 0.56± 0.01. the standard deviations were relatively low indicating the equal distribution of data across the means and uniformity in the results. the significant differences in the results of remaining phytochemicals within plants and between plants may be because of ecological factors such as climate, temperature, moisture, humidity, salinity, and water scarcity22. similarly, amount of moisture in the environment and rainfall rate may variate the phytochemical content in the plant sample and extracts23. flavonoids are the potential radical scavenging secondary metabolites that causes significant reduction in oxidative stress in cell lines, to generate immunity against diseases24. from the current study it can be seen that both plants extracts are rich in flavonoids content which concurs with the research outcomes of potential publications significantly25,26. the debatable point here is that both of the plant samples depicted least concentrations of alkaloids. a publication reported optimum levels of alkaloids in crude extracts of seeds which concurs with the present study27. alkaloids own low palatability and considered as one of the noxious food compounds in the in vitro phytochemical analysis & antioxidant assay of fruit extracts vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 56 hierarchy of the components, hence providing a validation for the safe consumption and application of fruit samples under study28. acacia fruit extracts showed higher ratios of terpenoids as compared to the sapindus showing more tendency to exhibit benefits for disease control and treatments. terpenoids are structural components in dietary and medicinal plants29. the higher quantities of terpenoids in acacia declare palatability of plant species. a research group examined different phytochemicals in plants samples30. the results exhibited the presence of terpenoids similar to current study which concurs with the standards set out by the previously reported scientific reviews. coumarins are derivatives of pyro benzene and possess potential biological antimicrobial activity, and least toxicity was evidenced in sapindus plant in the current study31,32. sapindus plant has higher palatability as compared to other competent plants, and our study is in line with the previously declared use19. anthraquinones and phlobatannins are rarely occurring secondary metabolites across plant species33,34, similar to current study where the metabolites were not evidenced. all of these outcomes indicated the selective proportions of metabolites in these plants and it can be a milestone in promoting selective extraction of these phytochemicals for further purposes. antioxidant assays total antioxidant assay (taoa) quantitative analysis of extracts was done by taoa standard protocol. results were compared with αtocopherol (standard), 0.513 and 0.476 bht, which validated that the sample qualities were quite similar to the said standards (α-tocopherol: 0.513, bht: 0.476, blank: 0.026) adsorption at 695nm. among all solvents, the value for distilled water added s. mukorossi gaertn. sample was 0.476 ± 0.096ab being same as the value of standard chemical bht, 0.476. hence, it can be replaced by standard chemical. in the case of a. concinna, distilled water indicated most extreme value i.e. 0.346 ± 0.050a while petroleum ether displayed least antioxidant agent value i.e. 0.263 ± 0.025a (fig. 2). all of the values are an average of three replicates in which ± is denoted standard deviation at 0.5% significance level (p ≤ 0.05) antioxidant analysis through radical scavenging activity of dpph results were being equated with standard antioxidant available i.e (bht, 0.190 and α-tocopherol, 0.095, blank, 0.03) absorption at 517nm. among all the solvent extracts estimated, only few showed in line results to the standard values. therefore, the closer solvents are recommended to be utilized as standards. chloroform extract of s. mukorossi gaertn. showed value 0.196 ± 0.029b which is close to the standard bht, 0.190 proving to be alternative to the standards (fig 3). all of the values are an average of three replicates in which ± is denoting standard deviation at 0.5% significance level (p ≤ 0.05). figure 2. antioxidant activity in plant extracts by taoa adsorption at 695nm (left: s. mukorossi; r.ight: a. concinna). in vitro phytochemical analysis & antioxidant assay of fruit extracts vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 57 figure 3. antioxidant activity of plant extracts by dpph analysis absorption at 517nm (left: s. mukorossi right: a. concinna). figures 2 and 3 reveal that antioxidant values of plants extracts concur to that of standards taken for the study. total antioxidant activity and dpph method values ranged from 0.203 to 0.576 at 695nm wavelength while 0.145 to 2.346 at 517nm wavelength which falls in line with comparative standard outcomes of previous research outcomes35. the generated outcomes from the assay displayed that plant extracts can scavenge the present radical up to a certain limit. a research group investigated the antioxidant potential by means of total antioxidant assay and free radical scavenging activity similar to the current study36. in our investigation methanolic extracts of s. mukorossi gaertn. displayed maximum antioxidant value such as 0.576 ± 0.096a. whereas, petroleum ether extracts had lowermost value i.e. 0.203 ± 0.085c at 695nm wavelength. similarly, distilled water concentration 0.476 ± 0.096ab which concurs with the value of standard bht 0.476. at 519nm wavelength, s. mukorossi gaertn. displayed highest antioxidant value in dis. water extract as 0.456 ± 0.169b and least with methanol such as 0.176 ± 0.035b. on the other hand, chloroform extract value was found as 0.196 ± 0.029b being very close to that of standard bht, 0.190. similarly, a. concinna (willd.) dc. also indicated maximum result with distilled water as 0.723 ± 0.195a and lowest with chloroform as 0.145 ± 0.045b. both outcomes lied closer to the standards utilized in the experimental approach that is bht, 0.190. the similar values led to the advantage that these extracts can be utilized as standards37. earlier reports showed that phytochemical compounds possess strong antioxidant activity for radical scavenging38. the radical scavenging ability leads to a reduction in oxidative stress and neutralizes the overall metabolism39. plant origin antioxidant and oxidative stress reducing bio-products are on huge demand in the market due to least side effects and long-term benefits due to easy adjustment within the human body drug translocations40. this study revealed that massive potential of acacia concinna and sapindus mukorossi evidently constitutes optimum capacities of antioxidant mediated oxidative stress release as the recommended proportions for active cell lines. most importantly, the polymer of quinic acid is reported as the strongest scavenger by the neutral radical dpph assay41. this polymer has been verified to be potent to that of bht used as a positive control42. hence, this study proves that it can be used as standard for further assays. c o n c l u s i o n it is concluded that s. mukorossi and a. concinna proved active in the presence of secondary metabolites e.g. alkaloids, saponins, terpenoids, coumarins, flavonoids and cardiac glycosides, and exhibited the absence of phlobotanins and anthraquinones. additionally, the antioxidant potential was perceived through total antioxidant assay (taoa) and dpph assays in which plant extracts especially distilled water extract of s. mukorossi gaertn., chloroform extract of s. mukorossi gaertn. and chloroform extract of a. concinna (willd.) dc. in vitro phytochemical analysis & antioxidant assay of fruit extracts vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 58 presented similar outcomes with standard antioxidants such as bht and α-tocopherol at two different wavelengths of 695nm and 517nm respectively. the results from current study showed s. mukorossi and a. concinna to be imperative and reliable for applications in chemical industry and pharmaceutical engineering. e t h i c a l a p r o v a l the manuscript does not comprise any animal and human based studies hence no ethical approval was needed. c o n f l i c t o f i n t e r e s t the authors declare no conflict of interest. f u n d i n g s o u r c e the research study was part of undergraduate thesis and did not receive any funding. a c k n o w l e d g e m e n t s the following study was done in collaboration with lahore college for women university, pakistan and united arab emirates university, uae. l i s t o f a b b r e v i a t i o n s % percentage anova analysis of variance bht butylated hydroxytoluene c centigrade dmso dimethyl sulfoxide dpph 2,2-diphenyl-1-picrylhydrazyl g gram ml mili liter mm mili molar n normal nm nano meter sc% scavenging taoa total antioxidant assay uv ultraviolet r e f e r e n c e s 1. wouters fc, blanchette b, gershenzon j, vassão dg. plant defense and herbivore counter-defense: benzoxazinoids and insect herbivores. phytochem rev. 2016; 15(6):1127-51. 2. egamberdieva d, mamedov n, ovidi e, tiezzi a, craker l. phytochemical and pharmacological properties of medicinal plants from uzbekistan: a review j med act. plants. 2017; 5(2):59-75. 3. petrovska bb. historical review of medicinal plants’ usage. pharmacogn rev. 2012; 6(11):1-9. 4. pan sy, zhou sf, gao sh, yu zl, zhang sf, tang mk, et al. new perspectives on how to discover drugs from herbal medicines: cam's outstanding contribution to modern therapeutics. evid.-based complement altern med. 2013; 1-7. 5. mcdaid d, sassi f, merkur s. promoting 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2015; 19(2):11724. 38. kedare sb, singh rp. genesis and development of dpph method of antioxidant assay. j food sci technol. 2011; 48(4):412-22. 39. lalhminghlui k, jagetia gc. evaluation of the freeradical scavenging and antioxidant activities of chilauni, schima wallichii korth in vitro. future sci oa. 2018; 4(2):272-8. 40. karimi a, majlesi m, rafieian-kopaei m. herbal versus synthetic drugs; beliefs and facts. j nephropharmacol. 2015; 4(1):27-9. 41. abozed ss, el-kalyoubi m, abdelrashid a, salama mf. total phenolic contents and antioxidant activities of various solvent extracts from whole wheat and bran. ann agric sci. 2014; 59(1):63-7. 42. hung tm, na m, thuong pt, su nd, sok d, song ks, seong yh, bae k. antioxidant activity of caffeoyl quinic acid derivatives from the roots of dipsacus asper wall. j ethnopharmacol. 2006; 108(2):188-92. vol6(1) (1).pdf introduction bacteria from clinical and non-clinical settings are becoming increasingly resistant to conventional antibiotics. the use of the antibiotics was started after some years of flemings’ discovery, who noticed the inhibition of staphylococcus species by penicillium spp. (sosa et al. 2010). soon after the breakthrough, this was not last for long and proved to be a misplaced belief that available antibiotics would always effectively treat all infections (sosa et al. 2010). increasing prevalence of resistance has been reported in many pathogens in different regions of the world including developing countries (byarugaba, 2005). however there are many factors which can influence bacteria to become insensitive to antibiotics; the two main factors are the dissemination of resistance genes and the extensive use of antibiotic. antibiotic-resistant pathogens are not more virulent than susceptible ones, but the resistant forms are harder to destroy. bacteria can acquire resistance genes through a few routes. many inherit the genes from their forerunners. other, from genetic mutations, which occur readily in bacteria, will spontaneously produce a new resistance trait or will strengthen an existing one (levy et al., 1998). infections caused by enterobacteriaceae are treated usually with antibiotics, such as fluoroquinolones, beta-lactams and aminoglycosides. however, they antibiotic resistance pattern and detection of newly emerging resistant gene new delhi metallo-ß-lactamase1(blandm-1) in escherichia coli and klebsiella pneumoniae erum mazhar 1, sayyada ghufrana nadeem 2, abdul basit khan 2, 3 1department of environmental science, jinnah university for women, karachi. 2department of microbiology, jinnah university for women, karachi. 3pcsir laboratories complex, off university road, karachi. abstract escherichia coli and klebsiella pneumoniae are the normal inhabitants of humans. most strains of e. coli are harmless and benefit their host but some of them may cause a wide variety of intestinal and extra-intestinal diseases, such as diarrhea, urinary tract infections, septicemia, neonatal meningitis and renal complications. several strains klebsiella may also cause bacteremia, pneumonia and urinary tract infections. these pathogenic strains are becoming resistant to antibiotic therapy. this antibiotic resistance is posing a major threat to public health and problem in treating various infections. the present study has been designed to evaluate antibiotic resistance pattern and detection of newly emerging gene new delhi metallo-ß-lactamase 1 (blandm-1) in escherichia coli and klebsiella pneumoniae clinical isolates. a total of 52 clinical isolates were collected from different hospitals of karachi in the period of august 2012 october 2012. antibiotic sensitivity test was performed by disc diffusion method using different antibiotics. in this study it was found that e.coli from diarrheal source and other sources showed highest resistance to ampicillin (upto 89%) and is highly sensitive to meropenem and imipenem (100%). while klebsiella pneumoniae showed highest resistance to ampicillin (100%) and showed some resistance to meropenem and imipenem (13%). in the pcr detection, 04 isolates out of 52 carried the resistant gene blandm-1. the presence of this blandm-1 gene was significantly higher in klebsiella pneumoniae as compared to escherichia coli strains. keywords: antibiotic, disc diffusion method, metallo-ß-lactamase 1, pcr. *corresponding author: sheikherum21@gmail.com 14 vol 5 (1), january 2014; 14-17 are now able to resist these antibiotics and can develop several mechanisms for resistance (kocsis and szabó, 2010). by 1983, some strains of klebsiella found to have extended spectrum ßlactamases (esbls) capable of inactivating extended-spectrum cephalosporins (pereira et al., 2011). emergence of carbepenamases further complicates the situation as carbapenems were used to treat cephalosporin resistant esbl-producing organisms (pereira et al., 2011). different studies indicated that the most common carbapenemase in the us is klebsiella pnuemoniae cabapenemase (kpc) (gupta, et al., 2011). recently, a new class b enzyme, new delhi metallo-ß-lactamase (blandm-1) was discovered which confers resistance to all antibiotic except fluoroquinolones and colistin (yong, et al., 2009). later studies confirmed the prevalence of this resistance factor pretty common in clinical isolates of india, bangladesh and pakistan (kumarasamy et al., 2010). cases have also been reported in other developed countries making it a global threat to human health (mochon, et al., 2011 & pfeifer, et al., 2011). these resistant traits are very serious in nature leaving very few or no remedial options. the aim of this study was to evaluate the resistance pattern of e. coli and k. pneumoniae isolated from clinical samples to different antibiotics. in addition, it was also intended to screen these isolates for the presence of newly emerging new delhi metallo-ß-lactamase1 (blandm-1) gene through polymerase chain reaction. material and methods study duration: 03 months (august 2012-october 2012). sample size: a total of 52 isolates belong to escherichia coli and klebsiella pneumoniae spp. were collected from different laboratories of karachi. these bacterial strains were isolated from diarrhea, sputum, urine and nasal swab. purification and identification of isolates: all isolates were initially processed to purify and confirm for their species identification. briefly, bacterial isolates were streaked on eosin methylene blue (emb) agar plates for isolated colonies and incubated for 24hrs at 37°c. all pure cultures were identified on the basis of their m o r p h o l o g i c a l , c u l t u r a l a n d b i o c h e m i c a l characteristics. antimicrobial susceptibility testing: kirby-bauer disk diffusion method as described earlier in many studies was used to determine the antibiotic resistance of the organisms (lalitha,2003). briefly, bacterial lawns were made on pre-incubated agar plates and antibiotic discs were placed aseptically. after incubation, zone of inhibitions were recorded and results were interpreted using clinical and laboratory standard institute, guidelines (clsi, 2011). polymerase chain reaction (pcr) for the detection of blandm-1: polymerase chain reaction (pcr) was used to enumerate blandm-1 resistant gene. the primer sequences and conditions as reported earlier were used in this study (kruttgen, 2011). briefly, initial denaturation was done at 95°c for 5 minutes followed by 35 cycles of denaturation at 95°c for 30 seconds, annealing at 60°c for 40 seconds, and extension at 72°c for 50 seconds. the thermal cycling was terminated with a final extension of 72°c for 5 minutes. these primers amplified an internal sequence of 206 base pairs of blandm-1 gene. agarose gel electrophoresis and ethidium bromide staining was performed to analyse pcr products. the sequences of the forward and reverse primers were: ndm-fm: 5’cttccaacggttt gatcgt c-3’, ndm-rm: 5’-attggcataagt cgcaa tcc-3’. results a total of 52 isolates were collected from different laboratories which belong to e. coli and klebsiella pneumoniae spp. antibiotic susceptibility testing was performed for all the isolates. a total of 7 antibiotics were used. all the strains showed high resistance to ampicillin. e.coli from diarrheal sources and other sources showed resistance to ampicillin as 82% and 89% respectively, and highly sensitive to imipenem and meropenem. while klebsiella 15 vol 5 (1), january 2014; 14-17 strains showed 100% resistance to ampicillin and 13% resistance to imipenem and meropenem. resistances to other antibiotics are shown in table: 1 and figure 1. pcr detection of blandm-1 gene: all the 52 isolates were subjected to pcr using primers for blandm-1 gene. these primers amplified an internal sequence of 206 base pairs of blandm1 gene. four isolates out of fifty two were positive and showed bands on the gel (figure 2 & 3). among the 04 positive strains, 03 strains were of k. pneumoniae and only 01 strain was of e. coli. discussion escherichia coli and klebsiella pneumoniae are the normal inhabitants of humans, but sometimes they become opportunistic pathogen and cause infections. these bacterial infections are often treated with antibiotics. now days, many bacterial strains are showing to have resistance against the drugs. this antibiotic resistance is posing a major threat to public health and in treating various infections. the present study has been designed to observe the sensitivity pattern through disc diffusion method and to detect the resistant gene blandm-1 in escherichia coli & klebsiella pneumoniae through pcr assay. a total of 52 isolates belong to e. coli and k. pneumoniae was included in this study. these isolates were collected from different laboratories of karachi in the period of august 2012october 2012. all the isolates were subcultured and identified on the basis of morphological, cultural and biochemical properties. antibiotic susceptibility test was performed for the detection of resistance pattern in escherechia coli and klebsiella pneumoniae. results showed that e. coli from diarrheal specimens showed 33% resistance, e. coli from various sources showed 35% resistance and klebsiella from different sources showed 32% resistance. all the strains showed high resistance to ampicillin, i.e e. coli strains showed (82%-89%) resistance, while klebsiella strains showed (100%) resistance. this resistance pattern is similar to the previous study (oplustil et al., 2001). the percentage of resistance among outpatients to ampicillin was 11.3% and, to ampicillin alone, the resistance was 94.5%. a similar profile was observed for inpatients, where we had 33.7% resistance to ampicillin /sulbactam, and 97% to ampicillin alone. all the e. coli and k. pneumoniae strains showed highest susceptibility to imipenem and meropenem i.e. 100% and 87% respectively. this pattern is similar to the study (alzahrani et al., 2005). esblproducing e. coli isolates showed highest susceptibilty to meropenem (95.8%) followed by amikacin (93.7%), and imipenem (91.7%). esbl16 vol 5 (1), january 2014; 14-17 figure 3: agarose gel electrophoresis k. pneumoniae. sm: 100bp dna size marker figure 2: agarose gel electrophoresis of e. coli from diarrheal samples. sm: 100bp dna size marker. table i: antibiotic resistance pattern of e. coli and k. pneumoniae key: amp=ampicillin, te=tetracycline, cip=ciprofloxacin, levo=levoflaxacin, c = c h l o r a m p h e n i c o l , i p m = i m i p e n e m , m e m = m e r o p e n e m figure 1: graphical representation of table 1.0 17 vol 5 (1), january 2014; 14-17 producing k. pneumoniae showed highest susceptibility to meropenem (94.4%) followed by gentamicin & piperacillin/tazocin (88.9%), and amikacin, ciprofloxacin & levofloxacin (83.3%). polymerase chain reaction (pcr) of all the 52 isolates was done and it was found that 04 isolates out of 52 carried the resistant gene blandm-1. the sample size was not so large, but still we were able to have some positive isolates carrying this resistant gene. the comparison of antibiotic susceptibility testing and pcr showed some contrast in the results. three k. pneumoniae and one e. coli were carrying blandm-1, but phenotypically they were sensitive to carbepenems. possible explanation of these types of results might be the presence of any point mutation or inclusion of insertion sequence which inhibit the expression of respective protein and thus no resistance in antibiotic susceptibility test. so with this study, it can be concluded that blandm-1 gene is prevalent in local clinical isolates which results in resistance to variety of antibiotics including carbepenems. references alzahrani a. j. et al. 2005. susceptibility patterns of extended spectrum ßlactamase (esbl)producing escherichia coli and klebsiella pneumoniae isolated in a teaching hospital. pakistan j. med. res.vol. 44, no.2. byarugaba d. k. et al. 2005. antimicrobial resistance and its containment in developing countries. in antibiotic policies: theory and practice, new york: springer. ed. i. pp 617–646. clinical and laboratory standards institute: p e r f o r m a n c e s t a n d a r d s f o r a n t i m i c r o b i a l susceptibility testing; tweny-first informational supplement. clsi document m 100-s 21 (isbn 156238-742-1). wayne, pa, usa: clinical and laboratory standards institute; 2011. gupta n, m. brandi et al. 2011.carbapenemresistantenterobacteriaceae epidemiology and prevention, clinical infectious diseases;53(1):60–67) pharmaceutical stability m. shakeel siddiqui1 and ghulam sarwar1 1jinnah university of women, karachi-74600, pakistan. 27 rads vol 2 (1), (2011); 27-30 introduction stability testing is the primary tool used to assess expiration dating and storage conditions for pharmaceutical produtcs. many protocols have been used for stability testing, but most in the insudtry are now standardzing on the recommendations of the international conference on harmonization (ich). these guidelines were delevoped as a cooperative effort between regulatory agencies and industry officals from europe, japan, and the united states (harman, 1999a). stability testing includes long-term studies, where the product is stored at room temperature and humidity conditions, as well as accelerated studies where the product is stored under conditions of high heat and humidity. proper design, implementaion, monitoring and evalution of the studies are crucial obtaining useful and accurate stability data. stability studies are linked to the establishment and assurance of safety, quality and efficacy of the drug production from early phase development through the lifecycle of the drug poduct. stability data fo the drug substance are used to determine optimal storage and packaging conditions for bulk lost of the material. the stability studies for the drug product are designed to determine the expiration date (or shelf life). in order to assess stability, the appropriate physical, chemical, biological and microbiological testing must be performed. usually this testing is a subset of the release testing. preclinical studies precinical studies include standard staility studies to assess shelf life, but also utilize special studies to study the drug and its degradation c h a r a c t e r i s t i c s . t h e d r u g s u b s t a n c e charcterizattion and stability is usually determine as part of pre-formulation studies. studies are chartacterzation degrade the solid drug substance and apporopriate solutions, allowing the determination of the degradation profile. the drug substance is usually challenged under a varitey of accelerated enviroment conditions to evaluate its intrinisic stability and degradation profile (harman,1999b). hplc is the predominant tool used to analyze the drug substance and the impurities, particularly for small molecules. frequently, the same hplc method may be used for drug substance and drug product, although different sample preparation method would normally be required. often the assay and impurity testing can be performed using a single hplc method. however, the assay and purity determinations may also be separate methods. at least in the u.s., full validation of the analytical method is not required until the end of phase 2 clinical trials, but the earliest stages, since verfication of stability hinges on a suitable method for seprating impurities from the active ingredient and at least quantifying the impurities realtive to the drug substance. stress studies at elevated temperature (e.g., 50 ac, 60ac and 70ac) for several weeks may be performed to assess thermal stability. provided the degradation mechanism is the same at the different tempreatures used, kinetic or corresponding author. e-mail: g-sarwar@hotmail.com 28 rads vol 2 (1), (2011); 27-30 statistical modles can be used to determine the rate of degradation at other tempretures (e.g., 25ac). the solid stability should also be performed in the presence and absence of water vapor to assess the dependence of stability on humidity. degration studies should also be performed in solution. the solvent for the solution testing will depend on the solubility of the drug substance and should include water, if the drug substance is water-soluble. other solutions or solvent system may be evaluated depending on the anticipated formulation or the synthethic process. a series of buffered solutions in the ph range 2-9 are useful in assessing the impact of solution ph on the degration. photostability should also be evaluted. a xenon light source can be used as a stress condtion. alternatively, one can use an accelerates version of ethier options 1 or 2 as discribed in the ich guideline determination of photostability. oxidation of the drug substance under accelerated conditoins (e.g., hydrogen peroxide), may also be performed to etablish oxidation product that could be formed and sensitivity to oxidative attack. early drug product stability studies are designed to help establish a suitable formulation for delivery of the drug substance. compatibility studies of the drug substance with excipients should be performed to eliminate excipients that are not compatible with the drug substance. clinical studies stability testing must be continued throughout clinical trials to support the safety, quality and efficacy of material relesed for clinical trials. stability data must be submitted as part of the ind filing prior to initiating the phase 1 clinical trial. prior to the first phase 1 stability study, the re-clinical studes should provide information on the appropriate long-term condition and the appropriate container/closure system. ich q1a provides the guidance for design of clinical stability studies. selection of batches, the container closure system, specifications, testing frequncy and storage conditions are the imporant issues to consider when designing a stability study. the container closure system must be evaluated for compatibility with the drug substance and drug product to ensure that the container does not contribute to degradation or contamination. the testing frequency represents the minimum data required for filing. it may be advisable to pull and test a one-month sample for each storage condition to ensure that the study is proceeding as expected. during phase 1, it may be necessary to evaluate multiple formulations, dosage strengths and container closure systems. using bracketing and/or matrixing can frequently reduce the resource allocation for these studies. these two design approaches are discussed in ich q1d. bracketing uses the extremes to provide data for the entrie study may include testing of all strengths at the 25, 50 and 100 mg are to be evaluated, the study may include testing of all strengths at the initial and final time points with only the 10 and 100 mg strengths being teswted at the intermediate time points. matrixing might be used to evaluate the same strenght in multiple container/closure system be selecting only certin container closure system for testing at each time-point. this selection is usually done in a random fashion. at the end of phase 1, the process for manufacture of the drug substance, and the drug product should be established (although 29 rads vol 2 (1), (2011); 27-30 refinements will typically continue for much longer). the time period in table 1 represents the minimun data required for the nda. the studies must continue until the long-term stability study is completed for the shelf life and retest period proposed in the nda submission. temperature cycling studies and in-use stability studies may be needed for certain types of formulations (particularly liqiud and semi-solid formulations). in early phase 3 studies one should expect to be placing the batchs on stalibity (at least three drug substance and drug product lots) that wil be used for filing the nda. these may be performed near the end of phase 3 and adequate stability data for these batches may not be available at the time of filing. shelf life and retest periods may be determine statistically with adequate quantitaive data. the period in table 1 represents the minimum data required for the nda. the studies must continue until the long term stability study is completed for the shelf life and retset period proposed in nda submission. post-approval (marketing phase) at least one lot of drug substance and one lot of each packaging type for drug product produced each year should be placed on longterm stability. addtional stability testing may be required to support process changes for drug substance and/or drug product. the filing requirements for changes are covered in multiple fda guidance documents addressing drug product changes (supac) and drug substance changes (bacpac). typically, this is an area that requires substanital regulatory understanding and experience to know how to proceed, and is beyond the scope of this article. concusion stability testing is interwoven through the entire fabric of the drug product life cycle. a detailed konwledge of the stability requirements and the impact on other areas (e.g., container closure, process shanges) is needed to properly design comments must cover retest or shelflife period at a minimum and includes storage, shipment and subsequest use must cover retest or shelflife period at a minimum and includes storage, shipment and subsequest use must cover retest or shelflife period at a minimum and includes storage, shipment and subsequest use must cover retest or shelflife period at a minimum and includes storage, shipment and subsequest use must cover retest or shelflife period at a minimum and includes storage, shipment and subsequest use must cover retest or shelflife period at a minimum and includes storage, shipment and subsequest use storage condition 25 oc + 2oc/60 % rh + 5% rh or 30oc + 2oc/65% rh + 5% rh 30 oc + 2oc/65 % rh + 5% rh 40 oc + 2oc/60 % rh + 5% rh 5 oc + 3oc 25 oc + 2oc/60 % rh + 5% rh --20 oc + 5oc study g e n e r a l c a s e : long-term g e n e r a l c a s e : intermediate g e n e r a l c a s e : accelerated r e f r i g e r a t i o n : long-term r e f r i g e r a t i o n : accelerated freezer: longterm m i n i m u m time period 12 months 6 months 6 months 12 months 6 months 12 months table 1 : ich q1a summary of stability parameters 30 rads vol 2 (1), (2011); 27-30 and evaluate stability studies in order to ensure minimal delays and minimize cost in developing a new drug product. references harman, rj. 1999a. the drug development process: 1. introduction and overview. pharm j., 262:334-7. harman, rj.1999b. the drug development process: 2. admiistrative processes and options for registration of medicines. ibid., 262: 928-31. the rules governing medical products in the erupean union. vol 3a. guidelines: medical products for human use; quality and boitechnology. luxembourg: european communities, 1998. ich guidlines. the rules governing medicinal products in the european unnion. vol 4. good manufacturing practies: medical products for human and veterinary use. luxembourg: eruopean communities, 1998. the rules governing medical products in the eruopean union. vol 2b. notice to applicants. luxembourg: eruopen communities, 1998. “rads” publishes articles within the whole field of physical science. generally these articles will be in or related to need of the country. manuscripts or data will be considered that have not been previously published or submitted elsewhere for publication. however, re-analysis of previously published data can be accepted. 1. all articles submitted for publication will be reviewed by referees. submit three copies of the article / manuscripts along with a floppy diskette (ibm computer) containing the manuscript in duplicate. while submitting diskette it should only the latest version of the manuscript. name the file clearly, label the diskette with the format of the file and the file name. provide information of the 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relative effect of vesicular arbuscular mycorrhizal fungi on growth and yields of soybeans. soil science society of american journal, 44, p. 528-532. 5. the manuscript should be accompanied by a covering letter signed by corresponding authors. instruction to authors 31 biology 2nd issue july-1 2016.pdf aflatoxin concentrations in rice varieties of punjab vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 39 op e n ac c e s s f u l l l e n g t h a r t i c l e aflatoxin concentrations in various rice varieties of punjab, pakistan nasir ishaque tahir1, shabbir hussain1,*, imran kalim2, amina asghar3, shahzada khurram syed4 muhammad amin idrees5, muhammad ameen ahmad1 1department of chemistry, lahore garrison university, lahore, pakistan. 2food & biotechnology research center, pakistan council of scientific and industrial research (pcsir) labs, lahore, pakistan. 3department of chemistry, dsnt, university of education lahore, pakistan. 4department of basic medical sciences, school of health sciences, university of management and technology, lahore, pakistan. 5department of physics, riphah international university, faisalabad campus, pakistan. a b s t r a c t background: mycotoxins are secondary fungal metabolites that are produced in rice, corn, nuts and several other cereals. the contamination of food stuff by mycotoxins is a cause of numerous diseases throughout the world. aflatoxins are potent mycotoxins that have chronic and acute toxicity. objectives: to evaluate the presence of aflatoxins b1, b2, g1 and g2 in branded and non-branded rice varieties available in punjab, pakistan. methodology: total 150 rice samples were collected in may 2019 from various cities (islamabad, rawalpindi, gujar khan, jehlum, kharian, gujrat, gujranwala, lahore, kasur, okara, sahiwal, mian channu, khanewal, multan, bahawalpur and rahim yar khan) of pakistan. all the rice samples were subjected to physical testing methods, followed by extraction (using chloroform extraction method) and qualitative and quantitative detection of aflatoxin using thin layer chromatography (tlc) technique and enzyme linked immunosorbent assay (elisa). results: no aflatoxins was detected in branded rice varieties which were stored in good packaging. the aflatoxin b1 (afb1) was detected in 65% of poorly stored rice varieties, with maximum contamination of 8.92ppb. the lowest moisture 9.4% content was found in (branded samples) and 10.9% (non-branded samples) while highest moisture content 10.8% found in (branded samples) and 16% found in (non-branded samples). the 54 out 100 non-branded samples found to have more than 13% moisture contents which is alarming. conclusion: with the implementation of effective strategies and special precautions during storage, harvesting, transportation and drying, we can prevent the contamination of rice product with fungi and stop aflatoxin production which is a major threat to country economy. keywords aflatoxins, branded, nonbranded, punjab, pakistan, rice. *address of correspondence dr.shabbirhussain@lgu.edu.pk article info. received: may 09, 2021 accepted: june 14, 2021 cite this article tahir ni, hussain s, kalim i, asghar a, syed sk, idrees a, ahmad aflatoxin concentrations in various rice varieties of punjab, pakistan. rads j biol res appl sci. 2021; 12(1):39-53. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. o r i g n a l a r t i c l e aflatoxin concentrations in rice varieties of punjab vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 40 i n t r o d u c t i o n the contamination of foodstuffs by mycotoxins is a cause of numerous diseases throughout the world1. mycotoxins are secondary fungal metabolites2 that are produced in rice, corn, nuts and several other cereals3. aflatoxins are potent mycotoxins that have chronic and acute toxicity4 comprised of closely related compounds e.g., aflatoxins b1, b2, g1, g2, m1 and m25-6. they exist ubiquitously in nature and may lead to several harmful and toxic effects on living beings5. they are produced in food commodities and animal feeds by the fungal strains (mainly by aspergillus. parasiticus and aspergillus. flavus; rarely by emericella venezuelensis, emericella astellata, aspergillus tamarii, aspergillus pseudotamari, aspergillus ochraceoroseus and aspergillus bombycis)5, 7. when animals consume aflatoxin-contaminated feed, then their meat, eggs, and milk are also contaminated8, 9. aflatoxins have mutagenic, carcinogenic and immunosuppressive effects10, 11. the presence of aflatoxins in diet severely affect kidneys, liver, growth rate, and reproduction, and can cause serious illness and even death. this problem is more common in developing countries5. uganda has higher cases of liver cancer due to aflatoxin-contaminated foods12. in kenya in 1981, twenty (20) people complained about stomach trouble and mild fever due to consumption of aflatoxin-contaminated food later they were diagnosed of livers damaged and 12 individuals were reported died1 3 . aflatoxins gain entry into food products when proper storage conditions (e.g., little moisture content and low temperature) are not applied14,15. the moisture and a hot climatic conditions are important factors contributing to toxin production16,17. plants are most valuable sources of food and food products throughout the world18-20. rice is one of the most important food crops21 having an excellent nutritional value22. it is the most popular staple food and an important source of vitamins, minerals, energy, fiber, and other biomolecules23. rice exports are the second highest source of income in pakistan24. rice is consumed worldwide as a food and food additive due to its unique fragrance, cooking advantages and taste. the diverse rice varieties include kernel, basmati, superkernel, super basmati, super shaheen, longgrain white and brown, guard supreme basmati, guard super kernal basmati, zainab basmati rice, falak premium basmati rice, mughal badshah rice, aroma super basmati rice, aroma world longest basmati rice, falak daily basmati rice, fine life super kernal basmati rice, kalaar basmati steam rice, zarafa super kernal basmati rice, ream awami basmati rice, hashmi super kernal basmati 1121, honeymoon basmati rice, shan shahi jalwa awami long grain rice family super kernel basmati rice, hassan 1121 basmati rice, khyber basmati rice, kausar basmati rice, naubhar nayab silla basmati rice, mehran basmati daily rice, jazaa premium basmati rice, motidana steamed 1121 basmati rice, al badar classic super kernal basmati and anmol super kernal basmati rice etc. these varieties are commonly used in asian dishes2 , 5 . after wheat, the rice family is pakistan’s second-largest food26. it is the third most cultivated crop in pakistan after wheat and cotton27. rice is a major export of pakistan and a main cash crop and a source of income for the pakistan, accounting for approximately 5.9% of total agricultural value-added and 1.3% of gdp27,28. the contamination in this costly crop create a huge impact on pakistan economy. in addition the existence of aflatoxins in rice-based products may cause severe toxicity especially in human genetic material i.e. dna and rna29. the impact of aflatoxins on human health is an important challenge today30,31; that is why the aflatoxin related research is catching attraction world wide32. the present study was conducted to evaluate the presence of aflatoxins b1, b2, g1 and g2 in branded and nonbranded rice varieties of punjab, pakistan. m a t e r i a l a n d m e t h o d s all the experimental work was performed in pcsir laboratories complex, lahore, pakistan. afb1, afb2, afg1 and afg2 standards were imported from the trilogy analytical laboratory (870 vossbrink dr, washington, mo 63090, usa), acetone (fisher scientific, usa), anhydrous ether (merck usa), chloroform (sigmaaldrich uk) and benzene acetonitrile (sigma-aldrich uk). tlc plates were procured from merck (290 concord road billerica massachusetts usa). elisa kit (model 680) bio-rad usa, reader grinding mill (romer series mill) romer lab singapore, wrist action shaker (model 75) burrell scientific singapore, hot plate (hot plate 180 pcsir pakistan), water bath (water bath aflatoxin concentrations in rice varieties of punjab vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 41 al/16, pcsir pakistan) and uv spectrophotometer (uv reader/280) redicom austria, were used. collection of rice samples total 150 rice samples were collected in may 2019 from various cities (islamabad, rawalpindi, gujar khan, jehlum, kharian, gujrat, gujranwala, lahore, kasur, okara, sahiwal, mian channu, khanewal, multan, bahawalpur and rahim yar khan) of punjab, pakistan. it is worth mentioning that rice crop planted from may-june and harvested in october-december in pakistan. sample preparation and testing physical testing homogenous rice samples (100g each) were collected from each rice variety, marked and stored for analysis. the defective, broken/smashed, other contaminations and rice weevils were removed before sample collection. grinding a total of 50g of each sample was carefully mixed for 10min. it was ground in a sample grinder (ilp, fbrc/al/05) and then passed through 20 mesh sieve to obtain a uniform (1mm particle size) and descriptive sample. the ground samples were stored in a sealed plastic pack to keep them safe before the experimental use. these samples were finally used for aflatoxins assessment. extraction of aflatoxins various methods of extraction and analysis are utilized for diverse types of raw materials because of their different chemical behaviors. researchers follow numerous extraction procedures depending upon their resources. in current studies, we have chosen the chloroform extraction method which has been considered an appropriate method for the aflatoxin extractions. extraction technique was applied by utilizing 50g of milled sample of rice in a 500ml erlenmeyer flask. twenty five (25ml) of water and 150ml of chloroform was added in the allocated flask. the erlenmeyer flask was mounted on a wrist shaker and shaken for 30min. the sample solution was then filtered by whatman grade no. 1 filter paper. extract (50ml) was positioned on a hot plate for evaporation33. it was finally subjected to analysis for the presence of aflatoxins b1, b2, g1 and g2. aflatoxin determinations by thin layer chromatography technique (tlc) dilutions were done up to per microliters for tlc spot applications. then 25µl of the test solution was applied on a thin layer chromatography plate, using a microscope syringe. total 5μl and 10μl concentrations of afb1, afb2, afg1 and afg2 were also applied as internal standards for aflatoxins on this plate. the tlc plate was placed in “first mobile phase” (anhydrous ether) in tlc container no. 1, until the solvent was reached on an average height. then tlc plate was taken out and dried before it was placed in the “second mobile phase” tank containing acetone and chloroform (2:1). when the mobile phase was reached to the required height, the tlc plate was taken out from tank 2 and then dried. the test plate was carefully observed under 365nm ultraviolet light for the presence/absence of aflatoxins. aflatoxins were evaluated by careful observation of fluorescence brightness and comparing the results with those of the aflatoxin standards applied on the tlc plates along with the samples34. the test solution was completely dried for quantitative determination. benzene and acetonitrile (98:2) were mixed to the test solution. then 3.5, 5.0 and 6.5µl spots (all of the same size) of the test solutions were put on the tlc plates. spots of the identical size of the standards were also put on the tlc plate. the tested and standard solutions were compared to identify the similarities between the two. fluorescent light used to examine the spots. when the sample spot and standard spot were superimposed, this indicates the presence of aflatoxin in the sample. in such situation, the color of the sample and rf values of the samples were corresponded to the aflatoxin standards35. calculations the concentration of aflatoxins in a sample can be calculated by the following equation36: concentration of aflatoxins (mg/kg) = s x v x y w x z s: volume (ml) of aflatoxin standard of equivalent intensity to z = ml of sample v: volume (ml) of solvents required for dilution of the final extract y: concentration (mg/ml) of aflatoxin standard aflatoxin concentrations in rice varieties of punjab vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 42 w: weight (g) of the original sample contained in the final extract z: volume (ml) of sample extract required to produce fluorescence intensity comparable to that of s = ml of aflatoxin standard. enzyme linked immuno sorbent assay (elisa) method sample preparation was preformed according to the instructions of the test kit manual of ridascreen aflatoxin b1 30/15 (r-biopharm gmbh, 2010)37. twentyfive milliliter (25ml) mixture of methanol: water (70:30) was added to 5g of rice followed by its vigorous stirring for 3min manually. the obtained extract was fil`tered through a filter paper and diluted with distilled water (1:1). at last, 50ml of the diluted filtrate was used for each well in a test. aflatoxin level was determined quantitatively by an elisa test procedure (ridascreen aflatoxin b1 30/15 (art no.: 1211) test kit manual) reported earlier38. to each well of a microtiter plate, 50ul of a solution (test solution and a standard solution) was added in duplicate followed by addition of 50ul each of the enzyme conjugate and the antiaflatoxin antibody to every well. the resultant solution was mixed gently and incubated at 20-25°c for 30min. then the wells were tapped upside down vigorously for the removal of their liquids into an absorbent paper. each empty well was then washed twice by 250ul washing buffer. subsequently, there was addition of 100ul of substrate/chromogenic solution to each well followed by its gentle mixing and incubation at 20-25°c for 15min in a dark place. then there was addition of 100ul of 1n h2so4 (stop solution) to each well. finally, the absorbance was noted in elisa plate reader at 450nm38. moisture analysis a known amount of fine and ground sample was taken in a well dried (by oven) petri dish. the petri dish was then heated in the oven at 100ºc overnight to ensure the effective removal of all the moisture. the dish was placed in a desiccator and cooled; it was heated again in an oven for 2hours again and weighed was taken to ensure the maximum removal of moisture from the sample. heating and weighing was continued until the constant reading of petri dish and sample was achieved39. % moisture = weight of the dried rice sample x 100 weight of the original sample r e s u l t s a n d d i s c u s s i o n the rice samples were collected and analyzed (qualitatively and quantitatively) for the aflatoxin contents by thin layer chromatography (tlc) and enzyme linked immunosorbent assay (elisa). the prepared samples were run through tlc plates and finally observed with a uv spectrophotometer. the results for tlc tests are compared in fig. 1 and 2 for branded (packed) and nonbranded (open or poorly stored) samples, respectively. aflatoxin concentration in open rice samples no aflatoxins were detected in all 50 branded rice samples which had their packaging according to the standard requirements. the corresponding tlc results are shown in fig. 1. the obtained data are displayed in table 1 and summarized in table 2. table 1. aflatoxin concentration in branded rice varieties available in local market (each rice sample = 500g). s. no. city and location from where the rice sample was picked company name & rice variety packaging conditions time in shelf (months) h2o content (%age) aflatoxin conc. (ppb) 1 kechery chowk, rawalpindi guard supreme basmati good 2 10 not detected 2 kechery chowk, rawalpindi guard super kernal basmati good 4 9.8 not detected 3 tulsa rd, opposite lalazar, rawalpindi zainab basmati rice good 5 10.2 not detected contd… aflatoxin concentrations in rice varieties of punjab vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 43 4 tulsa rd, opposite lalazar, rawalpindi falak premium basmati rice good 7 10.5 not detected 5 gt road, kotha kalan morgah, rawalpindi mughal badshah rice good 3 9.5 not detected 6 gt road, kotha kalan morgah, rawalpindi guard super kernalbasamati rice good 8 9.8 not detected 7 islamabad, islamabad capital territory, pakistan aroma super basmati rice good 6 10.1 not detected 8 islamabad, islamabad capital territory, pakistan aroma world longest basmati rice good 2 10.5 not detected 9 g.t. rd, islamabad, islamabad capital territory falak daily basmati rice good 3 10.7 not detected 10 g.t. rd, islamabad, islamabad capital territory fine life super kernal basmati rice good 5 9.4 not detected 11 national hwy 5, sector f dha phase ii, islamabad. kalaar basmati steam rice good 6 10.1 not detected 12 national hwy 5, sector f dha phase ii, islamabad zarafa super kernal basmati rice good 3 9.5 not detected 13 g.t. rd, islamabad guard super kernal basmati good 6 10.8 not detected 14 g.t. rd, islamabad guard super kernalbasamati rice good 4 10 not detected 15 g.t. rd, gujar khan, rawalpindi ream awami basmati rice good 7 9.9 not detected 16 g.t. rd, gujar khan, rawalpindi mughal pure basmati rice good 3 10.8 not detected 17 grand trunk rd, jada, jhelum hashmi super kernal basmati 1121 good 8 9.7 not detected 18 grand trunk rd, jada, jhelum hashmi super kernal basmati 1121 good 4 10.5 not detected 19 grand trunk rd, panjan kasana kharian honymoon basmati rice good 8 10.8 not detected 20 grand trunk rd, panjan kasana kharian honymoon basmati rice good 4 9.9 not detected 21 grand trunk rd, ali chak, gujrat shanshahi jalwa awami long grain rice good 6 10.2 not detected contd… aflatoxin concentrations in rice varieties of punjab vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 44 22 grand trunk rd, ali chak, gujrat shanshahi jalwa awami long grain rice good 2 10.5 not detected 23 askari homes rahwali cantonment, gujranwala falak easy cook daily basmati rice good 7 10.6 not detected 24 askari homes rahwali cantonment, gujranwala falak easy cook daily basmati rice good 3 9.9 not detected 25 hussain chowk, block b3, gulberg iii, lahore aroma world longest basmati rice good 9 10.4 not detected 26 hussain chowk, block b3, gulberg iii, lahore aroma world longest basmati rice good 7 10.6 not detected 27 fortress stadium, lahore family super kernel basmati rice good 8 10.1 not detected 28 fortress stadium, lahore family super kernel basmati rice good 4 10.4 not detected 29 aziz bhatti road, 4 st, cantt, lahore falak select super kernal basmati rice good 3 9.4 not detected 30 aziz bhatti road, 4 st, cantt, lahore guard super kernal basmati good 4 9.5 not detected 31 paf market, cantt, lahore hassan 1121 basmati rice good 5 9.9 not detected 32 ahmed bukhsh rd, r a bazaar cantt, lahore zainab basmati rice good 4 10.4 not detected 33 walton road, super town lahore falak daily basmati rice good 8 10.6 not detected 34 walton road, super town lahore fine old basmati rice good 11 10.3 not detected 35 walton road, madina colony, lahore hassan 1121 basmati rice good 5 10.2 not detected 36 walton road, madina colony, lahore mughal super sella basmati rice good 4 9.8 not detected 37 walton road, pir colony, lahore guard extreme basamati rice good 4 10.4 not detected 38 model town circular road, model town, lahore khyber basmati rice good 7 10.7 not detected 39 model town circular road, model town, lahore kausar basmati rice good 6 9.4 not detected 40 model town link rd, lahore guard super kernal basamati rice good 4 10.2 not detected contd… aflatoxin concentrations in rice varieties of punjab vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 45 41 model town link rd, lahore falak extreme basmati rice good 8 9.5 not detected 42 cash & carry, airport, lahore kalaar basmati steam rice good 6 10.4 not detected 43 cash & carry, airport, lahore mughal pure basmati rice good 8 9.8 not detected 44 cash & carry, airport, lahore naubhar nayab silla basmati rice good 9 9.4 not detected 45 cash & carry, airport, lahore mehran basmati daily rice good 3 9.9 not detected 46 y-block, dha phase 3, lahore jazaa premium basmati rice good 4 9.6 not detected 47 y-block, dha phase 3, lahore motidana steamed 1121 basamti rice good 7 9.8 not detected 48 street 5, h-block, dha phase 4, lahore al badar classic super kernal basmati good 6 10.4 not detected 49 abdul haque rd, johar town, lahore anmol super kernal basmati rice good 4 10.4 not detected 50 abdul haque rd, johar town, lahore falak easy cook sella good 8 10.6 not detected table 2. summary of results for the branded rice varieties. area rice brand time in shelf moisture content aflatoxin level islamabad, rawalpindi, gujar khan, jehlum, kharian, gujrat, gujranwala, lahore guard supreme basmati, gurad super kernal basmati, zainab basmati rice, falak premium basmati rice, mughal badshah rice, aroma super basmati rice, aroma world longest basmati rice, falak daily basmati rice, fine life super kernal basmati rice, kalaar basmati steam rice, zarafa super kernal basmati rice, ream awami basmati rice, hashmi super kernal basmati 1121, honymoon basmati rice. shanshahi jalwa awami long grain rice. faimly super kernel basmati rice, hassan 1121 basmati rice, khyber basmati rice, kausar basmati rice, naubhar nayab silla basmati rice, mehran basmati daily rice, jazaa premium basmati rice, motidana steamed 1121 basamti rice, al badar classic super kernal basmati, anmol super kernal basmati rice 2-11 months 9.4-10.8 not detected aflatoxin concentrations in rice varieties of punjab vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 46 figure 1. tlc results for the branded rice varieties. figure 2. tlc results of open rice (poorly stored samples) (left lanes-samples: 69-73, 75; right lanes: 1ppb, 2ppb, 4ppb-standards). aflatoxin concentration in open rice samples with poor storage conditions rice varieties which were collected from different cities and towns of punjab had shown the presence of aflatoxins. the corresponding tlc results are shown in fig. 2. out of 100 non-branded samples, the presence of aflatoxins was detected in 65 samples while the remaining 35 samples did not show the presence of aflatoxins. out of 65 total contaminated samples, 23 rice verities had shown the existence of aflatoxins below the limits recommended by the european authorities (2-4ppb) whereas 24 samples had shown aflatoxins in the recommended limits of 2-4ppb. 18 rice samples showed aflatoxin concentration above the recommended limits. the highest value of aflatoxin (8.92ppb) for observed for the rice sample 40 (niab ir-9). table 3 displays the aflatoxin concentration in the poorly stored rice samples whereas the final data are summarized in table 4 and fig. 3. aflatoxin concentrations in rice varieties of punjab vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 47 table 3. aflatoxins and moisture content analysis of local rice varieties. s. no. city and location from where the rice samples picked (minimum 500g each) variety of rice time in a shelf (months) h2o content (%age) aflatoxin concentration (ppb) 1 kechery chowk, rawalpindi basmati 370 4 12 not detected 2 bilal street, morgah, rawalpindi basmati pak 9 12.4 not detected 3 adamjee street, rawalpindi ks-282 12 14 1.86 4 g.t road, rawalpindi basmati 385 11 14.2 2.96 5 soan garden extension, islamabad basmati 2000 15 15 3.56 6 shalimar rd, block e soan garden, islamabad basmati pak 6 11.8 not detected 7 phase 1 jinnah garden, islamabad super basmati 2 12 not detected 8 gt rd, rawat, islamabad basmati 515 11 15.5 4.53 9 chamber more rawat, islamabad shaeen basmati 6 11.5 not detected 10 service rd, rawat, islamabad niab ir-9 9 15 2.62 11 bagga shaikhan, rawalpindi, islamabad ksk-133 6 15.1 1.73 12 banth, rawalpindi, punjab, pakistan basmati 2000 5 12 not detected 13 grand trunk rd, mandra, rawalpindi basmati 370 12 14.6 2.55 14 tharjiyal rd, mandra, rawalpindi super basmati 10 15.4 1.79 15 tharjiyal rd, rawalpindi ks-282 6 10.9 not detected 16 cheharikalyal, rawalpindi basmati 515 8 12.4 not detected 17 gujar khan, rawalpindi basmati 370 4 11.8 not detected 18 sandal road ward 14، gujar khan, rawalpindi super basmati 10 15.4 8.78 19 housing scheme 2, gujar khan, rawalpindi ksk-133 4 15.5 3.64 20 w7 gujar khan, pakistan basmati 2000 6 15.2 1.62 21 bhai khan, gujar khan, rawalpindi niab ir-9 10 11 not detected 22 sohawa city, jhelum ks-282 10 11.5 not detected 23 ehsan butt st, domelimohallah, dina basmati 515 9 14.9 2.48 24 mangla rd, dina, jhelum basmati 370 8 15.1 2.93 contd… aflatoxin concentrations in rice varieties of punjab vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 48 25 main bazaar, dina, jhelum ks-282 6 16 3.73 26 grand trunk rd, burha jungle, dina, jhelum niab ir-9 9 14.7 1.97 27 kala gujran, jhelum, punjab, pakistan basmati 370 5 12.3 not detected 28 g.t road, oppfauji mill ground، kala gujran, jhelum ksk-133 10 15.4 3.47 29 nawab colony, jhelum ks-282 6 11.6 not detected 30 sultan st, jada, jhelum niab ir-9 10 14.6 1.21 31 chak jamal rd, jada, jhelum basmati 2000 8 15.7 4.63 32 tufail rd, jhelum cantt, jhelum super basmati 6 12.4 not detected 33 jhelum cantt, jhelum 8 11.8 not detected 34 arain street, eidgah, jhelum basmati 2000 8 14.3 0.92 35 pinddadan khan jhelum rd, jhelum cantt, jhelum basmati 515 6 16 3.85 36 arsal town jhelum cantt, pinddadan khan jhelum rd ksk-133 10 15.4 6.43 37 bagga, jhelum pinddadan khan jhelum rd ks-282 8 12.9 not detected 38 nougran link rd, pinddadan khan jhelum rd, jhelum basmati 370 6 14.7 1.42 39 nougran, jhelum pinddadan khan jhelum rd super basmati 14 13.2 2.76 40 ghous plaza, grand trunk rd, sarai alamgir, gujrat niab ir-9 12 15.7 8.92 41 jalilpura, saraialamgir, gujrat basmati 2000 6 11.7 not detected 42 main bazaar, mohalla shaheedan, sarai alamgir, gujrat basmati 370 10 12.5 not detected 43 n5, mohalla shaheedan, sarai alamgir, gujrat super basmati 4 14.7 2.96 44 g.t. road, sarai alamgir, gujrat ks-282 5 14.4 5.32 45 bani mohalla kharian, guliana road, gujrat ksk-133 7 12.6 not detected 46 panjan kasana village, gujrat niab ir-9 2 14.5 1.62 47 grand trunk rd, thikrian, lala musa, gujrat basmati 370 4 14.7 6.12 contd… aflatoxin concentrations in rice varieties of punjab vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 49 48 asghar town haji asghar town, lalamusa, gujrat basmati 2000 6 12.8 not detected 49 karimpura, lalamusa, gujrat ksk-133 6 15.5 3.76 50 chota bazar road, lalamusa, gujrat ks-282 10 15.2 1.62 51 service rd w, mehermohalla, lalamusa, gujrat basmati 385 5 15.3 5.74 52 grand trunk rd, ellahi colony, gujurat super basmati 11 15.4 3.63 53 kalra khasa, g.t road, gujrat niab ir-9 6 14.6 0.75 54 muslim town, rahwali cantonments, gujranwala basmati 2000 10 15.1 8.75 55 meher colony, rahwali cantonments, gujranwala basmati 370 5 12.1 not detected 56 service rd, shaheenabad, gujranwala ks-282 4 15.4 1.52 57 safdar & asif rice trader, mohalla raitanwala krishan nagar, gujranwala basmati 515 9 12.5 not detected 58 civil lines, gujranwala super basmati 6 14.9 5.73 59 g.t. rd, near jama masjid bilal, eminabad, gujranwala ksk-133 5 15.4 1.55 60 mandiala rd, kamoke, gujranwala niab ir-9 10 15.5 2.73 61 canal road, sadhoke, gujranwala basmati 2000 6 14.9 1.7 62 mohalla faiz-e-madina, muridke, sheikhupura super basmati 5 12.4 not detected 63 ravi rayon, kala shah kaku, sheikhupura ks-282 4 14.4 1.22 64 main bazar, rana town ferozewala, sheikhupura basmati 370 6 15.7 6.86 65 g.t. road, shahdara town, shahdara, lahore basmati 2000 9 15.4 3.76 66 ahmed bukhsh rd, r a bazaar cantt, lahore ksk-133 6 11.6 not detected 67 akram park gurumangat gulberg iii, lahore ks-282 3 14.4 1.65 68 main bazar canal park, block o gulberg 2, lahore basmati 515 5 12.5 not detected 69 chahgallanwala, imran st, rasool park, lahore basmati 370 4 12.6 not detected 70 link ferozepur rd, new islamia park islamia park, lahore super basmati 6 14.8 1.76 contd… aflatoxin concentrations in rice varieties of punjab vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 50 71 multan rd, maraka village, lahore niab ir-9 10 11.4 not detected 72 canal bank rd, chimbran wali jhuggi, lahore basmati 2000 6 15.1 4.93 73 hospital road, manga raiwind road, manga mandi, lahore ksk-133 2 12.4 not detected 74 sherpur road, phool nagar, kasur ks-282 6 14.4 1.92 75 malik colony, phool nagar, kasur super basmati 7 15.4 5.76 76 jamber, lahore multan road, kasur niab ir-9 5 11.8 not detected 77 megha rd, pattoki, bilal colony, kasur basmati 2000 4 15.2 3.72 78 habibabad, habibabad road, okara basmati 370 3 15.7 1.47 79 g.t. rd, renalakhurd, okara basmati 2000 1 15.4 5.92 80 multan okara rd, sheikh basti, okara ksk-133 5 11.6 not detected 81 kot liaquat hayat, okara basmati 2000 2 12.4 not detected 82 wan bazar, sahiwal, sahiwal niab ir-9 6 15.1 3.65 83 liaqat chowk, sadman town sahiwal, sahiwal ks-282 6 15.4 2.73 84 block 5, chichawatni, sahiwal basmati 370 4 15.1 6.57 85 multan road, iqbal nagar super basmati 6 14.8 5.7 86 multan-mian channu road, mian channu basmati 370 5 12.6 not detected 87 ayoub chowk, ayoub road, khanewal basmati 2000 6 15.4 3.72 88 old kachehri rd, main bazar, kabirwala basmati 515 4 15.1 1.75 89 t block, new multan colony, multan ks-282 6 11.4 not detected 90 gulshan town, multan niab ir-9 7 12.4 not detected 91 chak raas, multan basmati 370 5 16 3.75 92 raiway road, shujaabād, multan ksk-133 4 15 3.42 93 permit road, jalalpur pirwala, multan super basmati 6 15.4 1.77 94 alipur road, uch sharif, bahawalpur basmati 2000 4 14.6 2.63 95 liaqat pur rd, janpur, rahim yar khan ks-282 7 15.1 6.83 96 khan bela, rahim yar khan basmati 370 5 14.4 1.65 contd… aflatoxin concentrations in rice varieties of punjab vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 51 97 n5, fateh pur kamal, rahim yar khan ksk-133 6 15 2.45 98 jamia masjid chowk zahir pir, nearby, khanpur rd, rahim yar khan niab ir-9 5 15.4 1.42 99 sardargarh, rahim yar khan ks-282 4 15.1 6.94 100 main road, gulshan e usman, rahim yar khan super basmati 6 12.4 not detected table 4. summary of open rice samples with poor storage. aflatoxin total no. of samples contaminated samples uncontaminated samples moisture content %age max ppb eu limits for contamination b1 in rice sample with poor storage 100 65 35 10.9 16 8.92 2-4ppb figure 3. comparison of aflatoxin concentration in open rice samples. aflatoxin b1 (afb1) which is most common aflatoxin, was detected (0.75-8.92 ppb) in all the 65 rice samples having poor storage. afb2, afg1 and afg2 were not detected in anyone of the tested samples. the recommended limit of aflatoxin b1 by european union (eu) is 2ppb for rice and other edible foods for humans; it is 4ppb for total aflatoxins. however, according to the pakistan standards and quality control authority (psqca), food and agriculture organization (fao), food and drug administration (fda), the allowed concentration of afb1 is 10ppb. so the observed afb1 concentration in the rice samples was found within the recommended limits of psqca, fao and fda. however, it was found beyond the limits of eu indicating a dire need to overcome aflatoxin contamination in rice. c o n c l u s i o n in the current study, detailed analysis has been conducted to monitor the presence of aflatoxins b1, b2, g1 and g2 in branded and non-branded rice varieties from various cities of punjab, pakistan. interestingly, all the branded rice samples were totally free form aflatoxins, however, 65% of non-branded and poorly stored rice samples had shown the aflatoxin contamination (0.75 to 8.92 ppb). moreover, in the remaining 35% samples, aflatoxins were found below the limits recommended by the european authorities (2-4ppb). highest concentration of aflatoxin was observed 8.92 ppbin few rice sample. in the branded rice samples the lowest moisture content detected were 9.4 and highest moisture content was 10.8%. non-branded (54%) rice samples have shown the moisture contents more than 13%, which is alarming. it is recommended that pakistani authorities should review periodically and analyze rice samples to ensure the absence of aflatoxins in foods. special precautions must be taken during storage, harvesting, transportation, drying and harvesting to stop aflatoxin production in cereals. if aflatoxins are present in aflatoxin concentrations in rice varieties of punjab vol. 12 (1), june 2021 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 52 food, their concentration should be minimized or eradicated by recommended de-toxifying technologies. c o n f l i c t s o f i n t e r e s t none. f u n d i n g s o u r c e none. a c k n o w l e d g m e n t s author is thankful to pakistan council of scientific and industrial research (pcsir) for providing necessary research facilities in their labs. l i s t o f a b b r e v i a t i o n s afb1 aflatoxin b1 eu european union fda food and drug administration fao food and agriculture 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of carrots during storage vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 168 op e n ac c e s s f u l l l e n g t h a r t i c l e effect of post-harvest treatments on quality characteristics of carrots during storage benish nawaz mirani1, shakeel hussain chattha2, shakeel ahmed soomro2*, bakhtawar wagan2, imtiaz ali dahri3, zaheer ahmed khan2, ghassan zahid4, babar mustafa ansari3 1institute of food sciences and technology, sindh agriculture university, tandojam, 70060, pakistan 2department of farm structures, sindh agriculture university, tandojam, 70060, pakistan. 3khairpur college of agricultural engineering and technology, khairpur, pakistan 4department of biotechnology, the university of azad jammu and kashmir, muzaffarabad, 13100, pakistan. a b s t r a c t background: carrot due to its versatility in culinary uses is considered to be one of the most preferred vegetable. the carrot in pakistan ranks third among winter vegetables, but due to adoption of improper postharvest techniques and storage, it leads to a great reduction in its quality. objective: the study was carried out to assess the effect of different treatments on quality characteristics of carrot. methodology: the study was carried out at the laboratory of farm structures, sindh agriculture university tandojam, pakistan. freshly harvested mature carrots free from damage and fungal attack were obtained from field. the carrot samples were subjected to different post-harvest treatments i.e. 40 ºc hot water dip for 2 minutes, 50 ºc hot water dip for 2 minutes, 0.4% concentration of calcium chloride (cacl2) dip for 2 minutes and tap water. results: the results revealed that moisture content and firmness decreased with increasing storage duration, whereas weight loss, fungal incidence and total soluble solids increased with increasing storage duration. the carrots treated with 0.4% concentration of cacl2 dip for 2 minutes had maximum moisture content (78.32%) and firmness (4.12 lbs), with minimum weight loss (41.33%), fungal incidence (38.14%) and total soluble solids (10.43%), followed by 40 ºc hot water dip for 2 minutes, 50 ºc hot water dip for 2 minutes and tap water. conclusion: the carrots treated with 0.4% concentration of cacl2 dip for 2 minutes showed better quality characteristics at the end of storage. the adoption of this treatment should therefore be encouraged in the developing countries for extending the quality characteristics of carrots. keywords calcium chloride, carrot, hot water dip, quality, storage. *address of correspondence shakeelsoomro@live.com article info. received: may 19, 2022 accepted: december 15, 2022 cite this article: mirani bn, chattha sh, soomro sa, wagan b, dahri ia, khan za, zahid g, ansari bm. effect of post-harvest treatments on quality characteristics of carrots during storage. 2022; 13(2):168-174. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n the carrot due to its versatility in culinary uses and its enriched healthy composition, such as protein, carbohydrate, fibre, vitamin a, potassium, and sodium is considered to be the most preferred vegetable1. the carrot in pakistan ranks third among winter vegetables2. to meet the increasing demand in future, researches are being carried to increase the quantity produced and to improve o r i g i n a l a r t i c l e effect of post-harvest treatments on quality characteristics of carrots during storage vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 169 post-harvest preservation3. the quality and acceptance of carrots at sale is affected at all the stages of the supply chain i.e. growing, harvesting, storage, cleaning, grading, packaging and distribution. in recent years, the black root rot caused by the fungi has become a key problem for the carrot production4. the disease symptoms, gray black lesions on the carrot surface, usually develop after storage during retailing when carrots are held at room temperature under moist condition5.after harvesting the carrot leads weight loss, discoloration, textural changing and structural break down which ultimately decrease shelf life of the carrots6. several chemical changes occur during the storage of carrots, including the conversion of polysaccharides to simple sugars and sucrose for reducing sugars, resulting in the release of off tastes, textural alterations, structural breakdown and colour change7. refrigeration with or without regulation of atmospheric composition is commonly practiced to maintain the quality of fruits and vegetables during storage; whereas it is not economically practical to apply such technologies8. fungicides prevent whole fruits from rotting after harvest, but they leave residues that can harm humans and the environment9. sulfites are an efficient chemical preservative, as they are both antibacterial and inhibited enzymatic browning. however, due to negative customer reactions, their use has been prohibited10. plastic sheets can also help to prevent moisture loss, although they are prone to microbial growth and disposal issues11. studies have shown that by employing a simple and ecologically friendly methods, ripening and colour can be delayed, water loss and decay can be reduced, and attractiveness can be improved12. edible films and coatings improves the shelf life of products, limit oxidation and respiration processes, maintain texture and sensory features, and are eco-friendly13. to increase the firmness and to extend the shelf life, calcium dips have been used as firming agents for a variety of fruits and vegetables after they have been harvested. pathogen germination, speculation and growth, as well as significant colour change, textural breakdown, and ripening and degradation, are all reduced by calcium treatments14. hot water immersion can also be used a heat treatment technique to control pests and diseases after harvest in fresh perishable commodities15. the overall quality of products when treated with hot aqueous solution and hot water treatment resulted better, when compared without hot treatment16. similar results for hot water treatment at different temperature has also been reported by hu et al.17. keeping in view the importance and necessity of the problem, and to provide a desired environment for maintaining the quality of carrot, the present study was carried out to determine the effect of post-harvest treatments on the quality of the carrot. m a t e r i a l s a n d m e t h o d s the study aiming to assess the effects of treatments on the quality of carrots was carried at laboratory of farm structures, sindh agriculture university tandojam. the carrot samples were collected from field at the time of maturity and transported to the laboratory. the sample of carrots were divided into four lots and treated according to the treatments i.e 40ºc hot water dip for 2 minutes (t1), 50 ºc hot water dip for 2 minutes (t2), 0.4% concentration of cacl2 dip for 2 minutes (t3) and control with tap water (t4).the treated samples were then placed at room temperature in the laboratory, and following observations were recorded (replicated thrice) at an interval of three days. ambient temperature and relative humidity the temperature and relative humidity were determined by using dry and wet bulb thermometers during the entire storage period. the ambient temperature was directly calculated from dry bulb, whereas relative humidity was determined by psychrometric chart using dry and wet bulb data. moisture content (%) a sample of 10g was taken and oven dried at 105ºc for 24 hours18. moisture content was then determined using the following equation. 100× sample wet ofweight sample of dry mattersample wet ofweight =content moisture weight loss (%) an electronic weight balance was used for evaluating the weight loss of carrot for all treatments. weight loss was determined according to the following formula19. 100× carrotfresh ofwight storageafter weight -carrotfresh ofwight = lossweight effect of post-harvest treatments on quality characteristics of carrots during storage vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 170 firmness (lbs) a fruit penetrometer with a probe of 15mm was used to measure the firmness of the carrot sample. the carrot was placed on a hard surface. the probe of the penetrometer penetrated the carrot tissue after a force was applied to the surface of the carrot. as a measure of carrot firmness, the force required to enter each tissue was measured20. fungal incidence (%) carrot samples were monitored daily at room temperature for signs of degradation. the amount of rotted roots as a percentage of the total number of carrots was then calculated21. total soluble solids (%) atc-1e hand-held refractometer (atago, japan) was used for determining the total soluble solids at a temperature of 20 ºc. two drops of carrot juice were placed on the refractometer plate, recording the tss percent on the scale accordingly22. statistical analysis the analysis of variance by statistics software (statistix ver. 8.1) was carried out using 2-factorial completely randomized design to examine the effect of treatments on quality of carrots. r e s u l t s a n d d i s c u s s i o n temperature and relative humidity of the experimental site the ambient temperature ranged between 27.5 to 32.4ºc throughout the storage period, with an average value of 29.45ºc, whereas the relative humidity ranged from 52.4 to 68.5% (figure 1). storage conditions such as temperature and relative humidity are the main factors influencing degradation of carrots during post-harvest preservation23. the temperature and humidity ranging between 0 to 1 ºc and rh of 9598% accordingly has been reported to be suitable by various researchers24. low relative humidity during storage and loss in weight of carrots resulted in deterioration of quality. in the present study, it was observed that the temperature and humidity conditions were not suitable for storage causing maximum damage and contamination. moisture content mean squares showed significant differences in moisture content for carrot under different treatments, duration and the interaction of treatment × duration (table 1). moisture content of carrot decreased with increasing days of storage. the maximum moisture content with 78.32% after 15 days of storage was observed in carrots treated with0.4% cacl2 dip for 2 minutes, followed by 40 ºc hot water dip for 2 minutes, 50 ºc hot water dip for 2 minutes and then with control throughout the storage duration (figure 2a). the decrease in moisture content may be due to high rate of respiration of carrots and low humidity. several methods have been used to reduce moisture loss from fruits and vegetables during storage i.e. refrigeration25, high humidity stores26, air tight storages27.mostofi et al.28reported that various chemical treatment have been used to slow down physiological changes and moisture loss. bahri & rashidi29 found a decrease in moisture level of carrot during 14 days storage after post-harvest treatment. 0 3 6 9 12 15 0 10 20 30 40 50 60 70 relative humidity temperature days after storage a m b ie n t c o n d it io n s figure 1. temperature and relative humidity during the study. effect of post-harvest treatments on quality characteristics of carrots during storage vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 171 table 1. mean squares of quality parameters of carrots as effected by different post-harvest treatments and storage duration. sov df weight loss moisture content fungal incidence tss firmness replication 2 0.6 6.350e-04 1.8 0.0138 1232.85 treatments (t) 3 240.5** 72.7388** 938.7** 5.3311** 1245.97* durations (d) 5 11848.1** 478.643** 13524.8** 42.4810** 1276.42* t*d 15 30.3** 5.93163** 77.7** 0.6498** 1234.69* error 40 0.9 4.565e-04 0.8 0.0085 1233.86 total 65 ** highly significant at p<0.01, * significant at p<0.05 0 3 6 9 12 15 0 20 40 60 80 100 days after storage m o is tu re c o n te n t (% ) 3 6 9 12 15 0 20 40 60 40oc cacl2 control 50 o days after storage w e ig h t l o s s ( % ) 0 3 6 9 12 15 0 1 2 3 4 5 days after storage f ir m n e ss ( lb s) (a) (b) (c) figure 2. effect of treatment and storage duration on moisture content (a), weight loss (b) and firmness (c). 3 6 9 12 15 0 10 20 30 40 50 60 70 40 o c cacl2 control 50 o c days after storage f u n g a l in c id e n c e ( % ) (a) (b) 0 3 6 9 12 15 0 3 6 9 12 days after storage t o ta l s o lu b le s o li d s ( % ) figure 3. effect of treatment and storage duration on fungal incidence (a) and total soluble solids (b). effect of post-harvest treatments on quality characteristics of carrots during storage vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 172 weight loss mean squares revealed significant variations in weight loss of carrot. the result were significantly different for treatment, storage duration and the interaction of treatment × duration (table 1). weight loss of carrots increased with increasing days of storage (figure 2b). the lowest weight loss with 41.33% was observed in carrots when treated with0.4% cacl2 dip for 2 minutes, followed by 40 ºc hot water dip for 2 minutes (48.32%), 50 ºc hot water dip for 2 minutes (52.94%) and then with control (57.37%). calcium chloride strengthens the cell walls of fruits and vegetables causing minimal loss when treated with [cacl2]0.4% dip for 2 minutes. the present results of the study are in agreement with the findings of niari et al.30, who stated that five minutes of calcium chloride dip of carrots sample was effective in maintaining the weight of the carrots. similar result has also been observed by marta et al.31 firmness the statistical analysis revealed a significant difference among treatment, storage duration and interaction between treatment × duration (table 1). a decreasing trend (4.96 to 3.41 lbs) with increasing duration was observed for the carrots in all treatments (figure 2c). the maximum firmness with 4.12lbs after 15 days of storage was observed in carrots when treated with0.4% cacl2 dip for 2 minutes, followed by 40 ºc hot water dip for 2 minutes (3.96lbs), 50 ºc hot water dip for 2 minutes(3.72lbs) and then with control (3.41lbs). the loss of firmness might be due to high rate of physiological process and attack of microorganisms, leading to deterioration and senescence. zudairea et al.32 stated that postharvest practices significantly affects the quality and physiological changes in products. koh & melton33stated that the chemical treatment i.e. calcium chloride reduces the ripening of fruits, resulting with increased firmness. heat treatment according to gil et al.34has the tendency of removing disease-causing spores that cause deterioration on the surface of fruits and vegetables. gonçalves et al.35reported that decay organisms soften fruits and vegetable tissues, which increases the rate of respiration and loss of moisture, and as a result decreases its hardness. fungal incidence the carrots were initially free from any fungal incidence, which then gradually increased during the entire storage duration as shown in figure 3a. mean squares revealed significant differences in fungal incidence of carrot under different treatments, duration and the interaction of treatment × duration (table 1). the highest fungal incidence with 71.53%was observed in control carrots, followed by 50 ºc hot water dip for 2 minutes, 40 ºc hot water dip for 2 minutes and lowest when treated with 0.4% cacl2 dip for 2 minutes (38.14%). augspole et al.36 stated calcium chloride is a chemical sanitizer that prevents fungal infections and their associated germinating spores. kaka et al.37for their study reported that hot water treatment is helpful in decreasing rot spores at the surface of fruits and vegetables. fallik38 similarly stated that heat treatment has direct effect on fungal pathogens by inactivating germination spores, which can also cause antifungal chemicals in the product that inhibit fungal growth. total soluble solids the mean squares for total soluble solids of carrots showed significant variations among treatment, storage duration and interaction between treatment × duration (table 1). total soluble solids of carrots increased with increasing days of storage (figure 3b). minimum values with 10.43% for tss among the postharvest treatments was observed for0.4% cacl2 dip for 2 minutes, followed by 40 ºc hot water dip for 2 minutes, 50 ºc hot water dip for 2 minutes and then washed with tap water (12.47%). bahri & rashidi29 while conducting an experiment on carrots observed an increase in total soluble solids with increasing day of storage. similar results has also been reported by rashidi et al.39 and gupta et al.40, whom reported that the total soluble solids level in carrots significantly increased with storage duration. c o n c l u s i o n s the calcium chloride and hot water treatments showed a significant effect on the quality characteristics of carrots during storage at ambient conditions for 15 days. carrots treated with 0.4% concentration of cacl2 dip for 2 minutes showed better quality (high moisture content, low weight loss, high firmness, least fungal incidence and lower total soluble solids) followed by 40 ºc hot water dip for 2 minutes, 50 ºc hot water dip for 2 minutes and with control. storage period significantly increased weight loss, fungal incidence and tss. however moisture content and firmness of carrots decreased with increasing storage effect of post-harvest treatments on quality characteristics of carrots during storage vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 173 period. the adoption of treatment viz. 0.4% concentration of cacl2 for 2 minutes should be encouraged in the developing countries for extending the quality characteristics of carrots. c o n t r i b u t o r s t a t e m e n t benish nawaz miran and shakeel hussain chattha contributed in study conception and design. benish nawaz miran, shakeel ahmed soomro and babar mustafa ansari contributed in performing the experiments. zaheer ahmed khan and ghassan zahid contributed in analysis of data. bakhtawar wagan, shakeel hussain chattha and imtiaz ali dahri supervised and contributed in interpretation of data. shakeel ahmed soomro contributed in writing the manuscript. c o n f l 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methods on the quality of onion bulbs. pakistan j agric agric eng vet sci. 2016;32(2):221–8. 20. rashidi m, ranjbar i, gholami m, abbassi s. prediction of total soluble solids and firmness of carrot based on carrot water content. int j agric biol. 2010;12(2):237–40. 21. isaac o, maalekuu bk. effect of some postharvest treatments on the quality and shelf life of three cultivars of carrot ( daucus carota l . ) during storage at room temperature. am j food nutr. 2013;3(2):64–72. 22. zou y, jiang a. effect of ultrasound treatment on quality and microbial load of carrot juice. food sci technol. 2016;36(1):111–5. 23. seljasen r, bengtsson gb, hoftun h, vogt g. sensory and chemical changes in five varieties of carrot (daucus carota l) in response to mechanical stress at harvest and post-harvest. j sci food agric. 2001;81(4):436–47. 24. ahmad t, cawood m, iqbal q, ariño a, batool a, tariq srm, et al. phytochemicals in daucus carota and their health benefits—review article. foods. 2019;8(9):1– 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rakcejeva t, kruma z, dimins f. shredded carrots quality providing by treatment with hydrogen peroxide. 9th balt conf food sci technol “food consum well-being” foodbalt 2014, jelgava, latv 8-9 may, 2014. 2014;150–4. 37. kaka ak, ibupoto ka, chattha sh, soomro sa, mangio hr, junejo sa, et al. effect of hot water treatments and storage period on the quality attributes of banana (musa sp.) fruit. pure appl biol. 2019;8(1):363–71. 38. fallik e. prestorage hot water treatments (immersion, rinsing and brushing). postharvest biol technol. 2004;32(2):125–34. 39. rashidi m, ranjbar i, gholami m, abbassi s. prediction of carrot total soluble solids based on carrot water content. am j agric environ sci. 2010;7(3):366– 9. 40. gupta dk, keerthika a, gupta ck, shukla ak, mohamed mbn, jangid bl, et al. climate change and its impact on fruit crops. hortic based integr farming syst. 2021;223–34. antibacterial activity of some household surface cleaners against common pathogens. vol 8 (1), jan 2017 issn: 2305 8722 23 r a d s j . b i o l . r e s . a p p l . s c i 23 op e n ac c e ss f u l l l e n g t h a r t i c l e antibacterial activity of some household surface cleaners against common pathogens sahifa naz1 and farkhanda afaque1* 1 department of microbiology, jinnah university for women a b s t r a c t surface cleaner has been widely used to control infections and its transmission. in hospitals antiseptics are used to control microbial growth on living tissues and inanimate objects. the activity of six commercial household surface cleaner, dettol, phenyl, harpic, max, sweepy and local surface cleaner were tested against common pathogens of 10 different species of each strains of staphylococcus aureus, escherichia coli, pseudomonas aeroginosa and bacillus subtilis. disc diffusion method were implanted with different concentrations of 100%,75%,50%,25% for each surface cleaner and applied on mha. after incubation of 24 hours inhibition zones were measured as the maximum zone of inhibition were observed by staphylococcus aureus at 75 % concentration and the minimum zone of inhibition were observed by bacillus subtilis at 25 % concentrations. this conducted study showed effectively killing of disinfectant in all test organisms and provide greatest protection in the transmission of diseases. keywords antimicrobial activity, surface cleaners, potential pathogens, health risk. address of correspondence farahasiddiqui@hotmail.com article info. received: april 3, 2017 accepted: may 29, 2017 cite this article: naz s, afaque f. antibacterial activity of some household surface cleaners against common pathogens. rads j. biol. res. appl. sci 8(1):23-25. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n the health of a man is the major part of life , health hazards can be very severe and more often leads to death (1). therefore, the environment should be clean and free from harmful microorganisms to prevent as much diseases as possible to remain healthy. this condition can be accompanied by using disinfectants and surface cleaners (2,3). the personal hygiene should be maintained by proper hand washing and using different cleaners and disinfectant to avoid the attack of bacteria that are the major cause of health hazards (4) and causes the git infections which are the main reason of food borne diseases. food borne diseases affect human by ingestion or drinking contaminated water and food with microorganisms. the kitchen utensils become contaminated with staphylococcus mainly s. aureus causes food borne gastroenteritis. they include nausea, vomiting, abdominal cramps, diarrhea, sweating, headache and prostration (5). in order to prevent infection it's highly important to use trusted detergents that kill pathogenic microorganism. according to osbore and grobe by using antiseptic surface 65 to 85% removal of bacteria could be attained from human skin (6). transient bacteria are attached to the skin and cause many infections these bacteria include pseudomonas aeruginosa (7) and staphylococcus aureus (8). cleaning surfaces are essential to obtain the germs free environment. many products are used to clean the surfaces but all products give the varying results. some products are designed to work best on specific surfaces and/or soils few of them are dettol, harpic, phenyl etc that can be effective to e.coli, s.aureus, pseudomonas and b.subtillus. o r i g i n a l a r t i c l e antibacterial activity of some household surface cleaners against common pathogens. vol 8 (1), jan 2017 issn: 2305 8722 24 r a d s j . b i o l . r e s . a p p l . s c i 24 using wel diffusion method, different disinfectants are evaluated on the human skin flora and surface flora (9). identification of bacterial species that are most resistant to the antibacterial surface cleaners of daily use was also made. in an analysis of the action of a disinfectant, there are some compounds that act upon different components of the cell such as acidic or alkaline compounds, chlorine derivatives, amphoteric compounds, phenolic compounds, hydrogen peroxides and ozone. it may often be difficult to distinguish between the primary stage (characteristic of the mode of action) and the secondary stage (merely a consequence of the action). precisely these compounds act upon cell wall, cell membrane, energy metabolism, cytoplasm, nucleus and even kill the spores (10). this study shows the comparison of different surface cleaners such as the branded surface cleaners against locally manufactured surface cleaners as well as this study also shows the effect of selected surface cleaners against different pathogens. m a t e r i a l s a n d m e t h o d sample collection and culture preparation: the samples are taken from different companies including dettol, phenyl, harpic, sweepy, max and local surface cleaner. nutrient broth used for each culture including s.aureus, e.coli, p.aeroginosa , b.subtilis and incubate for 24 hours. preparation of sterile disc: filter disks about 5 mm in diameter were made from whatman’s no.3 filter paper wrapped in aluminum foil. each sterile disc was incorporated individually with 40 μl of detergent with various concentrations (25%, 50% and 100%). assay of antimicrobial activity: using sterile forceps, discs impregnated with different dilution and different disinfectants were placed on each of the plates inoculated with the test organisms. the forceps was used to press down each of the disc gently against the agar surface so as to ensure good contact. the plates were incubated in an inverted position at 37oc for 24 hours. the zones of inhibition were observed. r e s u l t s the average sensitive zone for escherichia coli were 3.2cm, staphylococcus aureus were 2.7cm, pseudomonas aeruginosa were 2.6cm, bacillus subtilis were 2.4cm at 100%. the intermediate zones for escherichia coli were 2.4cm, staphylococcus aureus 2.8cm, pseudomonas aeruginosa 2.4cm, bacillus subtilis were 2.2cm at 75%. intermedaite zones for escherichia coli were 2.7cm, staphylococcus aureus 2.6cm, pseudomonas aeruginosa 2.4 cm, bacillus subtilus were 2.1cm at 50%. other zones for escherichia coli 2.6cm, staphylococcus aureus 2.7cm, and pseudomonas aeruginosa were 2.5cm, bacillus subtilus 0.8cm. whereas harpic, phenyl, sweepy were not show any zones. d i s c u s s i o n the effect of dettol® was assessed by (11) against some microorganisms associated with nosocomial infection including staphylococcusaureus, it was highly effective like this study results, results of this study regarding dettol® are similar to that obtained by (12) as the study recorded inhibition zones ranging from 28 to 17 mm for 100% to 5% concentration. the antimicrobial properties of surface cleaner are very helpful against some pathogenic organisms such as multidrug-resistant pathogens such as staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, and bacillus subtilis. the results of our research is that the antibacterial effects of antiseptics different pathogens are not only dependent on the types of antiseptics but also on their concentrations. sodium hypochlorite has a good activity against staphylococcus aureus and it is the most used antiseptic compound in homes cleaning. it also shows that different types of microorganisms vary in their response to different types of antiseptics. dettol, max, and local surface cleaner were having highest activity against all pathogens. antibacterial effect of dettol was more effective against staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, and bacillus subtilis. max was more effective against escherichia coli and bacillus subtilis. local surface cleaner was showed better antibacterial efficacy against common pathogens. antibacterial activity of some household surface cleaners against common pathogens. vol 8 (1), jan 2017 issn: 2305 8722 25 r a d s j . b i o l . r e s . a p p l . s c i 25 table i: antimicrobial activity of dettol and lemon max against different bacteria concentration dettol max s.aureus b.subtilus e.coli p.aeruginosa s.aureus b.subtilus e.coli p.aeruginosa 100% 2.7 cm 2.3 cm 3.2 cm 2.6 cm no zone 27 cm 27 cm no zone 75% 2.8 cm 2.2 cm 2.4 cm 2.5 cm no zone 26 cm 17 cm no zone 50% 2.6 cm 2.1 cm 2.7 cm 2.4 cm no zone 21 cm 16 cm no zone 25% 2.7 cm 0.8 cm 2.6 cm 2.6 cm no zone 16 cm 15 cm no zone table ii: antimicrobial activity of local tile ash, harpic, sweepy and phenyl against different bacteria concentration local tile wash harpic phenyl sweepy s.aureus b.subtilus e.coli p.aeruginosa 100% 2.1 cm 2.1 cm 2.0 cm 1.3 cm no zone no zone no zone 75% 1.9 cm 2.2 cm 1.3 cm 1.0 cm 50% 0.8 cm 1.6 cm 1.2 cm 1.0 cm 25% 0.8 cm 1.5 cm 1.2 cm 0.9 cm all strains of staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, and bacillus subtilis are resistant to phenyl, harpic, and sweepy . c o n c l u s i o n the research concludes that the surface cleaners and disinfectants can kill the bacterial pathogens and having bacteriostatic properties which is beneficial for preventing the people from many infections. the health care providers can use these cleaners to protect the immunocompromised patients from transmission of pathogenic or opurtunistic organisms. r e f e r e n c e s 1. awodele o, emeka pm, agbamuche hc, akintonwa a. the antimicrobial activities of some commonly used disinfectants on bacillus subtilis, pseudomonas aeruginosa and candida albicans. afr j biotechnol. 2007;6(8). 2. moses i, rosemary m, linda a and nsikak a. antimicrobial activity of some cleaning products against selected bacteria. int res j pharma appl sci. 2013; 3(4):175-179. 3. wang z, shen y, ma j, haapasalo m. the effect of detergents on the antibacterial activity of disinfecting solutions in dentin. j endod. 2012 jul 31;38(7):948-53. 4. bhat r, prajna ps, menezez vp, shetty p. antimicrobial activities of soap and detergents. adv biores. 2011 dec;2(2):52-62. 5. jay jm, loessner mj, golden da. modern food microbiology. springer science & business media; 2008. 6. osborne rc, grube j. hand disinfection in dental practice. clin prev den. 1981;4(6):11-5. 7. fluit ac, schmitz fj, verhoef j. frequency of isolation of pathogens from bloodstream, nosocomial pneumonia, skin and soft tissue, and urinary tract infections occurring in european patients. eur j clin microbiol inf dis. 2001;20(3):188-91. 8. higaki s, kitagawa t, kagoura m, morohashi m, yamagishi t. predominant staphylococcus aureus isolated from various skin diseases. j int med res. 2000;28(4):187-90. 9. cheesbrough m. district laboratory practice in tropical countries. cambridge university press; 2006. 10. barrette jr wc, hannum dm, wheeler wd, hurst jk. general mechanism for the bacterial toxicity of hypochlorous acid: abolition of atp production. biochemistry. 1989;28(23):9172-8.. 11. mahmood el. effect of dettolâ on viability of some microorganisms associated with nosocomial infections. afr j biotechnol. 2008;7(10):1554. 12. saha ak, haque mf, karmaker s, mohanta mk. antibacterial effects of some antiseptics and disinfectants. j life and earth sci. 2009;3:19-21. pathogens identification and evaluation of nigella sativa’s (kalonji) antibacterial activity urooj arif1*, sumaira javed2, sayyada ghufrana nadeem2, shazia tabassum hakim3 1department of environmental sciences, jinnah university for women, karachi-74600, pakistan 2medical mycology research and reference laboratory, department of microbiology, jinnah university for women, karachi -74600, pakistan. 3virology and tissue culture laboratory, department of microbiology, jinnah university for women, karachi -74600, pakistan. abstract to examine the presence of different pathogens capable of causing diseases in the humans, bacteria were isolated from clinical samples. 40 different clinical samples were collected from which different pathogens were isolated and identified by using conventional methods. in this article we also checked the antibacterial activity of nigella sativa. the nigella sativa commonly known as kalonji are use in herbal medicine, throught the world for the prevention and treatment of many diseases. the kalonji oil has many properties like antibacterial, antiparasitic, antioxidant etc. kalonji oil was used to study the antibacterial activity against 5 clinical isolates of bacteria. disc diffusion technique was applied on inoculated muellar hinton agar plates. among gram positive bacteria only staphylococcus aureus was used, among gram negative bacteria pseudomonas aeruginosa, klebsiella pneumoniae, proteus, and e.coli were used. the oil showed antibacterial activity against staphylococcus aureus and e.coli and pseudomonas aeruginosa. keywords: clinical samples, disc diffusion method, nigella sativa, n. sativa oil, thymoquinone. introduction pathogens isolation and identification is based on the cultivation of microbes with morphological and physiological characterization lasting 24-48 hours. however, early and accurate identification is always required for fast and targeted antimicrobial treatment. rapid and fast identification is mostly based on molecular based techniques but differentiation between closely related species is still difficult. identification of bacteria based on traditional methods relies on phenotypic identification of the pathogens using gram staining, culture and biochemical methods. (gobernado, 2003). mostly the body fluids are sterile like blood, urine, ear wax, nasal discharge, csf etc. normally the presence of microbes in these samples may be due to contamination or they may be the normal flora of surrounding area. when the microbes grow in different types of body fluids, they may cause infection in them. the blood is normally a sterile environment. these are the common pathogens of blood s. aureus, s. epidermidis, s. pyogenes, s. pneumoniae, m. tuberculosis, legionella, e. coli, k. pneumoniae, enterobacter, p. aeruginosa, proteus mirabilis, h.influenzae, and n. meningitidis. (herbert et al., 2007) pus is a yellowish viscous substance secreated by wounds in response to body’s natural immune system. the common bacterial pathogens were gram positive cocci like aureus, s. epidermidis and ßstreptococci, gram-negative pseudomonas, k.*corresponding author: uroojqazi@ymail.com 25 vol 5 (1), january 2014; 25-29 pneumoniae and e. coli. (bone r et al., 1992) sputum samples that may have some pathogens are usually used to look for infections by moraxella catarrhalis, m. tuberculosis, pneumoniae and h. influenzae. (annane et al., 2005) nigella sativa (klonjii) is used as food preservative and spice. in traditional medicine its oil and seed constituents have important medicinal properties (riaz et al., 1996). kalonji is used as food and medicines in many different countries including pakistan, iran, india, saudi arabia and egypt. it is used in conventional medicine for treatment or prevention of a variety of respiratory and gastrointestinal infection in the islamic world (riaz et al., 1996). it has been also used in many conditions related to kidney and liver function, respiratory, circulatory and immune system support, stomach, intestinal complaints, inflammatory diseases (malhotra, 2006; ramadan, 2007). they have insect repellent, antimicrobial, antitumor (khan et al., 2003) and anti diabetic, anti-h.pylorin, anti asthmatic, anti parasitic, antioxidant, antimicrobial, antiinflammation, hypoglycemic, antihypertensive, and anticancer activity (fararh et al., 2002). in nursing mothers seeds of kalonji are utilized for enhancing milk production, also improving digestion and in fighting against parasitic infections, anti-inflammatory antihistaminic, and antihypertensive (hajhashemi et al., 2004), and for the treatment of chronic colds. inhalation of its volatile oils is useful. it is very important in curing migraine, paralysis, facial palsy. its oil is effective in the treating skin infections like eczema, boils, earaches (khan, 2003; antuono et al., 2002; iqbal et al., 2011) and immune stimulation. in pakistan the crop of kalonji appears to be a potential and multi-purpose crop. recently, kalonji seeds have been shown for many biological activities, i n c l u d i n g : a n t i o x i d a n t , a n t i m i c r o b i a l , immunomodulatory, anti-inflammatory. its seed or extract inhibits fungal growth in dermatophytes (aljabre et al., 2005). the extract of kalonji seed and its derivative, thymoquinone, inhibits some opportunistic fungi including aspergillum niger, fusarium solani and scopulariopsis brevicaulis. kalonji contains approximately 100 valuable elements. it contains 21% protein, 35% fatty acids, 38% carbohydrates, vitamins such as a, b, b2, c and niacin, minerals like calcium, magnesium, potassium, selenium, iron, zinc and large amount of fixed oils (ko¨ kdil and yilmaz, 2005). kalonji have some active ingredients like fixed oils, nigellone and thymoquinone (aljabre et al. 2005). the aim of the present study was to evaluate the antibacterial activity of nigella sativa against different bacterial isolates from clinical samples. materials and methods sample size: 40 different clinical samples were collected from tahir shamsi lab, karachi which include 18 pus samples, 14 blood samples, 7 sputum samples and 1 pleuritic fluid sample. isolation of organisms: all samples were first inoculated on nutrient agar plates with the help of sterile wire loop for primary isolation and incubated at 370c for 24 hour. from the last day incubated plates, performed gram staining, biochemical tests that include sugar tubes (glucose, lactose, and sucrose), tsi agar, imvic and citrate. enzyme test were also performed that includes catalase and coagulase, to identify organism. for further confirmation some selective agar were also used. antimicrobial sensitivity testing: kalonji oil was purchase from local market of karachi, pakistan. disc (6mm in diameter) were punched out from whatman’s filter paper that placed in petri dish on a distance of 2-4mm. discs were sterilized in a hot air oven at 1600c for 1hour. 20µl of oil was pipette out on to each disc in order to soak the oil on disc properly and incubated at 370c for 1 hour. discs were then placed in air tight container and refrigerate at 40c until used. for antimicrobial testing of kalonji, kirby bauer disc diffusion method was followed by making lawn of bacterial culture on mueller hinton’s agar (mha) and placed oil soaked disc on it. incubated 26 vol 5 (1), january 2014; 25-29 the plate at 370c for 24. results and discussion forty different clinical samples were used for the isolation of pathogens. in pus samples out of 18, 6 staphylococcus, 3 klebsiella, 2 e.coli, 2 enterobacter, 2 bacillus, 2 proteus and 1 micrococcus were isolated. in blood samples out of 14, 5 salmonella typhi, 3 streptococcus, 2 proteus, 2 klebsiella, 1 pseudomonas and 1 e.coli were isolated. in sputum samples out of 7, 3 streptococcus, 2 pseudomonas, 1 staphylococcus aureus and 1 bacillus were identified (figure 1-3). in antimicrobial sensitivity testing, e.coli, aureus and pseudomonas were sensitive were found to be sensitive to nigella sativa and the zone of inhibition is about 120 mm, 150 mm and 210 mm respectively, while klebsiella and proteus were resistant to nigella sativa. antibiotic sensitivity testing was performed on five selected bacterial strains. proteus from pus sample was found to be resistant against cefactor, gentamycin and was sensitive to polymaxin b(18 mm), ceftrioxon (25 mm). s.aureus was sensitive to amikacin (13 mm) & gentamycin (14mm) while resistant to cefactor & penicillin g. e.coli was sensitive to polymaxin b (13 mm) gentamycin(14 mm) and resistant to cefactor & gentamycin. pseudomonas was sensitive to to ciprofloxacin (20 mm) and gentamycin (20mm) while resistant to ceftrioxon and ofloxacin. klebsiella was sensitive to gentamycin (13mm) while resistant to, polymaxin b, gentamycin and ceftrioxon. some studies (khan and ather, 2006) shows the oil was more effective on gram positive than gram negative bacteria because of gram negative have outer membrane that act as effective permeability barrier, which restricts the penetration of unnecessary compounds. kalonji oil showed pronounced activity against s. aureus, p. aeruginosa and e. coli, while dosen’t shows activity against klebsiella and proteus (figure 4-5). 27 vol 5 (1), january 2014; 25-29 figure 4: inhibition of s.aureus by antibiotics figure 1: distribution of isolated organisms in sputum samples. steprococcus pseudomona staph. aures bacillus figure 2: distribution of isolated organisms in blood samples. salmonella typhi streptococcus proteus klebsislla pseudomonas e.coli figure 3: distribution of isolated organisms in pus samples. s. aureus klebsilla e.coli enterobacter bicillus proteus micrococcus the out coming results may be due to the reason that kalonji oil was obtained from different commercial sources, that may contain different amount of thymoquinone that has antibacterial activity. references aboul-ela e.i., 2002, cytogenetic studies on nigella sativa seeds extract and thymoquinone on mouse cells infected with schistosomiasis using karyotyping mutation research 517: 11-17 aljabre s.h.m., randhawa m.a., akhtar n., alakloby o.m., alqurashi a.m. and aldossary a., 2005,antidermatophyte activity of ether extract of n i g e l l a s a t i v a a n d i t s a c t i v e p r i n c i p l e , thymoquinone.journal of ethnopharmacology, 101: 116-119. annane d, bellissant e, cavaillon jm 2005. "septic s h o c k " . l a n c e t 3 6 5 ( 9 4 5 3 ) : 6 3 – 7 8 . doi:10.1016/s0140-6736(04)17667-8. pmid bone, r.; balk, r.; cerra, f.; dellinger, r.; fein, a.; knaus, w.; schein, r.; sibbald, w. 1992. "definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. the accp/sccm consensus conference c o m m i t t e e . a m e r i c a n c o l l e g e o f c h e s t physicians/society of critical care medicine". chest 101 (6): 1644–55. doi:10.1378/chest.101.6.1644. pmid 1303622. d’antuono, l.f., a. moretti and a.f.s. lovato. 2002. seed yield, yield components, oil content and essential oil content and composition of nigella sativa l. and nigella damascena l. industrial crops & products, 15: 59-69. fararh, k.m., y. atoji, y. shimizu and t. takewaki. table ii: susceptibility pattern of bacteria against antibiotics resistant resistant sensitiveresistantantibiotics 20mm 20mm ciprofloxacin (cip5) gentamycin (cn10) ceftrioxone (cro30) ofloxacin (ofx5) p.aeruoginosa 28 vol 5 (1), january 2014; 25-29 figure 5: inhibition of s.aureus by n. sativa. table i: susceptibility pattern of bacteria against nigella sativa polymyxin (pb3oo) polymyxin (pb3oo) gentamycin (cn10) gentamycin (cn10) cefaclor (cec30) cefaclor (cec30) penicillin g ceftrioxon(cro30) ceftrioxon (cro30) polymyxin (pb3oo) ceftrioxon (cro30) cefaclor (cec30) cefaclor (cec30) gentamycin (cn10) ceftrioxon (cro30) ofloxacin (ofx5) 29 vol 5 (1), january 2014; 25-29 2002. isulinotropic properties of nigella sativa oil in streptozotocin plus nicotinamide diabetic hamster. res. vet. sci., 73: 279-282. fisher, c. 2002. spices of life. chem. br., 38: 40-42. gobernado m. 2003 .identification of bacteria by traditional method. enferm infecc microbiol clin. suppl 2:54-60. hajhashemi,v., a. ghannadi and h. jafarabadi. 2004. black cumin seed essential oil, as a potent analgesic and anti inflamatory drug. res,.18:195-199 herbert wm, klemens v, rudolf p, albert k. 2007. identification of human pathogens isolated from blood using microarray hybridisation and signal pattern recognition.bmc microbiology, 7:78 doi:10.1186/1471-2180-7-78 iqbal, m.s., a. ghafoor, inamullah, f.m. abbasi, a.s. qureshi and h. ahmad. 2011. study of nutritional characteristics, mineral nutrients and agro-biodiversity in black cumin (nigella sativa l.) genotypes from pakistan. afr. j. biotech., 10: 1475714766 ko¨kdilg. andyilmazh. 2005 —analysis of the fixed oils of the genus nigella l.(ranunculaceae) in turkey.biochemical systematics and ecology, 33:1209 khan, n., s. sharma and s. sultana. 2003. nigella sativa (black cumin) ameliorates potassium bromateinduced early events of carcinogenesis: diminution of oxidative stress. hum. exp. toxicol., 22: 193203. khan mth and a. ather,2006. the medicinal potential of black seed (nigella sativa) and its componentslead molecules from natural products. malhotra, s.k. 2006. nigella. in: handbook of herbs and spices. (ed.): k.v. peter, vol. 2. woodhead publishing ltd., cambridge, pp. 206-214. r a m a d a n , m . f. a n d j . t. m o r s e l . 2 0 0 2 . characterization of phospholipids composition of black cumin (nigella sativa).nahrung/food, 46: 240244. riaz, m., m. syed and f.m. chaudhary. 1996. chemistry of the medicinal plants of the genus n i g e l l a . h a m d a r d m e d i c u s , 3 9 ( 2 ) : 4 0 4 5 . antibacterial and antifungal activities of a.modesta, a. saspara and s. surattense vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 193 op e n ac c e s s f u l l l e n g t h a r t i c l e antibacterial and antifungal activities of acacia modesta, achyranthe saspara and solanum surattense used in folk medicine nazia sana1, rabia shaheen1, aneela sharif2, sehrish kalsoom2,*, zahida nasreen2 1department of botany, university of mianwali, mianwali, pakistan 2department of zoology, university of mianwali,mianwali, pakistan a b s t r a c t background: the antibiotic resistance is a grave concern across the globe due to inappropriate and over usage of antibiotics without prescription which could be fatal for the humans in the long run. therefore, the need of the hour is to look for more effective and economical options for treatment worldwide to reduce antibiotic resistance. objective: this study was targeted to assess the antibacterial and antifungal properties of leaf extracts of three plants (achyranthes aspara, acacia modesta and solanum surattense) against escherichia coli, salmonella and malassezia species. methods: the bioactive components were extracted using methanol and ethanol (70%) as solvents. the agar-well diffusion method was employed to assess the antibacterial and antifungal activities of leaf extract. the inhibitory zones were recorded. the bioassay studies of the crude extracts were undertaken at three different concentrations i.e., 30.0, 40.0, and 50.0 mg/ml. results and conclusion: the ethanol extracts of achyranthes aspara had shown the highest growth inhibitory effect at a concentration of 50.0 mg/ml with an inhibition zone of 13.2mm against e. coli. ethanolic leaf extracts of solanum surattense had shown maximum inhibition effects at a concentration of 50.0 mg/ml with 12.5mm zones of inhibition against salmonella. acacia modesta did not show good results against any bacterial and fungal strains as compared to the other two plants. keywords medicinal plants, ethanol extract, antimicrobial activity, a. saspara, s.surattense, a. modesta. *address of correspondence sehrish.kalsoom1820@gmail.com article info. received: june 26, 2022 accepted: october 07, 2022 cite this article: sana n, shaheen r, sharif a, kalsoom s, nasreen z. antibacterial and antifungal activities of acacia modesta, achyranthe saspara and solanum surattense used in folk medicine. 2022; 13(2):193-201. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n according to world health organization, 80% of the world’s population uses plant extracts and their constituents as folk medicine in traditional therapies. it has been estimated also that about 50% of all current theoretical drugs are from natural sources1. natural compounds in plants have antimicrobial properties that have been confirmed through several in vitro studies. a remarkable number of medicinal plants are used in the making of various drugs and the production of other conventional medicines2. it is estimated that out of o r i g i n a l a r t i c l e antibacterial and antifungal activities of a.modesta, a. saspara and s. surattense vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 194 422127 reported plant species, only 35,000 to 70,000 are involved in the antimicrobial activity3. plants are employed in drug manufacturing due to the prevalence of different phytochemicals such as flavonoids, terpenoids, polyphenols, carotenoids, vitamins, tannins, alkaloids, saponins, pigments, minerals and enzymes that have both antioxidant and antimicrobial activity. as medicinal plants have fungicidal and bactericidal effects, there is a dire need to evaluate plants for drug manufacturing and drug discovery4. the use of medicinal plants has a long history in the treatment of a range of diseases, including infectious diseases, and these days hundreds of thousands of plant species have been tested for their medicinal effect5. however, the phytochemical and pharmacological activities of many more plants remain to be studied. plant-derived substances are tolerated and accepted by patients and seem a reliable source of anti-microbial compounds6. the global impact of infectious diseases caused by fungi and bacteria is a major health problem worldwide. in recent years, antibiotic and multidrug resistance has become a grave concern in medical communities due to the increased incidence of infections. the world's leading cause of premature death is infectious diseases. therefore, it is the need of the hour to explore new antimicrobial compounds to resolve this problem7. malassezia, salmonella and escherichia coli (e. coli) species are the most common disease-causing pathogens for various infections, especially in those countries that have poor health care systems. the studies on these organisms and their antibiotic resistance pattern showed the quick spread of multidrug resistance in these pathogenic microbes. so, there is an urgent need for public education for healthcare providers and the general population regarding the careful utilization of antibiotics8. different plant parts (root, stem, leaves, and fruits) are useful for therapeutic and medicinal purposes. medicinal plants contain several chemical components which can be opted for the treatment of different diseases and to assess their inhibitory activity against different well-known pathogens9. the leaf extract of achyranthes aspera showed hepatoprotective activity in rifampicin. it is an antibiotic used to treat albino rats caused by salmonella. e. coli and cryptosporidium10. however, it is also investigated for invitro antimicrobial activity against escherichia coli and salmonella typhi by disc diffusion method. achyranthe saspera leaves extract inhibited the growth of salmonella typhi and e. coli11. acacia modesta leaves extract are used for the treatment of skin cancer as an astringent, antispasmodic and aphrodisiac. leaves contain carbohydrates, glycosides, phytosterols and another major compound that inhibit the growth of e. coli and fungal pathogens12. to show more positive results phytochemical screening (flavonoids, tannins, sterols, alkaloids and carbohydrates) was done by different extracts. these extracts inhibit the growth of e. coli13. m a t e r i a l s a n d m e t h o d s collection of medicinal plants three selected plants i.e., acacia modesta, solanum surattense, and achyranthe saspara have been used in the present study. these plants were collected from the surroundings of musa khel and swans in the district of mianwali. processing of medicinal plants collected plant leaves were washed, cleaned, and then dried under shade for two weeks. leaves were ground in a grinder to obtain a fine powder and stored in a container. culture of pathogens e. coli, salmonella, and malassezia spp were studied in the present work. the bacterial sample was taken from sewage water as it contains abundant pathogenic microbes and the fungal samples were obtained from the head of a student. these were cultured in the laboratory. biochemical tests and staining were used for the identification of e. coli, salmonella and malassezia spp. preparation of leaf extract 70% ethanol and 70% methanol were taken in 3 separate flasks (250 ml capacity each). 30.0 grams of plant material was poured into each of the flasks and mixed well to prepare methanolic & ethanolic extracts. to avoid contamination, these flasks were plugged into cotton and covered with aluminum foil. then these solutions were shaken from time to time via using an orbital shaker for 7 antibacterial and antifungal activities of a.modesta, a. saspara and s. surattense vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 195 days to get a homogenized mixture. extracts were filtered with whitman's filter paper no. 3. then filtrate was dried in a fume hood at room temperature. after 3 days crude leaves extracts were obtained. for dilution dmso (dimethyl sulfoxide) was added in crude extracts and then finally these extracts were stored in the refrigerator at 40c for further investigation. antimicrobial activity of plants extracts antimicrobial activities of selected ethanolic and methanolic leaf extracts of acacia modesta, solanum surattense, and achyranthes aspara were investigated by using the agar well diffusion method. for this purpose, cultured media was prepared. this media was autoclaved at1210c for 60 minutes. autoclaved media was poured into sterilized petri plates. when agar was solidified, 10.0 µl e. coli, salmonella and malassezia spp samples were taken with the help of sterilized micropipettes from culture broth and spread on petri plates. wells were formed with sterilized tips then these wells were filled with selected plant leaves crude extract. these plates were incubated for 24 hours at 370c. after the 24-hours zone of inhibition was formed, observed and measured. r e s u l t s the present study was designed to analyze the antimicrobial effect of different medicinal plants against salmonella, e. coli, and malassezia spp. overall results showed the most significant antimicrobial activity by using methanol extract of achyranthes aspara against e.coli and ethanol extract of solanum suratense against salmonella. methanol extract of solanum surattense shows the highest antimicrobial activity against malassezia spp. comparison of inhibition zone of three plant extracts against e. coli table 1 shows, an overall comparison of the antimicrobial activity of ethanol and methanol leaves extracts of selected plants. statistical analysis exhibit that the antimicrobial activity of achyranthes aspara was found to be very high as compared to acacia modesta and solanum surattense. the dilution of 1.0ml, ethanol and methanol extracts of achyranthes aspara shows the maximum zone of inhibition at the concentration of 50.0mg/ml against e. coli and a minimum zone of inhibition at 30.0mg/ml. ethanol extract have remarkable antimicrobial activity (fig 1) than methanol extract shown in series of: ethanol extract: 1.0ml=13.2mm> methanol extract: 1.0ml=12.6mm. the inhibition zone of solanum surattense shows a moderate effect against e. coli. ethanol extract of solanum surattense showed a maximum zone of inhibition that was 11.6mm at a concentration of 50.0mg/ml. table 1 show that acacia modesta leaves extracts show a minimal effect on e. coli growth as compared to the other two plants. the inhibitory zone of ethanol and methanol leaves extract of acacia modesta was 10.9mm at a concentration of 50mg/ml. fig 2 shows inhibition zones labelled 1,2,3,4 against e coli. figure 1. effect of different concentrations of ethanolic and methanolic leaves extracts of achyranthes aspara, acacia modesta and solanum surattense on growth of e coli in h ib it io n z o n e ( m m ) antibacterial and antifungal activities of a.modesta, a. saspara and s. surattense vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 196 table 1. inhibition zone of three plant extracts against e.coli. plant extract concentration (mg/ml) inhibition zone (mm) achyranthe saspara methanolic 30 12.0 40 12.2 50 12.6 ethanolic 30 11.3 40 12.5 50 12.2 solanum surattense methanolic 30 11.3 40 11.2 50 11.4 ethanolic 30 11.7 40 11.8 50 12.2 acacia modesta methanolic 30 9.8 40 10.8 50 10.9 ethanolic 30 9.6 40 10.5 50 10.9 figure 2. ethanolic leaves extract of achyranthes aspara against e. coli. concentration of ethanolic extracts: (1) 30 mg/ml (2) 50mg/ml (3) 30mg/ml (4) 40mg/ml over all comparison of ethanol and methanol leaves extracts of three plants shown in table 2. each plant, leaves extract show different activity against salmonella species. the inhibitory effects of leaves extracts were directly proportional to concentration of extracts. ethanol leaves’ extract of solanum surattense and achyranthes aspara showed excellent result as compared to methanol leaves’ extract against salmonella spp. 1.0ml, 0.8ml and 0.6ml dilution of different extracts showed varying degree of inhibitory effect. maximum zone of inhibition was obtained (12.5mm) at 50mg/ml concentration in ethanol extract of solanum surattense of 1.0ml dilution. 1 2 4 3 antibacterial and antifungal activities of a.modesta, a. saspara and s. surattense vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 197 table 2. inhibition zone of three plant extracts against salmonella spp. plant extract concentration(mg/ml) inhibition zone(mm) achyranthe saspara methanolic 30 8.5 40 9.7 50 11.4 ethanolic 30 10.0 40 10.9 50 12.5 solanum surattense methanolic 30 9.9 40 10.3 50 11.9 ethanolic 30 9.0 40 9.8 50 12.5 acacia modesta methanolic 30 8.6 40 9.4 50 11.9 ethanolic 30 8.8 40 10.2 50 12.1 figure 3. effect of different concentrations of ethanolic and methanolic leaves extracts of achyranthe saspara, acacia modesta and solanum surattense on growth of salmonella spp. 0 2 4 6 8 10 12 14 0.6 ml 0.8 ml 01 ml 0.6 ml 0.8 ml 01 ml 0.6 ml 0.8 ml 01 ml 0.6 ml 0.8 ml 01 ml 0.6 ml 0.8 ml 01 ml 0.6 ml 0.8 ml 01 ml methnolic ethanolic methnolic ethanolic methnolic ethanolic achyranthas aspara (ludhari) acasia modesta (phulaie) solanum suratense (mohakeri) in h ib it io n z o n e ( m m ) antibacterial and antifungal activities of a.modesta, a. saspara and s. surattense vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 198 figure 4. ethanolic leaves extract of solanum surattense against salmonella spp. concentration of ethanolic extracts: (1) 30 mg/ml (2) 40mg/ml (3) 30mg/ml (4) 50mg/ml table 3. inhibition zone of three plant extracts against malassezia spp. plant extract concentration(mg/ml) inhibition zone(mm) achyranthe saspara methanolic 30 12.0 40 9.0 50 7.7 ethanolic 30 11.3 40 12.5 50 10.8 solanum surattense methanolic 30 7.7 40 8.3 50 12.1 ethanolic 30 8.5 40 10.6 50 12.0 acacia modesta methanolic 30 10.1 40 10.8 50 12.0 ethanolic 30 9.3 40 9.7 50 10.8 the overall comparison of ethanolic and methanolic leaves extracts of three plants is shown in table 2. each plant leaves extract show different activity against salmonella species. the inhibitory effects of leaf extracts were directly proportional to the concentration of extracts fig 3. ethanol leaves extract of solanum surattense and achyranthe saspara shows excellent results as compared to methanol leaves' extract against salmonella spp. 1.0ml, 0.8ml, and 0.6ml dilution of different extracts showed varying degrees of inhibitory effect. the maximum zone of inhibition (fig 4.) was obtained 1 2 4 3 antibacterial and antifungal activities of a.modesta, a. saspara and s. surattense vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 199 (12.5mm) at 50mg/ml concentration in ethanolic extract of solanum surattense of 1.0ml dilution. figure 5. ‘effect of different concentrations of ethanolic and methanolic leaves extracts of achyranthe saspara, acacia modesta and solanum surattense on growth of malassezia spp.’. figure 6.methanolic leaves ‘extract of solanum surattense against malassezia spp. concentration of ethanolic extracts: (1) 50 mg/ml (2) 30mg/ml (3) 40mg/ml (4) 40mg/ml the overall comparison of ethanol and methanol leaves extracts of three plants is shown in fig 5. and table 3. each plant leaves extract shows different activity against malassezia species. the inhibitory effects of leaf extracts were directly proportional to the concentration of extracts. methanolic leaves extract of solanum surattense and ethanolic extract of achyranthe saspara showed excellent results against malassezia spp. 1.0ml, 0.8ml, and 0.6ml dilutions of different extracts showed varying degrees of inhibitory effect. the maximum zone of inhibition labelled 1 in fig 6. was obtained (12.1mm) at 50mg/ml concentration in methanol extract of solanum surattense of 1.0ml dilution 4 2 1 3 in h ib it io n z o n e ( m m ) antibacterial and antifungal activities of a.modesta, a. saspara and s. surattense vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 200 d i s c u s s i o n the present study was aimed at checking the antimicrobial activity of achyranthe saspara, acacia modesta, and solanum surattense against salmonella, e. coli, and malassezia species using the agar wells diffusion method. experiments were carried out in triplicates. zones of inhibition were analyzed using statistical terms. comparing mean ± standard deviation and statistical significance was calculated. a value of p < 0.05 was considered more significant statistically. achyranthes aspara leaves’ extract has been investigated for invitro antimicrobial activity against e. coli, salmonella, and malassezia species. the different concentrations (1.0ml, 0.8ml, and 0.6ml) of ethanolic and methanolic extract were used against selected bacteria and fungi. 1.0 ml dilution showed the highest antimicrobial activity against e. coli with a zone of inhibition of 12.6 mm at the highest 50.0 mg/ml concentration (table1, fig 1). naidu et al11also studied whole plant (leaves, stem, root, and inflorescence) extracts of achyranthe saspara by disc diffusion method. about 200.0 mg leaves powder was dissolved in 40.0 ml of solvents viz acetonitrile, methanol, chloroform, and n-hexane. leaves extract was used against e. coli and salmonella. they observed that methanolic extracts of whole plant powder inhibited the growth of salmonella typhi with an inhibition zone of 7.0mm at 50.0 mg/ml concentration but no activity showed against e. coli. only n-hexane inflorescences exhibit an inhibitory growth zone of 8.0mm against e. coli. when we matched our result (table 1, fig 1.) with naidu’s research, we noted that due to the use of different solvents and plant parts, our result is more significant and better than their experiment. solanum surattense displayed the highest antibacterial and antifungal activity against salmonella and malasseziaspp. the ethanolic leaves extract showed a maximum zone of inhibition of 12.5 mm at a concentration of 50 mg/ml against salmonella spp. while methanolic leaves extract displayed a maximum zone of inhibition of 12.1mm at a concentration of 50 mg/ml against malassezia spp (table 2,3. fig 3,5.). the data was compared with sheeba et al who also studied ethanolic leaves extract of solanum surattense, using 20.0 g to 30.0 g of leaf powder15. the antimicrobial activity of leaves was checked against pseudomonas aeruginosa, salmonella and e. coli. the leaf extracts showed highest antibacterial activity against salmonella spp with a zone of inhibition of 16.0 mm at 500.0μg concentration. but our experiment showed a minimum zone of inhibition of 12.5mm at the concentration of 50mg/ml against salmonella spp (table 2 fig 3.). due to the difference in the concentration of crude extract, our leaves extract showed less activity as compared to research presented by sheeba et al14. ethanol was found to be a suitable solvent for the extraction against e. coli and salmonella15. similarly are results showed that ethanol extract displayed maximum activity against salmonella spp (table 2, fig 3.). however, khalid et al16studied leaves and stem extracts of acacia modesta. the methanolic and water extracts were used against salmonella typhii and bacillus subtilis. they found that methanolic leaves and stem extract showed inhibitory growth zone of 8.0 mm and 10.0 mm. we observed that our results of methanolic leaf extract are highly effective against salmonella spp with a zone of inhibition was 10.2 mm at 50.0mg/ml concentration. c o n c l u s i o n the plant extracts under study showed antimicrobial activity when used against the test strains e. coli, salmonella, and malassezia species. these plant leaf extracts can be used in the formulation of new drugs used against bacterial and fungal strains. e t h i c a l a p r o v a l none. c o n f l i c t o f i n t e r e s t none. f u n d i n g s o u r c e none. antibacterial and antifungal activities of a.modesta, a. saspara and s. surattense vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 201 a c k n o w l e d g e m e n t s none. l i s t o f a b b r e v i a t i o n s dmso dimethyl sulfoxide r e f e r e n c e s 1. kirbag s, zengin f, kursat m. antimicrobial activities of extract of some plants. pak j bot. 2009;41(40): 2067-2070. 2. tahir l, n khan. antibacterial potential of crud of leaf, fruit and flower extracts of tagetesminutal. j pub health and bio sci. 2012; 1: 70-74. 3. renisheya jj, malar t, johnson m, mary um, arthy a. antibacterial activities of ethanolic extracts of selected medicinal plants against human pathogens.asian pac j trop med. 2011; 1(1): 76-78. 4. pandey g, madhuri s. some medicinal plants as natural anticancer agents. review article. pharm rev. 2009; 3 (6): 259-263. 5. górniak i, bartoszewski r, króliczewski j. comprehensive review of antimicrobial activities of plant flavonoids. phytochem rev. 2019; 18: 241– 272. doi:10.1007/s11101-018-9591-z 6. nigussie d, davey g, tufa tb, brewster m, legesse ba, fekadu a and makonnen e. antibacterial and antifungal activities of ethiopian medicinal plants: a systematic review. front pharmacol. 2021;12: doi: 10.3389/fphar.2021.633921 7. hossain ma, al-raqmi ka, al-mijizy zh, weli am, al-riyami q. study of total phenol, flavonoids contents and phytochemical screening of various leaves crude extracts of locally grown thymus vulgaris. asian pac j trop biomed. 2013 sep; 3(9):705-10. 8. afzal mams. antibiotic resistance pattern of escherichia coli and klebsiella species in pakistan. j micro biochem technol. 2017; 9(6): 277-279 9. periyasamy a, rajkumar k, mahalingam. phytochemical screening and antimicrobial activity from five indian medicinal plants against human pathogens. middle-east j sci res. 2010; 5 (6): 477482. 10. asif m, jabeen q, atif m, majid amsa, zaman mu. diuretic activity of achyranthe saspara linn crude aqueous extract in albino rats. j pharm res. 2012; 13 (12): 2039-2045. 11. naidu pvl, kumar kk, kumar mc, gunesh g, rao nm. antimicrobial activity of achyranthe saspara. j. bio-sci. biotech res asia. 2006;3(1a): 170-174. 12. sharma ak, kumar a, yadav s, rahal a. studies on antimicrobial and immunomodulatory effects of hot aqueous extract of acacia nilotica leaves against common veterinary pathogens. vet med int. 2014. http://dx.doi.org/10.1155/2014/747042. 13. eltegani s. phytochemical screening and antibacterial activity of acacia nilotica. european j bio & pharmaceutical sci. 2017;4(8): 906-912. 14. kumar pr, muralidharan pn. evaluation of the antibacterial potential of solanum surattense against streptococcus mutans. 2015 3 (3): 273-275. 15. sheeba e, parvathy s, palanivel s. direct regeneration from leaves and nodes explants of phyasalis minima. eur j app sci. 2013; 2(2); 58-61. 16. khalid h, nisar mf, majeed a, khalid n, bhatti kh. ethnomedicinal survey for important plants of jalalpur jattan, district gujrat, punjab, pakistan. eth leaf 2010; 2010 (7) https://opensiuc.lib.siu.edu/ebl/vol2010/iss7/11 bacteriological analysis of lipsticks sahahat saeed1 and khizran asif 1 1department of microbiology, jinnah university of women, karachi pakistan. 21 rads vol 2 (1), (2011); 21-26 abstract microbial contamination of cosmetic products is a matter of great importance to the industry and it can become a major cause of both product a n d e c o n o m i c l o s s e s . m o r e o v e r. t h e contamination of cosmetics can result in their being converted into products hazardous for consumers. the objective of the study was to evaluate the degree of bacterial contamination in lipsticks. fifty eight (58) samples of used lipsticks were included in the study. each sample was inoculated on macconkey's agar and nutrient agar plates for the isolation of bacteria. twenty eight (28) bacterial strains were isolated belonging to 6 different species or gram positive bacteria viz.. bacillu.s. spp. (8; 28.6%). m i c ro c o c c u s s e d e n t a r i u s ( 6 ; 2 1 . 4 % ) , streptococcus spp. (1: 3.6%), staphylococcus saprophyticus (8:28%) s. aureus (2: 7.1%) and s. epidermidis (3: 10.7%). it was concluded that lipsticks often contains preservatives but some are still subject to microbial contamination. key words: lipsticks, bacillus subtilis, micrococcus sederaarius, streptococcus spp., staphylococcus aureus introduction according to the european commission, 1993 “cosmetics” have been defined as “any substance or preparation intended to be placed in contact with the various external parts of the human body (epidermis. hair system. nails, lips and external genital organs) or with the teeth and the mucous membranes of the oral cavity with a view exclusively or mainly to cleaning them, perfuming them, changing their appearance and/or correcting body odors and/or protecting them or keeping them in good conditions (pieroni cal al., 2004). microbial contamination of cosmetic products is of great importance to the industry. it not only cause economic loss rather also results in the conversion of cosmetics into hazardous products for consumers. presence of water and nutrients in cosmetics favours the growth of microorganisms, although very few cases of human injury have been reported due to c o n t a m i n a t e d c o s m e t i c s . f u r t h e r m o r e , microorganisms also cause alterations in organoleptic properties, such as offensive odors. and changes in viscosity and color (orus and leranoz, 2005). preservatives used in cosmetics should be effective enough to prevent the multiplication of microorganisms with in the product. complete sterility is not feasible but it should not contain viable human pathogenic bacteria or fungi or cosmetic products must be inhibitory to pathogenic and nonpathogenic microorganisms. actively viable microorganisms can be deleterious to both the esthetics and to the functional characteristics of cosmetic products. microorganisms can affect on color. odor, emulsion stability, foaming, and clarity of cosmetics. ideally, cosmetics should be selfsterilizing against all microbes encountered during production, packaging. and usage. during corresponding author. e-mail: sabahatsaeed2003@yahoo.com 22 rads vol 2 (1), (2011); 21-26 production common sources of microbial contamination in cosmetic products are raw materials, equipment and air. water for batchmaking can also be the major source of contamination, therefore, control over the sanitary quality of this water will be emphasized. under summer temperature storage conditions, demineralized or deionized water can easily support bacterial populations. in a few cases as many as 106 bacteria/m1 have been observed. to prevent gross pollution of the batch water supply, the propagation of micro flora coming from the undeionized water, therefore, deionizer units and the storage tanks must be controlled (olson, 1967). methods to detect microbial contamination in cosmetics and their raw materials are usually based on traditional plate counts (orus and leranoz. 2005). lipsticks often contain preservatives but some are still subject to mould 'blooms'. mould grows on the lipstick while it is inside the lipstick case, often after the product has become moistened by breath during use (smart and spooner, 1972). keeping in view. the present study was undertaken to evaluate the degree of bacterial contamination in lipsticks. material and methods samples fifty eight (58) samples of used lipsticks were included in the study. collection of samples a sterile cotton swab was rotated and rubbed over the surface of each lipstick and then rotated and subjected to qualitative analysis. media for primary isolation nutrient agar medium (oxoid) and macconkey's agar medium (oxoid) were used for primary isolation. inoculation each swab was streaked onto the surfaces of 1 nutrient agar and 1 macconkey agar. incubation inoculated plates were incubated for 24 h at 37°c. maintenance of cultures after incubation, different types of colonies were picked and transferred to nutrient agar slants to get pure cultures. characterization and identification of organisms a l l p u r e c u l t u r e s w e r e s u b j e c t e d t o characterization by using different tests confirming to required standard diagnostic criteria (baron el al. 1994; cheesbrough. 2000). results a total of fifty eight (58) samples of used lipsticks were included in the present study. of these. 40 (69%) samples yielded no growth while only 18 (31%) samples yielded the growth of only gram-positive bacteria (table 1). table 1: lipstick samples positive for growth. the bacterial profile of eighteen lipstick samples is presented in table 2. total twenty eight (28) total number of lipsticks no growth growth 58 40 18 23 rads vol 2 (1), (2011); 21-26 bacterial strains were isolated belonging to 6 different species of gram positive bacteria viz.. bacillus spp. (8; 28.6%), micrococcus sederuarius (6; 21.4%). streptococcus. spp. (1: 3.6%), staphylococcus saprophyticus. (8: 28.6%). staphylococcus aureus (2; 7.1%), and staphylococcus epidermidis (3; 10.7%). table 2: bacteria isolated from lipsticks. discussion microbial spoilage of cosmetics is significant both from health and economic viewpoints and obviously should be prevented. it is clear that disease-causing organisms must be excluded, although some times it may be difficult to decide if an opportunistic pathogen will be troublesome in a specific product (smart and spooner. 1972). however. infections caused by contaminated cosmetics are relatively rare today, and the reported cases are all from hospitalized persons (lundov et al., 2009). in the present study, the aim was to study the lipstick samples that were used before and that have not an expiry date report in case of microbial contamination and preservative activity. fifty eight (58) samples of used lipsticks were included in the present study . the bacterial profile of eighteen lipstick samples is presented in table 2. total twenty eight (28) bacterial strains were isolated belonging to 6 different species of gram positive bacteria viz., bacillus spp., m. sedentarius,. streptococcus spp., s. saprophyticus, s.aureus, and s. epidermidis. in a past study, microbiological quality control of lipsticks has been investigated. in 81 samples, we found that 34 of them (42%) had total aerobic plate count and 19 of them (23.5%) were found to consist of mold and yeast which are not allowed by the cosmetic regulations. pathogen microorganisms such as s. aureus. p. aeruginosa. e. coli, salmonella spp. and shigella spp. were not detected from any of the samples (akin and altanlar. 1989). however, further literature about the bacteriological analysis of lipsticks is lacking. in the present study, bacillus spp. were found as predominant organism with an overall incidence rate of 28.6 % (table 1). most of the species of bacillus are air and soil contaminant: however, b. subtilis have also been reported to be involved in food poisoning (ostensvik et al., 2004). in addition to above mentioned gram-positive a species of micrococcus were also isolated in the present study. micrococcus species are commensal organisms colonizing the body surfaces of humans and are usually considered normal inhabitants of the skin (selladurai et a1., 1993). in the present study, among micrococcus species, m. sedentarius was observed with higher incidence rate i.e. 21.4% (table i ). they have been well recognized as o p p o r t u n i s t i c p a t h o g e n e s p e c i a l l y i n immunocompromised patients (yang et al., 2001) and have been reported to cause infections like endocarditis (old and mcneill. 1979). abscess (selladurai et al., 1993), localized cutaneous infections (folliculitis) (smith el al., 1985), meningitis (fosse et al., 1985), pneumonia (adang et al., 1992) and bacteremia (altuntas et al., 2004). organisms bacillus spp. micrococcus sedentarius streptococcus spp. staphylococcus saprophyticus staphylococcus aureus staphylococcus epidermidis total no. 08 06 01 08 02 03 28 % 28.6 21.4 3.6 28.6 7.1 10.7 100 24 rads vol 2 (1), (2011); 21-26 streptococci spp. were also isolated in the p r e s e n t s t u d y. h o w e v e r, t h e o v e r a l l incidence rate of streptococci was low i.e. 3.6% (table 1). streptococci are also associated with infective endocarditis (budzik and schneewind, 2006), orthopedic infections (arciola et al., 2007), neuro infections (benca et al., 2007), sepsis (maschieto et al., 2004), wound (heggers et al., 1998), genital infections (coque et al., 1995) and blood stream infections (routsi et al., 2000). the clinical significance of s. saprophyticus in urinary tract infection has been well documented in the literature (elmanama et al., 2006). in the present study. staphylococcus saprophyticus was also found as predominant organism with an overall incidence rate of 28.6% (table 1). s. epidermidis another coagulase negative staphylococcus. was isolated with the incidence rate of 10.7% (table 1 ). however, s. haemolyticus and s. saprophyticus are opportunistic bacterial pathogens that colonize human skin. beside s. saprophyticus and s. epidermidis, staphylococcus aureus a coagulase positive staphylococcus, was also isolated in the present study with the low incidence rate i.e. 7.1% (table 1). though s. aureus are the normal flora of the skin and mucous membranes (pour et al., 2007) their high incidence has clinical significance and they are considered wellrecognized pathogen. a number of studies have documented the clinical significance of s. aureus as a causative agent of urinary tract infections. s. aureus have also been reported to cause conjunctivitis (everitt et al., 2006). s. aureus can also cause bacteremia which may be complicated by endocarditis, matastatic infection or the sepsis syndrome (shurland et al., 2007). furthermore. s. aureus is also associated with toxic shock syndrome (naidu et at . 1991), fournier's gangrene, genital infections (kalorin and tobin. 2007), skin infections e.g. frunculosis (miller et al., 2007) and respiratory tract infections (yamaguchi et al., 2006). references adang,h.c. schouten. f.h. tiel and g.h. blijham. 1992. peumonia due to micrococcus spp. in a patient with acute myeloid leukaemia. lenkciniu,6(3): 224226. akin a. altanlar n. 1989. microbiological quality control of lipsticks which are ()11 the market in our country. mikrohiyol bul., 23(4): 369-378. altuntas, is.. o. yildiz.. b. eser. k. gundogan, b. sumerkan and m. cetin. 2004. catheter related bacteremia due to kocuria rosea in a patient 'undergone peripheral blood stem cell transplantation. bmc infect dis., 4: 62-65. arciola. c.r., d. campoccia. l. baldassarri, v. pirini, j. huebner and l. montanaro. 2007. the role of enterococcus facalis in orthopedic pert-implant infections demonstrated by automated ribotyping and cluster analysis. biomaterials. 26: 1528-1532. baron, e.j. l.r. peterson. and s.m. finegold. 1994. baily scotts diagnostic microbiology. 9th edition. the c.v. mosby company. pp: 333351. benca. a. ondrusova. m. huttova, r. rudinsky, p. kisac and f. bauer. 2007. netiroint-cctions due to enterococcus faecalls in children. neuro. enclocrihol. lett., 28(2): 37-33. budzik, j.m. and 0. schneewind. 2006. pili proue pertinent to enterococcal endocarditis. j . c l i n . i n v e s t . , 11 6 ( 1 0 ) : 2 5 8 2 2 5 8 4 . 25 rads vol 2 (1), (2011); 21-26 cheesbrough. m. 2000. district laboratory practice in tropical countries. part 1. pp. 370387. coque. t.m.. j.e. patterson. j.m. steckeeherg. and b.e. murray. 1995. incidence of hemolysin, gilatinase and aggregation substance among enterococci isolated from patients with endocarditis and other infection and from feces of hospitalized and community-based persons. j. infect. dis., 171: 1223-1229. elmanama. a.a.. n.m. elaiwa. a.e. el-ottol and f.h. ahu-elamreen. 2006. antibiotic resistance of uropathogens isolated from alshifa hospital in gaza strip in 2002. j. chemother., 18(3): 298-302. everitt, fla., p.s. little and w.f. smith. 2006. a randomized controlled trail of management strategies for acute infective conjunctivitis in g e n e r a l p r a c t i c e . b m j 3 3 3 : 3 2 1 3 2 3 . fosse. t., b. toga. y. peloux. c. -iranthil..1. bertrando and m. sethian. 1985. meningitis due to micrococcus luetus.. infection., 13(6): 280-281. heggers. .t.p., l,. mccoy. b. reisner, m. smith. p. edgar and r.j. ramirez. 1998. alternate antimicrobial therapy for vancomycin-resistant enterococci burn wound infections. burn car. rehabil., 19(5): 399-403. kalorin. c m. and eli. tobin. 2007. community associated methicillin and genital infections. j. urol., 177(3): 967-971. lundov, m.d., l. moeshy. c. zachariae and e.d. johansen. 2009. contamination versus preservation of cosmetics: a review on legislation. usage. infections. and contact allerg. contact dermatitis. 60: 70-78. maschieto, a., r. martinez. i.c. palazzo and a.l. darini. 2004. antimicrobial resistance of emerococcus sp. isolated from the intestinal tract of patients from a university hospital in brazil. mem. inst. om. cruz., 99(7): 763-767. miller, l.g.. c. quail. a. shay. k. mostafaie, k. bharadwa, n. i an. k. matayoshi. .j. cronin. .j. tan. g. tagudar and a.s. bayer. 2007. a prospective investigation of outcomes after hospital discharge for endemic, communityacquired methieillin resistant and susceptible staphylococcus aureus skin infection. clin. infect. dis., 44(4): 483-492. naidu a.s., .j. micdzobrodzki. musser. v.t. rosdahl. s.a. hedstrom and a. forsgren. 1991. human lactoferrin binding in clinical isolates of staphylococcus aurcus. the jaurual of medical microbiology., 34(6): 323-328. old. d.c. and g.p. mcneill. 1979. endocarditis due to micrococcus sedentarius in certac sedis. .j. clin. pathol., 32: 951-952. olson. s.w. 1967. the application of microbiology to cosmetic testing. j. soc. cosmetic 18: 191-198. orus, p. and s. leranoz. 2005. current trends in cosmetic microbiology. international microbiology, 8: 77-79. ostensvik. o., c. from b. heidenreich, k. o'sullivan and p.e. granum. 2004. cytotoxic bacillus spp. belonging to the b. cereus and b. subtili.ss groups in norwegian surface waters. journal of applied microbiology., 96(5): 987993. pieroni, a.. c.l. quave. m.l. villanelli, p. mangino, g. sabbatini, l. santini, t, boccetti, m. profili. t. ciccioli, l.g. rampa, g. antonini, 26 rads vol 2 (1), (2011); 21-26 c. girolamini, m. cecchi and m. tomasi. 2004. [thnopharmacognostic survey on the natural i n g r e d i e n t s u s e d i n l o l l : c o s m e t i c s , cosmeceuticals and remedies for healing skin diseases in the inland marches, central-eastern italy. journal of ethnopharmaciology., 91: 331344. pour. f.z.. s. miri. r. ghaseni. r. farid and j. ghenaat. 2007. skin colonization with staphylococcus aureus in patients with atopin dermatitis. the internet journal of dermatology., 5(11): 23-28. routsi, c., e. platsouka, o. paniara, e. dimitriadou, g. saroglou, c. roussos and a. arinaganidis. 2000. enterococcal infections in a greek intensive care unit: a 5-y study. scar d. j. inject. dis., 32(3): 275-280. selladurai. b.m., sikakumaran, s. aiyar and a.r. mohammad. 1993. intracranial suppuration caused by micrococcus luteus. br. j neurosurg., 7(2): 205-207. shurland. s.. m. zhan, d.d. bradham and m.c. roghmann. 2007. comparison of mortality risk associated with bacteriuria due to methicillinr e s i s t a n t a n d m e t h i c i l l i n s u s c e p t i b l e staphylococcus aureus. inject. control hasp. epiclemiol., 28(3): 273-279. s m a r t . s . a n d d . f. s p o o n e r. 1 9 7 2 microbiological spoilage in pharmaceuticals and cosmetics. j. soc. cosmetic chemist, 23: 721-737. smith, a.. r. neafie, s. yeager and s. skelton. 1985. micrococcus folliculitis in hiv-1 diseare. b r i t i s h . d e r m a t o l . , 1 4 1 ( 3 ) : 5 5 8 5 6 1 . yamaguchi. k.. a. ohno. and y. ishii. 2006. in vitro susceptibilities to levofloxacin and various antibacterial agents of 18, 639 clinical isolates obtained from 77 centers in 2004. jpn.antibiot., 59(6): 428-451. yang. s., s. sugawarna. t. monodane, m. nishijina, y. adachi. s. akashi. k. miyake. s. hare and h. takada. 2001. micrococcus !welts teichuronic acid activate human and murine monocytic cells in a cd14 – and toll-like receptor 4 dependent manner. infect. immune., 69(4): 2025-2030. effect of zinc on lentil (lens culinaris l.) metabolites and antioxidant enzyme activities. saima ibrahim1*, sidra shabbir1 1department of botany, jinnah university for women, karachi 74600, pakistan. abstract in the given study, the toxic effect of zinc (zn) was studied with special attention being given to the biochemical response of lens culinaris l., exposed to zinc toxicity at different concentration levels. the research carried out in a randomized complete block design with 3 replications of each treatment. the major change was observed in the production and accumulation of primary metabolites (i.e. protein and protease) and activity of antioxidant enzymes i.e. catalase (cat) and acid phosphatase (aps). the data showed that the low concentration of zn addition support soluble protein accumulation in lentil leaves, although protease activity greatly arrested the protein content and appeared as a negative factor with increasing zn levels. the response of antioxidant enzymes was also dose dependant, it increases their activity to suppress oxidative stress produced by heavy metal but at high dose they become ineffective due to increase in oxidation process controlled by metal application. the peak of activity appeared at 4ppm to 8ppm zn.a keywords: antioxidant enzymes, acid phosphatase (aps), catalase (cat), zinc toxicity. introduction zinc is an essential micronutrient required for optimum crop growth. numerous researches were conducted on zinc. nand & ram (1996) and selvi & ramaswami (1995) observed that, at higher concentrations zn leads to physiological and morphological disturbances and, eventually to decreased crop yield. higher concentration of zn in the plant tissue seriously affects activity of several enzymes and other fundamental metabolic processes. an excess of zn also reduced photosynthetic rate as a part of enzymes concerned in the photosynthesis. a toxic concentration of zn in plant tissue seriously affects activity of several enzymes and other fundamental metabolic processes. ali et al., (2000) carried out a broad study and affirmed that an excess of zn also reduced photosynthetic rate as a part of enzymes concerned in the photosynthesis. nitrogen metabolism is also affected in diverse ways by an excess of zn. the protein content is found to be reduced; nitrogen-fixation and nitrate reductase activity was also concealed by zn toxicity (phalson, 1989). excessive zn in plants can profoundly affect normal ionic homeostatic systems by interfering with the uptake, transport, osmotic and regulation of essential ions and results in the disruption of metabolic processes such as transpiration, photosynthesis and enzyme activities related to metabolism (broadley et al., 2007; abbas et al., 2009). zn phytotoxicity also induces oxidative stress by generating free radicals and reactive oxygen species (ros) (weckx & clijsters, 1997). these ros are highly reactive and cause the death of plants by damaging membrane lipids, proteins, pigments and nucleic acids. to cope up with the damages caused by the ros, cells possess their own comprehensive and integrated endogenous antioxidant defense system composed of both enzymatic as well as non-enzymatic components (miller et al., 2008). superoxide dismutase (sod),*corresponding author: ibrahim4sept@hotmail.com 20 vol 5 (1), january 2014; 20-24 peroxidase (pod) and catalase (cat) represent the endogenous defense of plant cells. these enzymes are present in different isoforms in several cell compartments and their expression is genetically controlled and regulated both by developmental environmental stimuli, according to the necessity to remove ros produced in cells (mittler et al., 2004). it is also well documented that flavonoids and polyphenols are natural antioxidants. flavonoids can directly react with superoxide anions and lipid peroxyl radical and consequently inhibit or break the chain of lipid peroxidation. this radical scavenging activity of extracts could be related to the antioxidant nature of polyphenols or flavonoids, thus contributing to their electron/hydrogen donating ability. plant phenolic compounds also participated in the defence system of plant against oxidative stress. the antioxidant action of phenolic compounds is due to their high tendency to chelate metals. phenolics possess hydroxyl and carboxyl groups, with ability to bind particularly fe+2 and cu+2 (jung et al., 2003). the roots of many plants exposed to heavy metals exude high levels of phenolics (wink elshirl, 2002). they may inactivate iron ions by chelating and additionally suppressing the superoxidedriven fenton reaction, which is believed to be the most important source of ros (arora et al., 1998). this general chelating ability of phenolic compounds is probably related to the high nucleophilic character of the aromatic rings rather than to specific chelating groups within the molecule (morgan et al., 1997). an enhancement of phenylopropanoid metabolism and the amount of phenolic compounds can be observed under different environmental factors and stress conditions (sakihama et al., 2002). the induction of phenolic compound biosynthesis was observed in wheat in response to ni+2 toxicity (diáz et al., 2001) and in maize in response to al+2 (wink el-shirl, 2002). phaseolus vulgaris exposed to cd+2 accumulate soluble and insoluble phenolics. similarly, an increase in soluble phenolics, such as intermediates in lignin biosynthesis can reflect the typical anatomical change induced by stressors: increase in cell wall endurance and the creation of physical barriers preventing response against harmful action of heavy metals (diáz et al., 2001). materials and methods the present research was to investigate the alleviating effect of different zn concentrations on growth inhibition and oxidative stress in lens culinaris l. (lentil). the experiments were carried out using plastic pots with holes at the bottom to facilitate water percolation. the zn was added to the soil at concentrations of 0 (control), 2 mg/kg (t1), 4mg/kg (t2), 6mg/kg (t3), 8mg/kg (t4), and 10mg/kg (t5). each treatment was replicated three times. ten seeds of lentil (lens culinaris) were sown per pot separately. the experiment was carried out in a greenhouse with a 14 h (26°c)/10 h (13°c) day/night cycle. soil water content was adjusted regularly. the plants were harvested after 21 days of treatment and subjected to the analysis of physiological and biochemical test to check the toxic effect of zn on primary, secondary metabolites and antioxidant contents of plant. total protein was estimated by bradford method (1976) respectively. protease activity was determined by the method of mcdonald and chen (1965). some important antioxidant enzymes assay including cat and aps, and secondary metabolites were also performed to investigate the oxidative stress produced in plants under zn alleviated concentration stress. cat activity was assayed by using method of maehly & chance (1959). activity of acid phosphatase (ap) was determined according to mcdonald and chen (1965). while, total phenolics estimation was done by methods used by ainsworth & gillespie (2007). flavanoid estimation was done by adapting methods from woisky, r. and salatino, a. (1998). results and discussion zinc effect on total protein and protease activity: table -1 showed that, with an elevated zn concentration, there was an increased in protease activity and decreased protein content was found in lentil crops (fig-1). similar results are reported by 21 vol 5 (1), january 2014; 20-24 barcelo et al., (1985). decrease in protein related with the heavy metal effect on protein accumulation and production. kastori et al., (1992) also reported in helianthus annus that content of soluble proteins decreases with high concentration of heavy metals. protein content under heavy metal influence may be affected due to enhanced protein hydrolysis (proteolysis) resulting in decreased concentration of soluble proteins (melnichuk et al., 1982). zinc effect on antioxidant enzymes activity: in the present research work, activity of antioxidant enzymes increased with zn supply up to certain level i.e. 4-6ppm, and also excess of zn produced oxidative stress in plants. antioxidant enzymes may alter the h2o2 to the h2o in the plant cells and counteract the toxicity effect of h2o2 (rezai and farboodnia, 2008). hence to shield cells against oxidative stress, antioxidant enzymes augmented proportionally, which is also consistent with our results (fig.2). rate of pod was highest among three antioxidant enzymes, followed by cat and aps at all treatments. the given data revealed the decreased activities of antioxidative enzymes in studied crop under high zn stress. maximum increase in lipid peroxidation was observed in plants treated with 6ppm zn as compared to non treated seedlings. similarly, the activity of cat was also increased by the treatment with 8ppm zn. catalase activity: cat is one of the major antioxidant enzymes that play a very important role in the protection against oxidative damage by breaking down hydrogen peroxide and plays an important role in plant defense, aging and senescence (mittler et al., 2004). the cat activities in shoot significantly increased with the zinc concentrations. the induction of this enzyme under zinc stress indicated that it helps in inhibiting the oxygen radical accumulation. similar to the present study, an increase in cat activity has been reported in other plant species exposed to zinc stress (prasad et al., 1999; mcgeer et al., 2000). our findings provide the evidence that cat may provide an additional protection against the oxidative damage induced by zinc stress. a gradual decline in cat activity in lentil plants grown under excess zn stress i.e. above 8ppm was observed. maximum cat activity (2.56 mm h2o2 min-1 mg22 vol 5 (1), january 2014; 20-24 figure 1: effect of zn on primary metabolites of masoor (lens culinaris) table-i: effect of zn on primary metabolite of lentil (lens culinaris) table-ii: effect of zn on antioxidant enzymes activity of lentil (lens culinaris) figure 2: effect of zn on antioxidant enzymes of masoor (lens culinaris) 1 protein) was observed in plant treated with 8ppm zn as compared to control plants (0.46 mm h2o2 min-1 mg-1 protein). this finding matches with the work of andrade et al., (2009). the decrease in cat activity observed in plant supplemented with excess zn might be due to inhibition of enzyme synthesis or a change in the assembly of enzyme subunits (radic et al., 2010). acid phosphatase (aps) activity: aps is the most important peroxidase in h2o2 detoxification operating both in cytosol and chloroplasts (mittova et al., 2000). initially it increase at 6ppm zn (p<0.005) in lens plant and then gradually decrease with the elevated concentration of the zn. increas in the acid phosphatase activity with high zn treatments might be due to the decline of phosphate (p) level in the cell of p starvation and that intracellular and extracellular acid phosphatases are integral components of plants response to p deficiency (duff et al., 1993). conclusion in conclusion, even though zn is an essential micronutrient for plants, higher zn concentrations (above 6mg/kg) reduced plant growth and induced oxidative damage. moderate zn supplementation (2mg/kg to 4mg/kg) played an important role in protecting plants from oxidative stress induced by excess zn exposure. although these antioxidant enzymes showed different patterns of activities exposed to zinc toxicity, the total activity of these enzymes were significantly enhanced, which reflect an increased degree of oxidative stress. our findings suggested that the defensive system of plant regulated the changes of enzyme activities in order to enhance the defensive function against excessive zinc. references abbas, g., m.q. khan, m. jamil, m. tahir and f. hussain, 2009. nutrient uptake, growth and yield of wheat (triticum aestivum) as affected by zinc application rates. int. j. agric. biol., 11: 389–396 ainsworth ea, gillespie km 2007. estimation of total phenolic content and other oxidation substrates in plant tissues using folin–ciocalteu reagent. nature protocols;2:875-877. ali g, srivastava ps, iqbal m. influence of cadmium and zn on growth and photosynthesis of bacopa monniera cultivated in vitro. biol. pl. 2000; 43:59901. andrade s.a., gratão p.l., schiavinato m.a., azevedo r.a., mazzafera p. 2009. zn uptake, physiological response and stress attenuation in mycorrhizal jack bean growing in soil with increasing zn concentrations. chemosphere, 75: 1363–1370 arora a., nair m.g., stras bur g g.m. 1998, structure-activity relationships for antioxidant activities of a series of flavonoids in a liposomal system. free radic. biol. med. 24, 1355. barcelo, j., m. vazquez, cn. poschenriender,1988. structural and ultrastructural disorders in cadmiumtreated bush bean plants (phaseolus vulgaris l.). new phytol., 108, 37–49. bradford m.a.1976. arapid and sensitive method for the quan¬tification microgram quantities of protein utilizing the principle protein dye binding. a n a l y t i c a l b i o c h e m i s t r y, 7 2 : 2 4 8 – 2 5 4 . broadley, m.r., p.j. white, j.p. hammond, i. zelko and a. lux, 2007. zinc in plant. new phytol., 173: 677–702 diáz j., bernal a., po mar f., merino f. 2001. induction of shikimate dehydrogenase and peroxidase in pepper (capsicum annum l.) seedlings in response to copper stress and its relation to lignification. plant sci. 161, 179, jung c.h., maeder v., funk f., frey b., stich er h., fros erd e. 2003. release of phenols from lupinus albus l. roots exposed to cu and their possible role in cu detoxification. plant soil 252, 301. 23 vol 5 (1), january 2014; 20-24 maehly, a. c. & chance, b. (1954). meth. biochem. anal. 1, 357. mcdonald, c.e. and l.l. chen, 1965. lowry modification of the folin reagent for determination of proteinase activity. ann. biochem., 10: 175. mcgeer, j.c., c. szebedinszky, d.g. mcdonald and c.m. wood, 2000. effects of chronic sublethal exposure to waterborne cu, cd or zn in rainbow trout. 1: lono-regulatory disturbance and metabolic costs. aqua. toxicol., 50: 231–243 miller, g., v. shulaev and r. mitter, 2008. reactive oxygen signaling and abiotic stress. physiol. plant, 133: 481–489 mitova v., volokita m., guy m., tal m. activities of sod and the ascorbate-glutathione cycle enzymes in subcellular compartments in leaves and roots of the cultivated tomato and its wild salt-tolerant relative lycopersicon pennellii. physiol. plant. 110, 45, 2000. mittler, r., s. vanderauwera, m. gollery and f. van breusegem, 2004. reactive oxygen gene network of plants. trends plant sci., 9: 490– 498 morgan j.f., klucas r.v., gray er r.j., abian j., becana m. complexes of iron with phenolic compounds from soybean nodules and other legume tissues: peroxidant and antioxidant properties. free radic. biol. med. 22, 861, 1997. nand, r., and n. ram (1996). amelioration of zinc stress by farmyard manure in a rice-wheat-cowpea system. acta-agronomicahungarica, 44 (1):3539. pahlson a-m. 1989. toxicity of heavy metals (zn, cu, cd, pb) to vascular plants. water air soil pollut 47:287–319. prasad, k.v,paradha saradhi and p. sharmila.1999. concerted action of antioxidant enzymes & curtailed growth under zn toxicity in brassica juncea. environ. exp. bot., 42:1–10 sakihama y., ya masaki h, 2002. lipid peroxidation induces by phenolics in conjunction with aluminium ions. biol. plantarum 45, 249. selvi, s. and p.p. ramaswami (1995). residual effect of integrated nutrient management in rice-rice-pulse cropping sequence. madras-agricultural-journal, 82 (!):24-26. weckx jej, ciljsters hm (1997) zn phytotoxicity induces oxidative stress in primary leaves of phaseolus vulgaris l. plant physiol.biochem. 35:405410. wink el-shirley b. biosynthesis of flavonoids and effects of stress. curr. opin. plant biol. 5, 218, 2002. woisky, r. and salatino, a.1998. analysis of propolis: some parameters and procedures for chemical quality control. j. apic. res. 37: 99-105. 24 vol 5 (1), january 2014; 20-24 introduction the war against infections is one that has been remuneration all through the history of human. in this centuries-long effort the worldwide population has prepared remarkable advancement in stopping infections, therapeutic infections, and enlarging living expectancies. up till now for all our successes, only once in history 30 years ago, with smallpox made completely eliminate an infection from the earth. now a days we are on the entrance of eradicating the earth of polio an infection able of crippling and killing many children. the story of polio is both lengthy and hesitant. the arrival of efficient vaccines in the 1950s facilitated polio prevention, and the resultant struggles were victorious wildly, however confronts still continue (plan, 2011). abbreviations: pv (polio virus), vapp (vaccine associated paralytic poliomyelitis), cvdpv (circulating vaccine derived polio virus). the polio virus polio is a well-recognized infection and has its roots bottomless in the history of various cultures and civilization (global eradication initiative, 2010b; valtanen, et al. 2000). the causative agents of poliomyelitis is polio viruses, characterize into three serotypes in the genus enterovirus (family picornaviridae). the virions have a single-stranded, messenger-sense rna and 60 copies of each of the 4 capsid proteins vp1 to vp4 (mick, et al. 1999). the rna genome of poliovirus is about 7,500 nucleotides (nt) in length and has the polarization of mrna, therefore describing it as positive stranded (belov, et al. 2012) . the positive-sense genome rna of pv, which is surrounded by a protein capsid shell, be able to be translated into a large polyprotein in a host cell, and after that the large polyprotein is sliced by viral proteases into a dozen of different proteins (shen, et al.2012). the proteolytic procedure flow produces about 10 mature proteins and numerous intermediate products, several of which carry out their self-determining jobs in the life cycle of virus (belov, et al. 2012). symptoms of polio the majority of illnesses are unapparent (72% or more). a more 5–24% may have an insignificant disease by flu-like symptoms including headache, gastrointestinal disorders, malaise, neck and back stiffness. about 4% build up nonparalytic poliomyelitis, below 1% in total, and 0.5% of children having infection, comprise paralytic infection. paralysis is may be spinal (79%), bulbar (2%), or mixed spinal bulbar (19%) (mayer and neilson, 2010). pathogenecity pv briefly lives in the git (gastrointestinal tract). the virus go into the mouth and make copies in the pharynx and gastrointestinal cells, after that moves towards the blood stream through local lymphoid tissue, then move towards the cns (central nervous system) and replicate in the motor neurons of anterior the global polio eradication initiatives: from past to present shazia tabassum hakim1, sayyada ghuffrana nadeem2, shaista malik1, urooj javed2, abdul basit khan3 1virology and tissue culture laboratory, department of microbiology, jinnah university for women, karachi 2medical mycology research and reference laboratory, department of microbiology, jinnah university for women, karachi 3pcsir labs. complex, off university road, karachi *corresponding author: g-sarwaar@hotmail.com 28 vol 3 (2), july 2012; 28-34 horn cells of the spinal cord and brain stem, cells of the roof of the cerebellum and the motor cortex, having the reason of their demolition (mayer and neilson, 2010). direct neural increase of pv may also arise in certain circumstances, for example in tonsillectomy with following bulbar paralysis or subsequent injection of an annoying matter into a limb leading to following paralysis of that limb (robertson, 1993). pv disease yields in various alters to the host cell, and perhaps one of the mainly prominent is the huge buildup of cytosolic doublemembrane vesicles and these vesicles are the hallmark of autophagy, a degradative lane of homeostasis and pressure response. poliovirus specially persuades autophagic signaling, and virus construction associates to the level of autophagic activity in cells (richards and jackson, 2012). a site consist of amino acid 89 to 100 of vp1 is a main immunogenic place for type 2 and type 3 polioviruses, as evaluated by monoclonal antibodies produced in mice (robertson, 1993). subsequent natural exposure, igm and igg emerge in the serum about 7 to 10 days after disease. adequately elevated levels can block poliovirus e n t r a n c e i n t o t h e c n s ( c e n t r a l n e r v o u s system).originally, the igm response is 2to 8-fold larger than the igg response (robertson, 1993). diagnosis can be done on the basis of clinical, with demonstration by stool testing or serology (mayer and neilson, 2010). epidemiology infection with polio virus was general globally, with seasonal max outs and epidemics in summer and autumn in temperate regions (cdc, 2010; mayer and neilson, 2010). polio has been filed in history for thousands of years, preliminary with very old egyptian that ranges from 1580-1359 b.c. this infection was first analyzed as an epidemic in the united states and europe in the late 1800s. while polio has influenced people as ancient times, it was solitary earliest medically accounted in 1840. unfortunately, only fifty years later in 1890, epidemics start to occur and polio was a main concern for doctors and researchers. through that time, polio was habitually exclusively observed in children; that’s why it was often submitted to as childhood paralysis (valtanen, et al. 2000). in 1952, more than 21000 cases of paralytic poliomyelitis were filed in the united states (alexander, et al. 2004; cdc, 1981). the worldwide documentation of smallpox eradication in 1980 made significant attention in additional infectious disease eradication attempts. subsequent the quick development towards break off aboriginal wild poliovirus spread in the americas in the early 1980s, the worldwide polio eradication initiative was launched with a declaration of the world health assembly (wha) in 1988 (global eradication initiative, 2010a; aylward and tangermann, 2011), and has since developed to turn into one of the biggest internationally corresponding health plans in history (aylward and tangermann, 2011) (fig. 1). though, imported wild polioviruses from regions where polio is endemic have reason of epidemics in countries previously reported to be polio free and in which specific subpopulations either show a weak general immunity, such as in finland in 1984 and 1985 (hovi, et al.1986), or lack immunity, such as in the netherlands in 1978 (bijkerk, 1979) and 1992 (oostvogel, 1994)), bulgaria (who, 1992), and romania (strebel, et al.1994). the reemergence of poliomyelitis has also been reported in countries where political and financial alterations have made it hard to retain immunization plans, for example various areas of the previous soviet union in the 1990s (oblapenko and sutter, 1997; patriarca, et al. 1997; wassilak, et al. 1997; fiore, et al. 1998).in spite of the extraordinary development of the who plans for worldwide eradication of infection, endemic poliovirus spread still continues in a lot of asian countries also in pakistan. patients with acute infection of polio from all over the province of sindh in pakistan have been admitted to the civil hospital karachi, and since 1989 they have been followed clinically and observed virologically (huovilainen, et al. 1995). pakistan started polio virus eradication work in 1994 and has had significant achievement (cdc, 2002). these actions are performed with the 29 vol 3 (2), july 2012; 28-34 continuing world health organization (who) prolonged plans on immunization, which search for to vaccinate children against poliomyelitis, measles, diphtheria, pertussis, tuberculosis, tetanus, and hepatitis b (lowther, et al. 2005). through 2011, following setbacks that yield in cases being exported to new countries, 650 verified cases of poliomyelitis were accounted provisionally from 16 polioinfluenced countries: 4 countries where the infection was endemic and 12 countries with restored diffusion (lasting =12 months) or outbreaks (lasting <12 months) after importations (cdc, 2011d; cdc, 2011e; cdc, 2012b)(fig. 2) . in 2010 worldwide reporting of children with 3 doses of oral trivalent vaccine was approximately 85% but is uneven at the national and sub national levels (cdc, 2011d; hopkins, 2013). in january 2012, india commemorated a complete year with no cases of poliomyelitis, except nigeria, afghanistan, and pakistan as countries with endemic infection here eradication was difficult for the reason that of political unsteadiness or frighten about immunization (cdc, 2011c; cdc, 2011a; cdc, 2011b)(fig.3). in 2011 among countries with restored spread chad and the democratic republic of congo identified the majority of cases (132 and 93, respectively (cdc, 2012b). recently the polio virus has been discovered in sewage in egypt which was thought to be imported from pakistan because genetic analysis revealed that egyptian virus linked to one that was last seen in pakistan in september 2012 however egypt has been polio free since 2004 this is the second time that polio virus from pakistan has infected any country other than neighboring afghanistan; the first was china where a virus from pakistan made an outbreak in 2011(roberts, 2013). the aim is to break off spread of poliovirus types 1 and 3 by december 2012. the major challenges to poliomyelitis eradication are contributor tiredness; political unsteadiness in areas of afghanistan, pakistan, and some other influenced countries; public tiredness with again immunizations against pv alone; and weak regular immunization systems. this plan is expected to expenditure of $9.5 billion for the period from 1988 to 2013 (global polio eradication initiative, 2010d; hopkins, 2013) 30 vol 3 (2), july 2012; 28-34 wild poliovirus 2011 wild polio virus type 1 wild polio virus type 3 importation countries endemic countries excludes viruses detected from environmental surveillance and vaccine derived polioviruse. fig. 2: reported cases of poliomyelitis as of 2011. (polioeradication.org) fig. 1: who polio initiative the world health organization spearheads an international program to eradicate polio around the world. polio cripples an estimated 350,000 children that year. (cdc.gov) data in hq as of 08 january 2013 wild poliovirus 2012 01 january 31 december wild polio virus type 1 wild polio virus type 3 wild polio virus type 1/3 excludes viruses detected from environmental surveillance and vaccine derived polioviruse. 1 wpv 1 case in gilgit balitstan, date of onset 11 august 2012, does not appear on the map. endemic countries importation countries fig. 3: reported cases of poliomyelitis as of 2012. (polioeradication.org) eradication by polio vaccine there are two polio vaccines which are utilized all over the world to eradicate pv. inactivated polio vaccine (ipv) was the first vaccine produced in 1955 by dr jonas salk also called the “salk vaccine”. it comprises of inactivated (killed) poliovirus strains of all 3 poliovirus types (global polio eradication initiative, 2010b) in which viral infection has been inactivated by treatment of formaldehyde. as a result, the utilization of ipv does not have the threats of vapp or cvdpv outbreaks. incubation with formaldehyde partly alters the structure of antigen of pv however inactivated vaccines have been observed to defend powerfully against the infection and have been the only vaccine utilized to control and eradicate pv in several countries (martin, et al. 2013). ipv is administrated by intramuscular injection and requires to be managed by an educated health worker (global polio eradication initiative, 2010b). this polio vaccine generates antibodies in the blood to all 3 types of pv. in the incident of disease, these antibodies stop the growth of the virus to the central nervous system and defend against paralysis. ipv produces very small levels of immunity in the intestine therefore, when an individual immunized with ipv is infected with wild poliovirus, the virus be able to still reproduce within the intestines and be shed in the faeces. it is risky carry on distribution and is above five times extra costly than oral polio vaccine (global polio eradication initiative, 2010e). the oral polio vaccine (opv) was developed in 1961 by albert sabin. it is also called “trivalent oral polio vaccine” or “sabin vaccine”. this polio vaccine consists of a mixture of live, attenuated (weakened) poliovirus strains of all three poliovirus types. opv produces antibodies in the blood to all three types of poliovirus. in the event of infection, these antibodies protect against paralysis by preventing the spread of wild poliovirus to the nervous system. it also produces a local, mucosal immune response in the mucous membrane of the intestines therefore these mucosal antibodies bound the replication of the wild poliovirus inside the intestine. this intestinal immune response to opv is thought to be the main reason why mass campaigns with opv can rapidly stop person-to-person transmission of wild poliovirus (global polio eradication initiative, 2010c). on the other hand, a serious consequence of the use of this live-virus vaccine, vaccine-associated paralytic poliomyelitis (vapp), was recognized as early as 1962 (luther, 1962; henderson, 1964). from 1961 through 1989, an average of 9 cases of vapp (range, 1-25 cases) were confirmed each year (schonberger, et al.1976; nkowane, et al.1987; strebel, et al.1992; alexander, et al. 2004) (fig.4). in immunocompetent individuals, the risk of vapp is very low evaluated with that of immunodeficient patients (yang, et al. 2005). susceptible individuals usually excrete polioviruses for two to six weeks and occasionally for up to 137 days after they have been immunized with oral poliovirus vaccine (sutter, et al.2003). the shedded viruses commonly show increased neurovirulence and are frequently transmitted to close contacts. although immunodeficiencies are listed as a contraindication for receiving opv, patients with these clinical conditions may sporadically receive the poliovirus live vaccine before their immunodeficiency is diagnosed and/or may be infected with opv strains excreted by a vaccinee or that is present in the community (galal, et al.2012). the duration and extent of spread are dependent on the magnitude of the immunity gap and the intensity of other risk factors favoring poliovirus circulation. this long-discussed hypothetical concern (fine and carneiro, 1999) has been realized by the recent occurrence of outbreaks of paralytic polio associated with circulating vdpvs (cvdpvs). though several important themes are common to all of the outbreaks, each outbreak has taught its own important lesson about the parameters for the safe 31 vol 3 (2), july 2012; 28-34 14 12 10 8 6 4 2 0 1980 1985 1990 1995 2000 2005 vapp imported c a s e s fig. 4 poliomyelitis united states, 1980-2009 administration of opv in a world free of circulating wild polioviruses (kew, et al. 2004). developed or rich countries are replacing opv with ipv to eliminate vapp. currently, 22 countries are using ipv exclusively and eight more have a sequential schedule of ipv and opv (d. wood, personal communication 2003). this situation has begun to evolve as rich–poor disparity. global public health leaders are divided on the acceptability of vapp in developing countries. some recognize the double standard, as developing countries will be exposed to a risk that the industrialized nations will avoid (nathanson and fine, 2002). conclusion polio is one of the diseases that can be eradicated. eradication is more than just bringing the number of cases to zero. eradicating polio means that polioviruses will be wiped off the face of the earth and that vaccination will no longer be necessary (who, 1999). in present we are facing polio endemic in developing and poor countries which include pakistan, afghanistan, chad, ethiopia, dr congo and nigeria because of large populations, high birth rates, overcrowding, lack of education, poor sanitary condition, poorly functioning immunization systems and various controversial rumors about vaccine. it is vital to finish polio eradication in pakistan, for the health of the nation, and for the whole global community. the highest levels of government have committed to finishing this job as a national responsibility (global polio eradication initiative, 2010f). so this is the time to educate the people about vaccine. all we need is to immunize every infant with the oral vaccine to eradicate this endemic and make the globe free of this infection. if the eradication initiative stops prematurely, the disease will return with a vengeance. references alexander ln, seward jf, santibanez ta, pallansch ma, kew om, prevots dr, strebel pm, et al. 2004. vaccine policy changes and epidemiology of poliomyelitis in the united states. jama, 292(14): 1 6 9 6 – 7 0 1 . d o i : 1 0 . 1 0 0 1 / j a m a . 2 9 2 . 1 4 . 1 6 9 6 . aylward b and tangermann r. 2011. the global polio eradication initiative: lessons learned and prospects for s u c c e s s . va c c i n e , 2 9 s u p p l 4 , d 8 0 – 8 5 . doi:10.1016/j.vaccine.2011.10.005. belov ga, nair v, hansen bt, hoyt fh, fischer er, ehrenfeld e. 2012. complex dynamic development of poliovirus membranous replication complexes. j virol, 86(1): 302–312. bijkerk h. 1979. poliomyelitis in the netherlands. dev. biol. stand., 43:195– 206. cdc. 1981. annual summary: reported morbidity and mortality in the united states. atlanta, ga: centers for disease control. cdc. 2002. progress toward poliomyelitis eradication— pakistan and afghanistan, january 2000–april 2002. mmwr, 51(24):523–4. cdc. 2010. travelers’ health: yellow book. in: cdc health information for international travel 2010. atlanta: u.s. department of health and human services, public health service, 2009. cdc. 2011(a). progress toward poliomyelitis elimination — nigeria, january 2010–june 2011. mmwr, 60: 1053-7. cdc. 2011(b). progress toward poliomyelitis eradication — afghanistan and pakistan, january 2010–september 2011. mmwr, 60:1523-7. cdc. 2011(c). progress toward poliomyelitis eradication — india, january 2010 – september 2011. mmwr, 60:1482-6. cdc. 2011(d). progress towards interrupting wild poliovirus transmission worldwide: january 2010–march 2011. wkly. epidemiol. rec., 86:199204. cdc. 2011(e). tracking progress toward global polio eradication — worldwide, 2009–2010. mmwr, 60: 32 vol 3 (2), july 2012; 28-34 441-5. cdc. 2012(a). poliomyelitis: epidemiology and prevention of vaccine-preventable diseases (pink book). available from: cdc. 2012(b). progress towards global interruption of wild poliovirus transmission, january 2011–march 2 0 1 2 . w k l y. e p i d e m i o l . r e c . , 8 7 : 1 9 5 2 0 0 . fine pem and carneiro iam.1999. transmissibility and persistence of oral polio vaccine viruses: implications for the global poliomyelitis eradication initiative. amer j epidemiol, 150:1001-1021. fiore l, genovese d, diamanti e, catone s, ridolfi b, avoort hg, et al. 1998. antigenic and molecular characterization of wild type 1 poliovirus causing outbreaks of poliomyelitis in albania and neighboring countries in 1996. j clin microbiol, 36 (7): 1912-1918. galal nm, bassiouny l, nasr e and abdelmeguid n. 2012. isolation of poliovirus shedding following vaccination in children with antibody deficiency disorders. j infect dev ctries, 6(12):881-885. global polio eradication initiative. 2010 (a). global eradication of poliomyelitis by the year 2000 : world health assembly resolution 41.28. available from: global polio eradication initiative. 2010 (b). inactivated polio vaccine (ipv). available from:. global polio eradication initiative. 2010 (c). polio eradication. available from: global polio eradication initiative. 2010 (d). financial resource requirements 2012–2013. available from: . global polio eradication initiative. 2010 (f). augmented national emergency action plan for polio eradication 2012, available from:. global polio eradication initiative. 2010 (e). oral polio vaccine (opv). available from: . henderson da, witte jj, morris l, langmuir ad.1964. paralytic disease associated with oral polio vaccines. jama, 190:41-48. hopkins, d. r. 2013. disease eradication. new engl j medi, 368(1): 54–63. doi:10.1056/nejmra1200391. hovi t, cantell k, huovilainen a, kinnunen e, kuronen t, lapinleimu k, et al. 1986. outbreak of paralytic poliomyelitis in finland: widespread circulation of antigenically altered poliovirus type 3 in a vaccinated population. lancet i: 1427–1432. huovilainen a, mulders m, agboatwalla m, tuija p, stenvik m and tapani h.1995. genetic divergence of poliovirus strains isolated in the karachi region of pakistan, j gen virol, 76: 3075-3088. kew om, wright pf, agol vi, delpeyroux f, shimizu h, nathanson n & pallansch ma. 2004. circulating vaccine-derived polioviruses: current state of knowledge/ bull world health organ, 82(1): 16 – 23. lowther sa, mir t, bile mk, abdul hafiz r & mounts aw. 2005. characteristics of districts in pakistan with persistent transmission of wild poliovirus, 2000-2001. east mediterr health j, 10(4-5): 582–590. luther t.1962. the association of cases of poliomyelitis with the use of type iii oral polio vaccines. washington, dc: us dept of health, education, and welfare: 1-8. martin j, crossland g,wood dj and minor pd. 2003. characterization of formaldehyde-inactivated poliovirus preparations made from live-attenuated strains. j gen virol, 84: 1781–1788. mayer ca and neilson aa. 2010. poliomyelitis prevention in travelers. aus fam physician, 39 (3): 122 – 125. mulders m, reimerink j, stenvik m, alaeddinoglu i, van der avoort hg, hovil t, et al. 1999. a sabin vaccine-derived field isolate of poliovirus type 1displaying aberrant phenotypic and genetic 33 vol 3 (2), july 2012; 28-34 34 vol 3 (2), july 2012; 28-34 features,including a deletion in antigenic site 1. j gen virol, 80: 907–916. nathanson n and fine p. 2002.poliomyelitis eradication – a dangerous endgame. science, 296:269-70. nkowane bm, wassilak sg, orenstein wa, et al. 1987. vaccine-associated paralytic poliomyelitis. jama, 257:1335-1340. oblapenko, g., and r. w. sutter. 1997. status of poliomyelitis eradication in europe and in central asian republics of former soviet union. j infect dis, 175(suppl.1):s576–s581. oostvogel pm., van wyngarden jk, van der avoort hg, mulders m, spaedonck m, rumke hc, et al. 1994. poliomyelitis outbreak in unvaccinated community in the netherlands, 1992–93. lancet, 344:665–670. patriarca pa, sutter rw, and oostvogel pm. 1997. outbreaks of paralytic poliomyelitis, 1976–1995. j infect dis, 175(suppl.1):s165–s172. plan, s. 2011. polio eradication. 1–12. richards al and jackson wt. 2012. intracellular vesicle acidification promotes maturation of infectious poliovirus particles. plos pathog, 8(11): e1003046. doi:10.1371/journal.ppat.1003046 roberts l. 2013. polio virus spread from pakistan to egypt. science aaas. available from: robertson, s. 1993. the immunological basis for immunization series.êmodule 6: poliomyelitis.êworld h e a l t h o rg a n i z a t i o n . g e n e v a , s w i t z e r l a n d . schonberger lb, mcgowan je, gregg mb.1976. vac cine-associated poliomyelitis in the united states, 1 9 6 1 1 9 7 2 . a m j e p i d e m i o l , 1 0 4 : 2 0 2 2 11 . shen h, sun h, li g. 2012. what is the role of motif d in the nucleotide incorporation catalyzed by the rna-dependent rna polymerase from poliovirus? p l o s c o m p u t b i o l , 8 ( 1 2 ) : e 1 0 0 2 8 5 1 . doi:10.1371/journal.pcbi.1002851 strebel pm, aubert-combiescu a, ion-nedelcu n, biberi-moroeanu s, combiescu m, sutter rw,et al. 1994. paralytic poliomyelitis in romania, 1984–1992: evidence for a high risk of vaccine-associated disease and reintroduction of wild-virus infection. am j epidemiol, 140: 1111–1124. strebel pm, sutter rw, cochi sl, et al.1992. epidemiology of poliomyelitis in the united states one decade after the last reported case of indigenous wild virus associated disease. clin infect dis, 14:568-579. sutter rw, kew om, cochi sl. 2003. poliovirus vaccine. in: plotkin sa and orenstein wa, editors.vaccines.4th ed. philadelphia: w.b. saunders company. 651-705. valtanen s, roivainen m, piirainen l, stenvik m, and hovi t. 2000. poliovirus-specific intestinal antibody responses coincide with decline of poliovirus excretion.êj infect dis,ê182: 1-5. wassilak s, oblapenko g, dittmann s. 1997. progress in europe towards the goal of poliomyelitis eradication. eurosurveillance, 2:39–41. world health organization (who). 1992. expanded program on immunization. poliomyelitis outbreak, bulgaria. weekly epidemiol. rec. 67:336–337. world health organization (who). 1999. a practical guide with polio eradication as a case study. available from:. yang c, chen h, jorba j , sun hc, yang sj, lee hc, et al. 2005. intratypic recombination among lineages of type 1 vaccine derived poliovirus emerging during chronic infection of an immunodeficient patient. j virol 79: 12623-12634. “rads” publishes articles within the whole field of physical science. generally these articles will be in or related to need of the country. manuscripts or data will be considered that have not been previously published or submitted elsewhere for publication. however, re-analysis of previously published data can be accepted. 1. all articles submitted for publication will be reviewed by referees. submit three copies of the article / manuscripts along with a floppy d i s k e t t e ( i b m c o m p u t e r ) c o n t a i n i n g t h e manuscript in duplicate. while submitting diskette 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in which the article appeared (in abbreviated form), journal’s volume,number and finally first and the last page number such as carling de & brown mf. 1980. relative effect of vesicular arbuscular mycorrhizal fungi on growth and yields of soybeans. soil sci. soc. amer. j., 44: 528-532. 5. the manuscript should be accompanied by a covering letter signed by corresponding authors. instruction to authors a study of bacterial profiling on currency notes and coins vol 8 (1), jan 2017 issn: 2305 8722 1 r a d s j . b i o l . r e s . a p p l . s c i 1 op e n ac c e ss f u l l l e n g t h a r t i c l e a study of bacterial profiling on currency notes and coins asra qayyum1 & hira batool1* 1 department of microbiology, jinnah university for women a b s t r a c t paper currency notes and coins act as fomite, containing microorganism on their surface which is transferred from one person to another. currency notes and coins are heavily contaminated by microbes, playing a chief role in the spread of microorganisms which include normal saprophytic micro flora to pathogenic microorganisms. accordingly, several studies demonstrated the fact that currency notes has been identified as one of the principal vehicle that convey pathogens which lead to specific health hazard to community. a number of sources that may contribute to currency contamination include counting machine, atmosphere, during storage, usage, managing and production. for the undertaken research, a total of 12 samples were collected from different sources from public sector. microbial analysis was performed by serial dilution conducted in tsb broth and the inoculated cultures transferred into the selective agar medium. isolation and identification of bacteria was performed by gram staining, colonial morphology and biochemical characteristics. the current study revealed that the frequency of microorganisms recovered from currency as 66.66% e.coli, 33.33% shigella spp and 100% was s.aureus. antimicrobial susceptibility test performed for the confirmation of bacterial isolates. these all isolates are associated with potential infection. currency notes and coins identified as a prospective health danger because pathogen would spread through circulation of notes and coins. by the help of personal hygiene standards we can reduce the risk of potential bacterial infection. keywords bacterial profiling, currency, health hazard address of correspondence hb_heer@hotmail.com article info. received: april 3, 2017 accepted: may 29, 2017 cite this article: qayyum a, battol h. a study of bacterial profiling on currency notes and coins. rads j. biol. res. appl. sci 8(1):1-5. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n pathogenic microorganisms are agents that attack the body and alter the immune system (1). they are prokaryotic and unicellular, causing serious lifethreatening infections (2) microorganisms are ubiquitous in nature found in soil, radioactive waste, water, biomass and even organic matter. the habitat of microorganism the bodies of life forms, like plants and animals and also exist as a normal flora in the human body, and thrive on the skin and within the digestive tract. they also play a vital role in recycling of nutrients. while a body’s immune system contribute to the elimination of these bacteria (3). microorganisms that are present in air, water, and food etc., are easily spreading from one person to another. the most common mechanism of the spread of pathogens is by fomites (4). fomites or insentient objects play a major role in indirect spread of infections like diphtheria, trachoma, gastroenteritis, whooping cough and diarrhea cause by pathogenic microorganism. currency contaminated by microbes might also represent as fomites, playing a key role in the communication of microorganisms (5). money is used as a medium for goods exchange, payment of debts and for overdue expenses in financial activities. a number of sources that may contribute to currency contamination include; counting machine, the atmosphere, in course of storage, usage, managing or production. (6). currency notes could o r i g i n a l a r t i c l e a study of bacterial profiling on currency notes and coins vol 8 (1), jan 2017 issn: 2305 8722 2 r a d s j . b i o l . r e s . a p p l . s c i 2 be contaminated by air droplets during coughing, sneezing, touching with previously contaminated hands or other components. people when they count the money they usually tongue-wet the currency notes for convenience which thereby contaminate their hands as well as currency notes. so, it is obvious that anything that gets on hands may be transferred to money and viceversa (7). currency notes are often handled by various groups of individual during transaction (8). many disease causing microorganisms serve as likely sources of enteropathogens that might survive on the currency notes and to be the reason of infections and potential sporadic cases of food borne diseases (9). some studies showed that the bacterial survival on coinage and copper seems to be affected regarding their growth size. for this reason it has been concluded that coins begin to bear opportun¬istic bacterial pathogens, but contain very lesser amount of microorganisms (10). some previous studies show the occurrence of bacteria on bank notes and coins of a large number of developed countries. although the studies conducted from pakistan, united state, india, saudi arabia, nigeria, kenya, mexico, burma, china and turkey but few of all found significant amount of contamination on their currency. the major pathogens that are present on money are escherichia coli, vibrio spp., klebsiella. pneumonia , serratia spp., enterobacter spp., salmonella typhi, acinetobacter spp , enterococcus spp., staphylococcus aureus, bacillus spp., staphylococcus epidermidis, streptococcus pneumoniae, proteus spp., pseudomonas aeruginosa, shigella spp., corynebacterium spp., lactobacillus spp., burkholderia spp., micrococcus spp. and alcaligenes (12) . contact of a hand with a contaminated surface can effect in an inconsistent degree of pathogen transmission. nosocomial pathogens act as a reservoir in hospitals materials such as beds and keyboards that fall in contact with the person and may transmit these microorganisms and also serve as infectious agent for cross transmission (13). currency notes and coins are mainly carrying pathogenic microorganism on it. moreover, various fomites in the operation theater which is another source of direct or indirect association with surgical methods were found to be diversely contaminated with familiar microbial pathogens (14). all these pathogens associated with threatening infection ranging from skin infection to food born infection. however, because of the lack of statistical importance, occurrence of invalidate factors or lack of randomization, the disinfection and hygiene preventive studies regulate thus far could not establish a definitive underlying determinants. an important evaluation for preventing food-borne diseases is hygiene training for food handlers. many food markets greatly rely on the interchange of money for food. due to lack of appropriate hand hygiene that causing the condition uncertain. additionally, foodhandling tools can ease prevent cross-contamination present between currency and food through contact with the hands it might be probable to lessen the cross contamination by the training of workers, who cannot wash hands between venture. for that reason the reduction of food borne illnesses through the transmission of these pathogens on currency, regular vaccination of food handlers for hepatitis a virus infection can lessen the spread of food borne infection caused by hepatitis a virus (15). m a t e r i a l s a n d m e t h o d sample collection and preparation: the 12 samples of notes and coins are taken from different sources. the samples (coins and notes) were aseptically placed in to a sterile tube containing 10ml tsb broth to dislodge the micro organisms and then removed the notes and coins. serial dilution was made up to ten fold dilutions in tsb broth to obtain the discrete colonies. plating and culturing: 0.1ml was taken from 10-1 dilution and by using streak plate method inoculates the culture on nutrient agar, blood agar and macconkey’s agar, these plates were then incubated at 31 ºc for 24 hours. identification of microorganisms: the colonies were identified by microscopic examination and physical examinations by interpreting colony morphology and biochemical tests such as imvic, catalase and coagulase tests. a study of bacterial profiling on currency notes and coins vol 8 (1), jan 2017 issn: 2305 8722 3 r a d s j . b i o l . r e s . a p p l . s c i 3 r e s u l t s table i: the percentage of the isolated microorganisms form the currency notes and coins table ii: antibiotic susceptibility test for confirmation of organism in the present study, 12 samples are subjected to microbial analysis includes the culturing all the notes and coins obtained from various sources from public sector were contaminated with bacteria. three different species were isolated. escherichia coli were isolated from 8 samples as (66.66%), shigella spp from 4 samples as (33.33%) and staphylococcus aureus from 12 samples as (100%) shown in (figure 1) and (table i). the colonial morphology of e.coli on macconkey’s agar appear as pink color lactose fermenting colonies as shown in (figure 2) and non lactose fermenting colonies indicated the presences of shigella (figure 3). the growth on nutrient agar shows yellow pigmented pin pointed colonies and beta hemolysis on blood agar indicated the presence of s.aureus (figure 4). for the further confirmation, biochemical test were performed for gram negative bacteria include; tsi and cirtate and for s.aureus catalase and coagulase were performed. the tsi for e.coli interpreted as butt and slant (acidic) gas production is positive and h2s negative and citrate utilization test is also negative. the tsi of shigella spp interpreted as slant (alkaline), butt (acidic) and gas production and h2s negative and citrate utilization test is also negative. for s.auerus coagulase and catalase test is positive. the gram reaction of e.coli and shigella are gram negative rods and s.aureus are gram positive cocci in bunches. for the further confirmation of s.aureus. the antibiotic susceptibility test was performed on mha by using novabiocin. the zone of inhibition is 20mm and 22mm which indicated the presences of s.aurues because it is sensitive to novabiocin (table ii). fig 1: the microorganisms isolated from currency notes and coins fig 2: (lactose fermenting colonies of e.coli on macconkey agar ) fig 3: non-lactose fermenting colonies of shigella spp. on macconkey agar. organisms percentage shigella spp 33.33% e.coli 66.66% s.aureus 100% organisms zone of inhibition results s.aureus 20mm and 22mm sensitive a study of bacterial profiling on currency notes and coins vol 8 (1), jan 2017 issn: 2305 8722 4 r a d s j . b i o l . r e s . a p p l . s c i 4 fig 4: colonies of s.aureus are surrounded by a zone of beta hemolysis. d i s c u s s i o n the recent study was conducted in accordance to investigate the isolation & identification of bacterial pathogens on the circulation currency notes & coins. total of 12 samples were preceded for this purpose. isolation and identification of pathogenic bacteria i.e. s.aureus, e.coli & shigella was conducted. however, these bacteria are associated with potential and pathogenic diseases or infection. many people do not concern about their sullied fingers when they deal with money; the butcher with their hands contaminated with bloody fingers, the street food vendors with their wetly-oily fingers and others collect or hold the paper currency with contaminated hands, these all conditions can lead to the contamination of currency with pathogenic microorganisms. it is quiet common that individuals who handle the notes discharge some of their body’s normal flora on the currency note leading to the dissemination of microbes between the handlers. moreover, the contamination of the notes can be detected through dust, soil, water, micro flora of the different handlers (hand, skin, etc.). some people usually use their saliva when tally the notes. majority of people does not bring money in wallets and pinching of paper currency is common, especially among market women, motorcyclists, bus drivers and bus conductors, butchers and meat sellers, restaurant operators, etc. currency notes made up of cotton provide a fibrous surface, which bear great opportunity for bacterial attachment, and more long a currency notes stays in circulation, the more chances of contamination are there. on the other hand, the occurrence of a significant amount of copper in coined metal alloys appears to be restricting the bacterial survival on coins (11). currency notes and coins identified as a likely health hazard due to the spread of pathogen through circulation of notes and coins but because of public education on proper handling and caution of currency notes and coins are advocated in association to minimize currency contamination (15). c o n c l u s i o n it can be concluded that currency is potential source of contamination such as pathogenic bacteria and the people are getting microorganisms form the currency notes easily that causing the serious problems among people. awareness is highly recommended related to the improvement of personal hygiene and money handling practices to reduce the risk of infection. r e f e r e n c e s 1. madigan m; martinko. brock biology of microorganisms (13th ed.). pearson education, 2006. 2. rybicki e. the classification of organisms at the edge of life or problems with virus systematics. sou afr j sci. 1990;86(4):182-6. 3. lwoff a. the concept of virus. j. gen. microbiol. 1956;17 (2): 239–253. 4. barolia sk, verma s, verma bk. coliform contamination on different paper currency in ajmer, rajasthan, india. universal journal of environmental research and technology. 2011;1(4): 552-556. 5. sharma a, dhanashree b. screening of currency in circulation for bacterial contamination. current science. 2011;100(6). 6. neel r. multidrug resistance of isolates of methicillin resistant staphylococcus aureus (mrsa) in paper currency notes from restaurants and hotels in lusaka in zambia. int. j. pharm. sci. 2012;5(1): 363-366. 7. pal k, das ns, bhattacharya s. bacteriological profile of indian currency circulating in a tertiary care hospital in rural bengal. ijrrms. 2013; 3(2). 8. neel r. isolation of pathogenic microorganisms from contaminated paper currency notes in circulation from different market places in korogwe and mombo towns in tanzania. j. microbiol. biotech. res. 2012; 2 (3):470-474. 9. tagoe dna, adams l, kangah, vg. 2011. antibiotic resistant bacterial contamination of the ghanaian currency note: a potential health problem. j. microbiol. biotech. res. 2011;1(4):37-44. 10. espirito santo c, morais pv, grass g. isolation and characterization of bacteria currency & transmissible diseases resistant to metallic copper surfaces. appl. environ. microbial. 2010;76(5), 1341–1348. a study of bacterial profiling on currency notes and coins vol 8 (1), jan 2017 issn: 2305 8722 5 r a d s j . b i o l . r e s . a p p l . s c i 5 11. food science australia (fsa). money handling in food service operations. food safety and hygiene. a bulletin for the australian food industry, 2000. available from: http://www.foodscience.csiro.au/fshbull/fshbull20c.htm. 12. anderson rm, may rm. infectious diseases of humans, dynamic and control. oxford university press, new york, 1991. 13. catalano m, quelle ls, jeric pe, di martino a, maimone sm. survival of acinetobacter baumannii on bed rails during an outbreak and during sporadic cases. j. hosp. infect. 1999;42(1); 27–35. 14. bures s, fishbain jt, uyehara cf, parker jm, berg bw. computer keyboards and faucet handles as reservoirs of nosocomial pathogens is intensive care unit. am. j. infect. control. 2000;28(6); 465–471. 15. spivack n. the threat of contaminated money: proposed solutions, 2005. available from: http://www.novaspivack.com/best-articles/the-threat-ofcontaminated-money-proposed-solutions. prevelance of overweight and obesity among young female students in association with bmi vol 8 (2), july 2017 issn (print): 2305 – 8722 issn (online): 2521 – 8573 1 r a d s j . b i o l . r e s . a p p l . s c i 1 op e n ac c e ss f u l l l e n g t h a r t i c l e prevalence of overweight and obesity among young female students in association with bmi farha aziz*, madiha muzaffar ali & farah jabeen department of biochemistry, jinnah university for women a b s t r a c t the study is designed to obtain the occurrence of obesity among 180 young female students of university, age 19-23 years in association with body mass index (bmi) according to who classification. a questionnaire was prepared to collect information, including age, height, body weight, and socioeconomic status. among 180 girls 18.33% (33) of girls were under weight, 58.88 % (106) were normal, 16.66 % (30) were overweight and 6.11% (11) of girls were obese. overweight was more common among female students than obesity. the dietary practices and choices of young female girls have been evaluated by using food frequency questionnaire and found majority of students were used to skipped breakfast and frequent consumption of fast food including burgers, pizzas and coca cola due to lack of knowledge on healthy balance diet and adverse effect of obesity. healthy living, eating healthy balanced diet and regular exercise help to maintain standard body weight and health. keywords overweight, obesity, bmi, prevalence, food frequency questionnaire, female students address of correspondence farahtariq2@hotmail.com article info. received: april 3, 2017 accepted: may 29, 2017 cite this article: aziz f, ali mm, jabeen f. prevelance of overweight and obesity among young female students in association with bmi. rads j. biol. res. appl. sci 8(2):01-04. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n adolescence is a period for growth spurts and puberty changes between childhood and adulthood. during this period accumulation of excess fat among young girls are tend to persist into adulthood and has become a great threat in the development of constant diseases and conditions like type 2 diabetes mellitus, hypertension, dyslipidemia, and cancer in later age. (1, 2). it is an independent risk factor for cvd and correlated with morbidity and mortality high risk associated with reduced life. (1, 3, 4). obesity and overweight are defined as unnecessary or abnormal fat accumulation in the body due to high caloric intake or less physical activity (5). obesity is rapidly becoming a major medical and public health problem for last two decades in different parts of the world including pakistan to be found 9th out of 188 countries (6-8). the pakistan national health survey (nhs) documented that approximately 1% of the people is obese and 5% is overweight in the young age (15-24 years) in pakistan. according to nhs (1990–94), the prevalence of obesity for men and women are 22% and 37% respectively in big cities. according to pakistan demographic health survey report 2013, pakistani females live in urban areas are more susceptible to obesity than males (9). genetic tendency, traditions, hormonal imbalances, metabolic and eating disorders, and physically inactive living are major factors to cause obesity. several studies revealed that overweight and obesity among children and adolescent were significantly related with unhealthy modern lifestyle, improper knowledge of healthy balanced diet and physical inactivity o r i g i n a l a r t i c l e prevelance of overweight and obesity among young female students in association with bmi vol 8 (2), july 2017 issn (print): 2305 – 8722 issn (online): 2521 – 8573 2 r a d s j . b i o l . r e s . a p p l . s c i 2 (10). globally the idea of nutritionally healthy balanced diet among the general population has been completely changed with addition of higher amount of saturated bad fats, trans-fatty acids and added sugar. in pakistan food consumption trends and healthy dietary patterns has been replaced by high intake of fast food, processed foods, junk food and sugary fizzy drinks in pakistan (11). m a t e r i a l s a n d m e t h o d s a descriptive cross-sectional study of 180 female students aged 19 to 23 years was carried out during the year of (2015 – 2016) in the jinnah university for women, nazimabad, karachi. a total number of 180 girls were randomly selected from university campus to obtain relevant information on anthropometric, socioeconomic, dietary condition of the adolescent girls. a questionnaire was developed to obtain general information, sign and symptoms regarding anemia, dietary habits, and data for bmi. height and weight were measured using standardized protocols (11). weight measurements were made without shoes to the nearest 0.1 kg using a single portable weighing scale. height was measured without shoes and recorded to the nearest 0.1 cm with a height rod fixed on a wall. a questionnaire was developed to obtain general information, sign and symptoms regarding anemia, dietary habits, and data for bmi. the general information including parent’s education, occupation, income, family structure and socio economic status of participitants. data was analyzed by using t-test and presented as mean ± sd. r e s u l t s in the present study, 180 female students of age 18-24 years of jinnah university for women, karachi, pakistan during semester 2016 were randomly selected. weight in kilogram and height in meter of all participitants were measured by using digital weighing machine and measuring tape respectively (table 1; fig 1). according to who classification for bmi among 180 girls 17.77% (33) of girls were under weight, 19.53% (106) were normal, 29.59% (30) were overweight and 30.46% (11) of girls were obese (table 2; fig 2). eating habits of the students were analyzed by using a food frequency questionnaire. majority of female students were used to skip breakfast. high intake of burgers, french fries, junk food, soft drinks etc were observed during university hours. fewer intakes of fiber rich food, fruits vegetables, seeds, legumes and water consumption was also restricted to 4-5 glasses per day. table i: mean ± sd values of weight, height and bmi of female students categorize of bmi weight (kg)/mean ± sd height (m)/mean ± sd bmi(kg/m2) under weight 40±12.4 1.5±10.4 17.77±3.5 normal weight 50±11.5 1.6±11.7 19.53±2.8 over weight 58±10.2 1.4±12.5 29.59±4.1 obese 76±11.6 1.6±10.8 30.46±3.2 table ii: prevalence of over weight and obesity in young girls according to who bmi classification classes of bmi (kg/m2) no of female students bmi (kg/m2) percentage (%) under weight 33 17.77±3.5 18.33±3.5 normal weight 106 19.53±2.8 58.88±2.8 over weight 30 29.59±4.1 16.66±4.1 obese 11 30.46±3.2 6.11±3.2 prevelance of overweight and obesity among young female students in association with bmi vol 8 (2), july 2017 issn (print): 2305 – 8722 issn (online): 2521 – 8573 3 r a d s j . b i o l . r e s . a p p l . s c i 3 fig 1: categorize female young girls according to who bmi classification fig 2: percentage prevalence of obesity among female university d i s c u s s i o n the aim of this study was to assess eating habits, behaviors and the occurrence of overweight and obesity in young university girls. according to who classification for bmi results showed that majority of university females (58.88%), age 18-24 yr were belong to normal weight (bmi 19.53). 16.66 % (bmi 29.59) were overweight and 6.11% (30.46) of girls were obese. several studies showed majority of students don’t take breakfast regularly due to shortage of time in the morning. breakfast is considered as a very important meal to start a better day (13, 14). consuming a regular breakfast can be helpful in maintaining a normal body mass index (bmi). (8,5). university students generally do not follow healthy eating behaviors. adolescents are more likely to eat fast food frequently. high intake of fast food, soft drinks, coca cola, tea in adolescent diet was generally observed. all these unhealthy eating habits are more likely to attain overweight and obesity among young girls. usually university student diet is high in saturated fat and low in fibers, fruits and vegetables (15, 16, 17, 18). students commonly choose fast food due to its flavor, aroma and tastiness. globally healthy balance diet and dietary patterns have been replaced by unhealthy irregular eating behaviors in youth. adolescents are more probably like to eat fast food frequently. high intake of fast food, soft drinks, coca cola, tea in adolescent diet showed strong correlation with bmi (13, 19, 20). adolescents have inadequate knowledge and information about dietary sources, recommendations, diet-disease relationships, and dietary habits or choices (21). c o n c l u s i o n adolescence obesity is a common serious health problem among young girls. health awareness programs to get better health and nutritional condition of the university female girls should be implemented to focus on the improvement of eating habits as well as quality and quantity of the diet. moreover, students awareness programs should be held on regular basis to aware the concept of balanced diet and health risk of obesity among young girls. r e f e r e n c e s 1. engeland a, bjørge t, tverdal a, søgaard aj. obesity in adolescence and adulthood and the risk of adult mortality. epidemiology, 2004;15(1):79-85. 2. mohammadpour-ahranjani b, rashidi a, karandish m, eshraghian mr, kalantari n. prevalence of overweight and obesity in adolescent tehrani students, 2000–2001: an epidemic health problem. public health nutrition. 2004;7(5):645-8. 3. dietz wh. critical periods in childhood for the development of obesity. the am. j. clin. nutr., 1994;59(5):955-959. 4. must a, strauss rs. risks and consequences of childhood and adolescent obesity. int. j. obes. relat. metab. disord., 1999;23(2):2-11. 5. sherin a. obesity: how to prevent pakistani people from getting heavier?. khyber medical university journal, 2014;5(2):59-60. 6. hasnain s. the fatty acid binding protein 2 (fabp2) polymorphism ala54thr and obesity in pakistan: a prevelance of overweight and obesity among young female students in association with bmi vol 8 (2), july 2017 issn (print): 2305 – 8722 issn (online): 2521 – 8573 4 r a d s j . b i o l . r e s . a p p l . s c i 4 population based study and a systematic metaanalysis. gene. 2015 dec 10;574(1):106-11. 7. deliens t, clarys p, de bourdeaudhuij i, deforche b. determinants of eating behaviour in university students: a qualitative study using focus group discussions. bmc public health, 2012;4(1):53. 8. silliman k, rodas-fortier k, neyman m. a survey of dietary and exercise habits and perceived barriers to following a healthy lifestyle in a college population. cal. j. health promot., 2004;18:281. 9. balcı yi, karabulut a, gürses d, çövüt i̇e. prevalence and risk factors of anemia among adolescents in denizli, turkey. iran. j. pediatr., 2012;22(1):77. 10. tanzil s, jamali t. obesity; an emerging epidemic in pakistan-a review of evidence. j ayub med coll abbottabad, 2016;28(3):597-600. 11. yahia n, achkar a, abdallah a, rizk s. eating habits and obesity among lebanese university students. nutrition journal. 2008 oct 30;7(1):32. 12. lohman tg, roche af, martorell r. anthropometric standardization reference manual. human kinetics books; 1988. 13. adolphus k, lawton cl, dye l. the effects of breakfast on behavior and academic performance in children and adolescents. front. hum. neurosci., 2013;7:425. 14. butler sm, black dr, blue cl, gretebeck rj. change in diet, physical activity, and body weight in female college freshman. am. j health behav., 2004;28(1):24-32. 15. debate rd, topping m, sargent rg. racial and gender differences in weight status and dietary practices among college students. adolescence., 2001;36(144):819-833. 16. de la hunty a, gibson s, ashwell m. does regular breakfast cereal consumption help children and adolescents stay slimmer? a systematic review and meta-analysis. obes. facts, 2013;6(1):70-85. 17. duffey kj, gordon-larsen p, jacobs dr, williams od, popkin bm. differential associations of fast food and restaurant food consumption with 3-y change in body mass index: the coronary artery risk development in young adults study. am. j. clin. nutr., 2007;85(1):201-208. 18. milosavljević d, mandić ml, banjari i. nutritional knowledge and dietary habits survey in high school population. coll. antropol., 2015;39(1):101-107. 19. szajewska h, ruszczyński m. systematic review demonstrating that breakfast consumption influences body weight outcomes in children and adolescents in europe. crit. rev. food sci. nutr., 2010;50(2):113119. 20. james pt, leach r, kalamara e, shayeghi m. 2001. the worldwide obesity epidemic. obes. res., 9 suppl. 4:228s–233s. 21. world health organization. obesity and overweight. world health oranization, 2009. assessment of basic nutritional profile and antioxidant potential of pakistani pomegranate (punica granatum l.) varieties vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 123 op e n ac c e s s f u l l l e n g t h a r t i c l e assessment of basic nutritional profile and antioxidant potential of pakistani pomegranate (punica granatum l.) varieties naila siraj1*, saima tehseen1, muhammad asim shabbir2 1department of food science and technology, government college women university, faisalabad, 38000, pakistan. 2national institute of food science and technology, university of agriculture, faisalabad, 38000, pakistan. a b s t r a c t background: pomegranate seeds are the remains of industrial processing of pomegranate fruit into various products such as juice, jam, and jelly that cause environmental pollution and a loss of lipid and water-soluble bioactive compounds. the seeds comprise 1525% of the whole fruit weight. its lipid fraction has a distinct fatty acid profile with high polyunsaturated fatty acid contents such as punicic acid (pa) (50-80%), tocopherols, and phenolic compounds. objective: the objective of the current study was to characterize the three main pakistani pomegranate varieties (desi, kandhari, and bedana), supporting waste exploitation strategies. the physicochemical characteristics, fatty acid profile, antioxidant potential of all three varieties were studied. methodology: the extracted oils samples were investigated for their lipid contents, fatty acid composition, physicochemical properties, and antioxidant potential on the varietal base. results: total lipid contents ranged from 8.70-15.9% dry matter with conjugated linolenic acid (68.5-76.7% of total fatty acids) as the predominant fatty acid in all samples, followed by linoleic, oleic acid, stearic acid, and palmitic acid. further characterization revealed that desi was superior regarding acidity and oxidation stability over the other two varieties associated with higher total phenolic and tocopherol contents with potent antioxidant activities. conclusion: the present results demonstrate a preferable fatty acid pattern in pomegranate seed oil (pso), which may be potential functional ingredient for product development in food and non-food applications in combination with high antioxidant contents. keywords pomegranate, fatty acids, antioxidant potential, seed oil, waste utilization *address of correspondence nailaateeq@gcwuf.edu.pk article info. received: january 26, 2022 accepted: december 27, 2022 cite this article: siraj n, tehseen s, shabbir ma. assessment of basic nutritional profile and antioxidant potential of pakistani pomegranate (punica granatum l.) varieties. 2022; 13(2):123-133. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n the earliest known fruit plant is pomegranate (punica granatum l.), instigating from ponum granatum, which means seeded or grainy apple, native to central asia and persia1. it is grown in india, turkmenistan, pakistan, north africa, china, afghanistan, and some mediterranean countries. usaid (the united states agency for international development) estimates global production to be between 3.80 million tons in 2017, with an expected increase in the next years following the risen global awareness of pomegranate fruit’s health benefits2. in pakistan, the pomegranate’s central cultivation area is in baluchistan, whereas punjab and khyber pakhtunkhwa o r i g i n a l a r t i c l e assessment of basic nutritional profile and antioxidant potential of pakistani pomegranate (punica granatum l.) varieties vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 124 produce pomegranates on a small scale on isolated farms. the best-known varieties in pakistan are desi, kandhari, and bedana, with the primary harvest season in september but are available around the year when stored under appropriate conditions3. the industrial transformation of fruit and vegetables into food products generates enormous by-products, many of which are considered high value due to their high content of different bioactive compounds4. pomegranate seeds are the remains of industrial processing of pomegranate fruit into various products such as juice, jam, and jelly that cause environmental pollution and a loss of lipid and watersoluble bioactive compounds. the seeds comprise 15-25% of the whole fruit weight. its lipid fraction has a distinct fatty acid profile with high polyunsaturated fatty acid contents such as punicic acid (pa) (50-80%), tocopherols, and phenolic compounds. omega-6 and omega-3 pufas are the two primary groups of pufas that are relevant to human health. in most diets, linoleic acid (la, 18:2-6) and α-linolenic acid (ala, 18:3-3) are the most abundant pufas. la and ala are considered animals that cannot produce essential fatty acids since these. because these pufas are produced in plants, la and ala are mostly found in high concentrations in plant-based foods. low essential fatty acid intakes have been linked to dermatitis, renal hypertension, mitochondrial activity problems, cardiovascular disease, type 2 diabetes, impaired brain development, arthritis, depression, and decreased body response to infection. recent research on pomegranate peels has demonstrated good antioxidant potential and antimicrobial activity. until recently, pso considered superior nutritional quality for usage in food applications. however, it has been suggested to have great potential to reduce the risk of cardiovascular disease, inflammation, and cancer, as well as relieve menopausal symptoms and support immune functions5. although the optimization of pso extraction and characterization of fatty acid profiles have been reported in several studies, there is only limited evidence available on the physicochemical characterization of pso6. whereas some data were published on phytochemical and antioxidant characteristics of pso from varieties grown in the usa, iran, india, turkey, and china7, there is a lack of knowledge regarding seed oils from pakistani pomegranate varieties. therefore, this study explores the pomegranate seed residues (psrs) of three pakistani pomegranate varieties as a material with potential economic value by referring to its lipid yield, fatty acid profile, physicochemical properties, and antioxidant potential. m a t e r i a l s a n d m e t h o d s materials approx. 15 kg fresh, fully ripe, and good-quality pomegranate varieties (desi, kandhari, and bedana) were procured from the local market of district faisalabad, pakistan. a certified fatty acid methyl ester (fame) reference standard mixture was obtained from sigmaaldrich (bellefonte, pa, usa). standards of gallic acid, tocopherols (α (≥96%), β (≥97%), γ (≥96%), δ (≥97%)), folin-ciocalteu reagent, 2,2'-azino bis (3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts), 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2carboxylic acid (trolox), and 2, 2-diphenyl-1 picrylhydrazyl radical (dpph) purchased from sigma-aldrich (st. louis, mo, usa). all tests used ultra-pure water generated with < 5 ppb toc and 18.2 m x resistivity (merck, ma, usa) by integrated water purification system milli-q. all other chemicals and solvents were obtained from fisher scientific co. (nepean, ontario, canada) of the american chemical society (acs). pomegranate seed oil extraction sugars and other clinging components were removed from pomegranate seeds by introducing ju 2000 vitae (moulinex, barcelona, spain) in a blender for juice extraction. the pomegranate seed was ground in a regular domestic coffee grinding machine and rinsed and dried in the sun (2-3 days) by passing through 30–40 mesh screens. the pomegranate seed powder was dried at 60ºc for 48 h and kept at -18°c until further use (7±1% moisture content). the oil was extracted from the pomegranate powder using the standard soxhlet extraction method for 24 h (3 * 8 h) for each variety with hexane as a solvent. a rotary evaporator was used to evaporate the hexane at reduced pressure, followed by drying under nitrogen to remove the remaining hexane. the collected oil samples were preserved at -20oc in amber glass jars for further research. the extraction yield (%) was determined by assessment of basic nutritional profile and antioxidant potential of pakistani pomegranate (punica granatum l.) varieties vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 125 dividing the volume of obtained oil (ml) by the original amount of dry matter (g) multiplied by 1008. fatty acid profile fatty acid methyl esters (fame) of oil samples generated utilizing the koh-methanol method according to jing et al. (2012)7 procedure. a 20 mg oil sample was combined with 2 ml iso-octane and 0.2 ml koh-methanol. after 5 minutes at room temperature, add 2 ml iso-octane and 3 ml water to the mixture. the supernatant was then drained and thoroughly rinsed with water. the uppermost isooctane layer was recovered and used for gc-ms (model: 14-a, shimadzu, japan). a db-5ms column (30 m x 0.25 mm with a 0.25 µm film thickness) used with carrier gas helium flowed at a 1.0 ml/min rate. the sample was injected as 0.5 µl at a split ratio of 5:1. initially, the temperature was set to 70°c for 5 minutes, up from 10°c/min to 200°c and then to a final temperature of 275°c at the rate of 5°c/min. ms spectra were obtained with an ion source temperature of 200°c at an ionization energy of 70 ev and an interface temperature of 260°c. an integrator was used to quantify fatty acid contents by estimating the area under individual peaks compared to the total area under all peaks. fame preparation of pa standard following hashempour et al. (2010)9, the methyl esters of the pa standard were produced independently. trimethylsilyl diazomethane reagent interacted with the standard. 10 mg of pa were combined with 1 ml of methanol/toluene (1:4, v/v), and then 2 mol/l trimethylsilyl diazomethane (100 l) were added. following 30 minutes at room temperature, the mixture was added to 2 ml isooctane and 5 ml water, with the supernatant subjected to gc-ms after phase separation. physicochemical analysis a sopelem series 3296 refractometer (sopelem, france) was used to calculate the refractive index (ri) according to the method of godswill et al. (2018)10 with some modifications. the specific gravity (sg), acid value (av), iodine value (iv), and saponification value (sv) of recovered oil samples were determined using the aocs protocols (2006)11. the unsaponifiable matter was estimated following the technique outlined in aoac12 (2016). the viscosity was measured using a viscometer (model lv dvii-brookfield, middleboro, ma, usa). the values recorded as pa.s. finally, the melting and flashpoints were determined by following the aocs (2006) methods11. tocopherol contents following katsanidis and addis’s method (1999)13, the tocopherol contents were determined using hplc (model: perkin elmer series 200, usa). the analysis was carried out using an isocratic mode normal phase hplc column (250mm x 4.6mm, 5.0m particle size) with a mobile phase of iso-octane/ethyl acetate (96:4 v/v). simultaneously, the detector was tuned to 290 nm excitation and 400 nm emission wavelengths, with a run time of 30 minutes and a flow rate of 1.0 ml/min. the temperature in the column was 35°c. individual oil samples or standards were dissolved in hexane, sterile filtered, and injected in a 2.0 µl volume. for every standard (α, β, γ, and δ tocopherol), an external calibration curve was created and utilized to estimate the volume of tocopherol present in the oil samples. after eight (08) injections, the silica column was re-activated using a 10% isopropanol in a hexane solution to increase its efficiency14. duplicate samples were prepared. total phenolic content (tpc) after oil extraction, the residual psrs from all three types were dried at room temperature overnight. approximately 1 g of dried defatted psr was extracted in ultrasonic waves with 10 ml of 80% methanol at room temperature and centrifuged for 10 minutes at 2500 g/s. the supernatants were collected and kept at 4°c in the dark until further examination. according to mohdaly et al. (2011)15, tpc was measured using the folin-ciocalteu reagent. the prepared methanolic pomegranate seed extracts (50 mg) were combined with 0.5 ml folin reagent and 7.5 ml deionized water, then incubated for 10 minutes at room temperature with 20% sodium carbonate (w/v). the mixture was placed in a water bath at 40°c for 20 minutes before cooling in an ice bath. the absorbance was measured using a spectrophotometer at a wavelength of 765 nm (u-2001, hitachi instruments inc. tokyo, japan). the tpc was measured using a 10-100 g/ml gallic acid standard curve (r2 = 0.990). gallic acid equivalents (gae) were calculated in mg/g of oil. both samples were tested three times. assessment of basic nutritional profile and antioxidant potential of pakistani pomegranate (punica granatum l.) varieties vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 126 determination of antioxidant activity radical scavenging activity was measured according to siddiq et al. (2013)16 using a dpph solution in methanol. dpph stock solution (2.4 g/l methanol) was diluted with 800 ml/l methanol to create a working solution (a515 w1.1). blank, standard, or sample (0.6 ml) was combined with 3.0 ml of dpph working solution, maintained in the dark for 20 minutes, and measured absorbance. blank (ethanol acetone, 7:3) was utilized to calculate radical scavenging activity. using 50-250 mmol/l trolox solution, a standard curve was created. the trolox equivalent antioxidant capacity (teac) was determined using a modified abts radical cation decolorization test16. briefly, 7 mmol/l abts solution and 2.45 mmol/l potassium persulfate were combined in a 1:1 ratio and left in the dark for 12-16 hours to form abts.+ radical cation (abts.+). this solution was diluted with methanol (800 ml/l) so that its absorbance at 734 nm was between 0.700 and 0.020. the diluted abts.+ solution (3 ml) was combined with 30 ml of blank, standard, or sample, and the absorbance was measured at 734 nm using a spectrophotometer after a 6-minute reaction. the blank was processed with ethanole: acetone (7:3). using trolox solution (0.3-1.5 m mol/l), the standard curve for calculating antioxidant capacity was generated. the experiment indicated the antioxidant capabilities of the samples as m mol trolox equivalents (te)/100 g fresh weight (fw). oxidative status analysis the peroxide (pov) and para-anisidine (p-av) values were estimated according to the aocs (2006) method11. conjugated dienes (cd) and conjugated trienes (ct) were determined at 232 and 270 nm, respectively. oil samples were diluted with iso-octane to change the absorbance within limits, as per the standard iupac form. statistical analysis and data interpretation a one-way analysis of variance anova was used in the statistical study, and duncan’s multiple range test was used to assess the significant difference between means. differences at p≤0.05 were considered statistically significant. the results are presented as mean±sd of triplicate measurements unless indicated otherwise. r e s u l t s a n d d i s c u s s i o n lipid contents and fatty acid profile the economically viable pso exploitation depends on extraction technology, yield, and oil characteristics and quality. that might determine the potential of the oil source for downstream applications, such as food and non-foodbased products (figure 1). the total oil content in the current investigation ranged from 8.7-15.9% in the three varieties (figure 2), which is comparable to previous studies conducted on pso from iranian cultivars (6.6-19.3%)18, turkish cultivars (7.616.2%)19 and indian cultivars (7.0-19.0%)20. the kind, number, and placement of fatty acids within the glycerol moiety determine the oil’s nutritional and physicochemical properties. therefore, they can vary considerably in plant oils, depending on their source. while comparing different extraction methods for pso extraction, the highest oil yield was observed by the soxhlet method (15.66%)21. while six primary fatty acids could be identified in pso samples (table 1), the predominant fatty acid is conjugated linolenic acid (clna, c18:3), also known as pa, considered one of the principal and active pso components responsible for its antioxidant potential. the average pa values of bedana (68.5%), kandhari (75.5%), and desi (76.7%) are following previous studies in cultivars from georgia (78-83%)22, china (73-79%)7, italy (72.20-84.10%)19, india (31.8086.60%), turkey (70-76%)20. other sources for pa have, on average, a lower pa content, such as seed oils from catalpa bignonioides (28%), momordica charantia (59%), prunus mahaleb (28%), trichosanthes kirilowii (36%), and calendula officinalis (30%)23. assessment of basic nutritional profile and antioxidant potential of pakistani pomegranate (punica granatum l.) varieties vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 127 figure 1. external view and cross section of the selected pakistani pomegranate varieties (a) desi (b) kandhari (c) bedana. figure 2. oil recovered from three pakistani pomegranate varieties (a) desi (b) kandhari (c) bedana table 1. lipid contents and fatty acid composition of pomegranate seed oil values in the same row with different letters are significantly different (p < 0.05). sfa, saturated fatty acids; usfa, unsaturated fatty acids. oleic (c18:1; 10.4-12.8 %) and linoleic acid (c18:2; 5.412.0 %) are prominent second and third fatty acids and are somewhat higher compared to other studies investigating pmo-composition growing in various countries (spain, israel, turkey, iran, tunisia)19. the total saturated fatty acids (sfas) were between 6.08.5%; the main sfas are palmitic, stearic, and myristic acid, as seen in table 1, which is significantly lower than other edible oils, such as 15% in soybean oil and pumpkin seed oil24, 25. stearic and palmitic acid values are comparable with turkish (1.4-2.8%, 1.4-2.0%), chinese (2.8-3.6%, 1.6-2.8%), and georgian pomegranate cultivars (2.8-4.8%, 2.1-3.6%)22, 20. consequently, saturated to unsaturated fa (sfa/ufa) ratios align with data on cultivars from spain, iran, turkey, israel, and tunisia19. besides cultivar variation, differences in pso oil content and lipid profiles are most likely due to different climate conditions, local growth, and environmental influence. high pomegra nate varieties lipid contents (g/100g) c14:0 c16:0 c18:0 c18:1 c18:2 c18:3 sfa ufa sfa/uf a desi 15.90±0.0 4 a 0.51±0 .01 a 4.60±0. 04 c 0.75±0. 01 b 10.61±0. 33 a 6.73±0.0 9 b 76.66±1. 12 a 6.00±0. 15 c 94.00±0. 20 a 0.06±0. 02b kandhari 11.00±0.0 5 b 0.34±0 .02 b 5.30±0. 08 b 2.92±0. 50 a 10.40±0. 19 c 5.42±0.4 0 c 75.50±0. 50 b 8.50±0. 28 a 91.50±0. 78 c 0.09±0. 01ab bedana 8.70±0.0 c 0.15±0 .01 c 6.18±0. 50 a 0.67±0. 10 c 12.75±0. 22 b 12.00±1. 02 a 68.52±1. 63 c 6.80±0. 13 b 93.20±0 .70 b 0.07±0. 02b assessment of basic nutritional profile and antioxidant potential of pakistani pomegranate (punica granatum l.) varieties vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 128 quantities of unsaturated fatty acids, such as linolenic, linoleic acid, and pa, are preferred for human consumption, which may enhance pso usage in human nutrition. physicochemical properties physicochemical characteristics are significant in evaluating edible oil’s present condition and quality. therefore, several parameters, such as iv, sv, and av, are used to monitor oil quality and determine the required level of refinement26 (table 2). the iodine value is useful for determining unsaturation but does not identify the individual fatty acid27. a relatively high iodine value was observed in the desi variety, while other varieties showed a lower iodine value in kandhari and bedana, respectively. due to the high level of punicic acid in oil samples, the higher iodine value indicates a greater number of unsaturated bonds. besides, the av, a measure of free fatty acids and inversely proportional to the degree of edibility of an oil25, was significantly lower in desi than in the other two varieties. the iv and av of iranian pomegranate varieties and cold-pressed oil were 179.4215.90 and 216.5-220.34g i2/100g and 3.78-8.36% oleic acid, while own data on av are more comparable with coldpressed pso oil with 0.55-3.78% oleic acid28. short-chain fatty acids have a higher sv than longer-chain fatty acids, with a higher number of carbon atoms in triacylglycerols29. the sv was moderately significant; the highest was observed in bedana following kandhari and desi, similar to dadashi et al. (2013)27 findings. pso has a substantial percentage of high molecular weight fatty acids, as seen by the greater sv of oil samples, equivalent to other culinary oils used in everyday life. the differences in unsaponifiable materials reported across three cultivars could be due to the distinctive morphology of pomegranate fruit and the different timing between horticultural maturity for commercial harvesting and physiological ripening. the physical characteristics of oils depend on the chemical and functional bodies present with glycerol moiety that ultimately influence the behavior of lipids in food and the procedures for processing and manipulating these lipids. the ri varies significantly with earlier studies conducted on pso possessing 1.47-1.51 and 1.46-1.52 ri by elfalleh et al. (2007)31; dadashi et al. (2013)27, respectively. sg was observed highest in bedana, relatively higher than iranian pomegranate varieties and cold-pressed oil (0.92 to 0.93 and 0.91-0.92 g/cm3)27. different studies conducted on pso and evaluated their physicochemical properties agree with our results27. different extraction methods exert no significant effect on the physicochemical attributes of pso21. tocopherol contents tocopherols (α, β, γ, δ) are naturally occurring antioxidants enriched in vegetable & fruit seed oils with varying contents, depending on their source and with a generally more considerable difference between different oils compared to within cultivar variation. tocopherol contents in seed oils usually range between 0.20-80 mg/100g, and α-tocopherol is the main tocopherol except for 𝛽 and γ. the total tocopherol content of pakistani pso, ranging from 2.5-3.8 µmol/g, is comparable with other natural sources like blueberry, marionberry, onion, cardamom, and milk thistle seed oils23. table 3 shows that the three cultivars differed in total tocopherol content and composition. desi has significantly higher overall tocopherol contents than the other cultivars (p≤0.05). the tocopherol contents observed in different pomegranate cultivars worldwide showed different concentrations in seed oil; elfalleh et al. (2011)31 observed the following pattern α-tocopherol> γ-tocopherol> δtocopherol for tunisian pomegranate seed oils. jing et al.7 (2012) indicated the order δ-tocopherol> α-tocopherol> γtocopherol for seed oils of chinese pomegranates, and caligiani et al. (2010)32 found the δ-tocopherol> αtocopherol> γ-tocopherol pattern in pomegranate oil samples. the varying tocopherol content of pakistani pomegranate cultivars could be attributable to genetic differences or environmental factors. the α-tocopherol contents are analogous to 718.70-1388.30 µg/g in chinagrown pomegranate cultivars seed oil7, 1576-1786 µg/g tunisian cultivars31, and 1612-1737 µg/g georgia cultivars21. the γ-tocopherol was much lower than studies conducted earlier; 814-928 µg/g in georgia-grown cultivars and 957-1154.5 µg/g in tunisian-grown cultivars31. also, δtocopherol is comparable with tunisian cultivars (241.90299.10 µg/g)31 and georgia cultivars (214-238 µg/g)7. results are parallel with liu et al. (2022), who compare different extraction methods for pso extraction21. assessment of basic nutritional profile and antioxidant potential of pakistani pomegranate (punica granatum l.) varieties vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 129 table 2. physicochemical analysis of pomegranate seed oil pomegranate varieties desi kandhari bedana specific gravity 0.98±0.01 0.97±0.03 0.99±0.01 viscosity (pa.s; 25ºc) 0.12±0.03 0.13±0.02 0.11±0.01 refractive index 1.47±0.03b 1.43±0.01c 1.50±0.04a density (g/cm3) 0.91±0.03b 0.90±0.21b 0.92±0.01a melting point (ºc) 41.50±0.66b 42.00±0.81b 45.00±0.71a flash point (ºc) 100.0±1.61c 103.0±1.19b 106.00±2.36a acid value (% oleic acid) 3.77±0.05b 3.98±0.18a 4.00±0.08a saponification value (mg koh/g) 181.10±1.08b 181.50±1.50b 184.30±1.05a unsaponifiable matter (%) 1.04±0.04a 0.90±0.12b 0.80±0.07bc iodine value (g i2/100 g oil) 134.90±2.25a 123.50±1.60b 120.40±1.95c odor characteristics pomegranate odor characteristics pomegranate odor bland color dark yellow dark yellow light yellow state at ambient temperature liquid liquid liquid vapor properties negligible at ambient temperature negligible at ambient temperature negligible at ambient temperature values in the same row with different letters are significantly different (p < 0.05). table 3. tocopherol composition of pomegranate seed oil pomegranate varieties desi kandhari bedana α-tocopherol (µg/g) 1211.10±18.03a 890.80±10.11b 768.63±20.09c γtocopherol (µg/g) 50.21±2.30a 42.50±1.45b 35.10±2.39c δ-tocopherol (µg/g) 300.51±4.31a 256.90±3.90b 245.71±4.50c total tocopherols (µg/g) 1561.82±7.90a 1190.20±10.87b 1049.44±7.01c values in the same row with different letters are significantly different (p < 0.05). antioxidant activity the natural food sources like fruits and vegetables are enriched with phenolic compounds that function as imperative antioxidants; these exhibit the ability to donate an electron or hydrogen atom to a free radical to form a stable radical and prevent the auto-oxidation chain reactions in biological membranes. table 4 shows the tpc range between 3.4-4.9 mg gae per g dw. desi is offering the highest tpc, following kandhari and bedana. pakistani pomegranate seeds contain higher tpc than other oils, e.g., soybean, sunflowers, maize, grapes, hemp, flax, and pumpkin34. however, khoddami et al. (2014)35 found higher tpc in cold-pressed pso of three cultivars of 10.44 mg gae g-1 oil. the dpph• radical system is frequently used to evaluate antioxidant activity in vitro and in vivo studies. rojo-gutiérrez et al. (2021) found 91.29 and 98.28% scavenging activity against dpph and abts radicals, respectively, in pso extracted through green extraction methods (microwave-assisted and ultrasound-assisted extractions)36. assessment of basic nutritional profile and antioxidant potential of pakistani pomegranate (punica granatum l.) varieties vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 130 table 4. antioxidant potential and oxidative stability of pomegranate seed oils pomegranate varieties desi kandhari bedana total phenolic contents (mg gae/g oil) 4.94±0.58 a 4.30±0.83 b 3.40±0.38 c dpph. (µm trolox/g oil) 67.21±2.82 a 56.36±3.06 b 52.70±2.59 c abts.+ (µm trolox/g oil) 71.90±3.27 a 65.10±3.73 b 61.35±2.03 c peroxide value (meq of o2/kg) 1.21±0.05 c 1.54±0.30 b 1.98±0.10 a para-anisidine value 0.30±0.01 c 0.55±0.15 b 0.77±0.08 a conjugated dienes (λ232 nm) 0.22±0.24 c 0.75±0.05 b 0.99±0.12 a conjugated trienes (λ270 nm) 0.04±0.02 b 0.07±0.01 ab 0.09±0.02 a values in the same row with different letters are significantly different (p < 0.05). because several approaches might produce significantly divergent results, no single method is suitable for measuring the antioxidant potential of foods. various methods must be utilized, each based on a different mechanism. table 4 represents the dpph. scavenging activities of seed oil of three pakistani pomegranate cultivars dependent on the concentration by quenching the dpph• depicting increased contents increased the dpph. scavenging activity of oil samples. desi had considerably higher dpph• scavenging activities (p≤0.05) than kandhari and bedana. desi variety showed the highest dpph. scavenging activity (67.78±2.82%) follow by kandhari (56.83±3.06%) and bedana (52.38±2.59%). table 4 represents the abts•+ scavenging activity of oil samples and shows the antioxidant activity comparable with some artificial antioxidants. compared to other oil samples, the desi variety oil samples had the highest abts•+ intensity (71.48±3.27%). the variation in results may be due to various factors like radical’s stereoselectivity or solubility of the sample in different radical scavenging systems reported to upset natural extracts’ ability to scavenge diverse radicals. pomegranate cultivars from sri lanka showed tpc in the range of 1.19-2.39 mg gae g-1 with a higher 96.70% dpph, 93.1±2.0% abts radical scavenging activity35. he et al. (2012)37 extracted the phenolic compounds from pomegranate seed residues and found the tpc in the range of 3.12-27.52 mg gae g-1 with 4265.7 mmol/100 g dw dpph and 2692.0 mmol/100 g dw abts radical scavenging activity. the scavenging assay’s consequences recommended that pso enriched with phenolic contents, gifted to capture the free radicals in a system through hydrogenor electron-denoting mechanisms. as a result, pso can scavenge free radicals in vulnerable matrices, such as biological membranes, and prevent fatal free radical arbitrated chain reactions from starting. these studies confirmed the pso’s ability to capture free radicals in various systems, suggesting that these molecules could be helpful therapeutic mediators for treating free radical-induced pathology. the effect of extraction on pso using different extraction methods, e.g., soxhlet, stirring, microwave & ultrasonic irradiation, and supercritical fluid extraction on the phenolic contents, showed significant results with other experimental conditions. the highest phenolic contents were observed in soxhlet > microwave > stirring > ultrasound with high pressure and lower temperature38. higher tpc with antioxidant activity of pakistani pomegranate seeds supported by jing et al. (2012)7. oxidative status the pov, p-av, and cd & ct were used as lipid peroxidation indexes to measure the antioxidant abilities for the present study. universally, pov is considered a standard method for measuring lipid peroxidation extent in fat/oil-based products. the pov measures the concentration of peroxides and hydro-peroxides generated in the early phases of lipid oxidation. pov is one of the most common methods for determining oxidative rancidity in oils and fats. in our study, the lipid oxidation level of oil samples was determined by placing oil samples at 70ºc for 72 h in a hot air oven. table 4 shows the pov of oil samples with a significant difference (p≤0.05). the results revealed that the pov of the present study samples were higher than 0.39-0.48 meq o2/kg of four (04) iranian assessment of basic nutritional profile and antioxidant potential of pakistani pomegranate (punica granatum l.) varieties vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 131 pomegranate cultivars39 while lower than 1.00-4.40 meq o2/ kg oil in seven (07) turkish pomegranate cultivars oil. the p-av for the three cultivars’ oil samples ranged from 0.30±0.01-0.77±0.08% shown in table 4. chemical structures can account for the difference in p-av. the oxidative stability of lipids is improved by the stability of phenoxy radicals, limiting the degree of proliferation40. the results showed that the desi variety has more antioxidant capability than kandhari and bedana varieties. the present findings are supported by carvalho et al. (2016)41. the cd and ct values of pomegranate oil samples are shown in table 4, ranging from 0.22±0.240.99±0.12 λ232 and 0.04±0.02-0.09±0.02 λ270, respectively. because the increase in cd and ct is related to the degree of oxidation, the oil sample’s os will be lower42. our study’s oxidative status results showed that pakistani pomegranate seed has more antioxidant potential than tea leaves. the relatively lower pov, av, and cd & ct values of oil samples showed that pakistani pso could categorize as stable oils. the antioxidant activity of vegetable and fruit-based oils is mainly ascribed to the phenolic contents found in plant seed oils. c o n c l u s i o n based on the present study results, the seeds of pakistani pomegranate cultivars as a by-product after juice extraction can be a potential source of essential fatty acid (clnas), biologically active compounds. findings also showed substantial connections between total phenolic contents and antioxidant activity, representing the influence of phenolic compounds in oil antioxidant activity with higher oxidative stability. the lipid content of pomegranate seeds was probably insufficient for commercial use; on the other hand potential constituent for particular consumption or medical usage. although, it could be an economical source of a natural antioxidant for different food products due to the antioxidant activity of conjugated linolenic acid. the findings support the use of pomegranate seeds in manufacturing pomegranate oil or developing nutraceutical foods for human nutrition. c o n t r i b u t o r s t a t e m e n t all authors contributed equally in preparation and evaluation of the manuscript. c o n f l i c t s o f i n t e r e s t no conflict of interest. f u n d i n g s o u r c e the current research projected was funded by higher education commission, islamabad, pakistan. a c k n o w l e d g e m e n t s ns is grateful for the stipend provided by the higher education authorities. l i s t o f a b b r e v i a t i o n s none r e f e r e n c e s 1. verma n. mohanty a. and lal a. 2010. pomegranate genetic resources and germplasm conservation: a review. fruit, vegetable and cereal science and biotechnology. 4(s2): 120-125. 2. kahramanoglu, i. 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(icaebs’16) (pp. 105-109). 31. elfalleh w. tlili n. nasri n. yahia y. hannachi h. chaira n. ying m. and ferchichi a. 2011. antioxidant capacities of phenolic compounds and tocopherols from tunisian pomegranate (punica granatum) fruits. journal of food science. 76(5): c707-c713. 32. caligiani a. bonzanini f. palla g. cirlini m. and bruni r. 2010. characterization of a potential nutraceutical ingredient: pomegranate (punica granatum l.) seed oil unsaponifiable fraction. plant foods for human nutrition. 65(3): 277-283. 33. wu g. chang c. hong c. zhang h. huang j. jin q. and wang x. 2019. phenolic compounds as stabilizers of oils and antioxidative mechanisms under frying conditions: a comprehensive review. trends in food science & technology, 92: 33-45. 34. khoddami a. man y.b.c. and roberts t.h. 2014. physico‐chemical properties and fatty acid profile of seed oils from pomegranate (punica granatum l.) extracted by cold pressing. european journal of lipid science and technology. 116(5): 553-562. 35. bopitiya d. and madhujith t. 2015. antioxidant potential of pomegranate (punica granatum l.) cultivars grown in sri lanka. tropical agricultural research. 24(1). 36. rojo-gutiérrez e. carrasco-molinar o. tiradogallegos j.m. levario-gómez a. chávez-gonzález m.l. baeza-jiménez r. and buenrostro-figueroa j.j. 2021. evaluation of green extraction processes, lipid composition and antioxidant activity of pomegranate seed oil. journal of food measurement and characterization, 15: 2098-2107. 37. he l. zhang x. xu h. xu c. yuan f. knez ž. novak z. and gao y. 2012. subcritical water extraction of phenolic compounds from pomegranate (punica granatum l.) seed residues and investigation into their antioxidant activities with hplc–abts+ assay. food and bioproducts processing. 90(2): 215-223. 38. vernès l. vian m. and chemat f. 2020. ultrasound and microwave as green tools for solid-liquid extraction. in liquid-phase extraction. (pp. 355-374). elsevier. 39. mirzaee s. 2014. studying seed and oil physicochemical characteristics of four iranian pomegranate (punica granatum l.) varieties. international journal of biosciences (ijb). 4(8): 78-86. 40. zhang q. saleh a.s. chen j. and shen q. 2012. chemical alterations taken place during deep-fat frying based on certain reaction products: a review. chemistry and physics of lipids. 165(6): 662-681. 41. carvalho ebtd. yoshime l.t. sattler j.a.g. pavan r.t. and mancini-filho j. 2016. characterization of constituents, quality and stability of pomegranate seed oil (punica granatum l.). food science and technology (campinas)(ahead): 0-0. 42. ramadan m.f. 2013. healthy blends of high linoleic sunflower oil with selected cold pressed oils: functionality, stability and antioxidative characteristics. industrial crops and products. 43: 6572. bacteriological analysis of nile tilapia fish (oreochoromis niloticus) roquia fatima1, qudsia hussain2, sayyada ghufrana nadeem2, shazia tabassum hakim3 1department of microbiology, jinnah university for women, karachi 74600, pakistan. 2medical mycology research and reference laboratory, department of microbiology, jinnah university for women, karachi -74600, pakistan. 3virology and tissue culture laboratory, department of microbiology, jinnah university for women, karachi -74600, pakistan. abstract fish is a major food in great demand throughout the world. fish protein is better and safer than meat (animal protein) because it contains lower cholesterol. a research was conducted to analyze the bacterial habitat and content of tilapia fish. total seven fishes were taken. the samples were examined in microbiology laboratory by serial dilution method. sections of the skin, gills and intestine of fishes were aseptically removed by means of a sterile scalpel and pair of sterile scissors. a serial dilution was prepared and from last dilution 0.1ml was plated on nutrient agar plates. total eighteen different species of bacteria were isolated and identified. gram negative include pseudomonas sp. and enterobacter sp., while gram positive species include staphylococcus aureus and streptococcus sp. some of these pathogens have tendancy to transmit to man (who eat fish meat or deal with fish and fish products). streptococcus infection was detected in high prevalence among cultured fresh water fishes, especially during summer seasons. the most common signs of streptococcosis in fish were septicemia, skin ulcers, hemorrhages of the eye, in some cases changed cloudy and destructed (pop –eye) and hemorrhages on the skin especially in the base of fins and tail. hence it is considered that a variety of bacterial s p e c i e s c a n b e a s s o c i a t e d w i t h f re s h ti l a p i a f i s h re l a t e d p a t h o g e n t o h u m a n s . keywords: cultural fish, gills, intestine, nile tilapia introduction fish has been one of the main source of foods for humans for many centuries and still constitute an important part of the diet in many countries (leisner, et al. 2001). as a result, there is a considerable increase in the demand for fish being the cheapest source of animal protein (ladipo, et al. 1981). the advantage of fish is its easy digestibility and high nutritional value. these important attributes makes the commodity readily susceptible to microbial attack particularly bacteria. fish flesh naturally contains very low levels of bacteria (adam & tobaias, 1999). disease out breaks in fish tank spreads very quickly and you have to first identify the type of disease before you can take action. the bacteria are transmitted by fish that have made contact with other diseased fish. bacterial fish disease and infections are very common and are one of the most difficult health problems to deal with. bacteria can enter the fish body through the gills or skin or it can stay on the surface of the body (douglas, 2007). shell fish such as tilapia have a particular large pool of nitrogenous extractives and are even more prone to rapid spoilage, a factor which accounts for the common practice of keeping them alive until immediately prior to consumption. the speed with which a product spoils is also related to the initial microbial load on the product. the natural flora of the environment may be contaminated with organisms associated with man such as members*corresponding author: qudsiahussain@hotmail.com 30 vol 5 (1), january 2014; 30-35 of the enterobacteriaceae and staphylococcus aureus which can grow well at 30-37oc (micheal, et al. 2007) fish is among the most important sources of protein to human consumption, thus the study of the signs and lesions, induced by fish diseases, helps the protection in our national economy. infectious diseases of cultured fish are among the most notable constraints on the expansion of aquaculture and the realization of its full potential (plumb, 1994). the organisms from the environment around the fish may become closely associated with the external surfaces of the fish. there may be accumulation of the organisms at sites of damage, such as missing scales or abrasions. the organisms may enter the mouth with water or food and pass through and/or colonies the digestive tract (austin & austin, 1987). scrutiny of the available literature suggests that fish have only low bacterial populations on the skin. bacterial populations of skin is 102 to ~104 cm-2. muscle has been considered by some to be sterile (apun, et al. 1999), whereas other investigators have reported the presence of bacteria. also, some workers have found bacteria in kidney and liver of healthy fish (evelyn and mcdermott, 1961). gill tissue has been found to harbour high bacterial populations, e.g., up to 106. enterobacteria, gram-positive cocci, pseudomonads, and vibrios have been recovered from the gills of healthy juvenile trout (nieto, et al. 1984). some taxa, such as pseudomonas, have been implicated as causes of fish spoilage (gillespie, 1981; malle, 1994 ) by the production of histamines (kim, et al. 2001) principally during storage of fish. some of these pathogens that isolated from the skin and the internal organs of o. nilotica were aeromonas sp., pseudomonas sp., streptococcus sp. (abd ellatif and adawy, 2004; laila, et al. 2004; el-refaee, 2009; attia, 2004). some of these pathogens could transmit to man who eat fish meat or deal with fish and fish products (goncalves, et al. 1992; weinstein, et al. 1997; zlotkin, et al. 2003). aeromonas sp. and pseudomonas sp. isolated from o. niloticus by 35.96% and 16.88% respectively (abou el-atta, 2003). pseudomonas was widely distributed in ecosystem and was recognized as one of the primary cause of bacterial hemorrhagic septicemia in fish, pseudomonas septicemia, usually is associated with environmentally stressful conditions such as overcrowding, low temperature, injuries (aly, 1994; allen, et al. 1983). it may be a secondary invader of damaged fish tissue (roberts and home, 1978). p. fluorescens considered the causative agent of red spot disease attack all kinds of cultured fishes where the disease raised in running water ponds, stagnant water ponds as well as in cages (angka and lioe, 1982), the disease favored by stressor as low temperature, injuries and recorded that the incidence of pseudomonas septicemia was 11% (eissa, et al. 1996). the infected fish showed dark body coloration, exophthalmia with corneal opacity and hemorrhage in the eyes, loss of balance, frayed and torn tail and fins, scale detachment and skin discoloration with scattered hemorrhages all over the body surface with slight ascites, petechial hemorrhages were seen on the ventral abdominal wall and the base of the fins (badran & eissa, 1991; el-altar and moustafa, 1996). tilapias are known to harbour bacterial flora in their guts (sugita, et al. 1985; al harbi and uddin, 2005) showed that bacteria species isolated from the intestine of a tilapia species are predominantly gramnegative rods (87%). the main aim of the present study is to analyze the bacterial load of tilapia fish. materials & methods sample collection: we took seven samples of tilapia fish. one fish was healthy and three were infected. preserved in sterile polythene bag in fridge. fish sample was cut from the gill, skin, and intestinal region with a sterile knife. preparation of stock cultures: the cut sample was crushed into small pieces in sterile mortar with about 10ml sterile water. take 1ml crushed sample and 9ml distilled water to make up 1:10 dilution. isolation and identification of bacteria: inoculate 31 vol 5 (1), january 2014; 30-35 0.1ml of the diluted solution in nutrient agar and streak with the help of wire loop. all plates were incubated at 34oc for 24 hrs. the developed colonies on the plates were examined after the incubation period. these colonies were then isolated and identified according to their colonial, morphological and biochemical characterstics.. results & discussion in the present study, isolates from seven specimens of nile tilapia, (oreochromis niloticus) have shown in table i. one specimen of nile tilapia was fresh while six others were infected and used to isolate gram positive and gram negative bacteria. both fresh and infected specimens were used in the study because sometimes apparently without any symptoms the fresh specimens may carry the pathogenic diseases. the findings of the study revealed the isolation of both gram – p o s i t i v e b a c t e r i a : s t re p t o c o c c u s s p . a n d staphylococcus aureus and gram – negative: enterobacter sp. and pseudomonas aeruginosa and occluded that heavy contamination of water affects the health of fishes and aquatic organisms. the study of bacteriological analysis from tilapia fish revealed the presence of possible pathogenic micro-organisms as well as high coliform bacterial counts from fish samples. in the light of these major findings, the following recommendations are made; public health authorities and food inspection authorities of the country should 32 vol 5 (1), january 2014; 30-35 table i: cultural & biochemical characteristics of isolated grampositive bacteria from fish samples ensure adequate supervision and monitoring of food handling and sales especially ready to eat products like fried fish and cultured fish/fresh water fish. this research has brought to light those bacterial species associated with fresh tilapia fish and has shown that they are potentially pathogenic to humans. hence adequate measures should be taken in processing the fish before consumption. references abd el-latif, m.m. and r.s.m. adawy. 2004. studies on oreochromis niloticus in manzala lake, suez canal vet.med.j.v 11 (2) 403-410. abou el-atta. m. e. 2003. efficiency of polymerase chain reaction (p.c.r.) in diagnosis of some bacterial fish pathogens. ph.d.thesis in vet. sci (bacteriology, immunology and mycology) 44-63. adam a.j. tobaias w.j (1999) red mang rove proproot habitat as a finfish nursery area; a case study of salt rivea bay, st. croix, usvi. proc gulf caribb fish inst 46: 22-46 al-harbi, a. h. and uddin, n. m. (2005). bacterial diversity of tilapia (oreochromis niloticus) cultured in brackish water in saudi arabia. aquaculture, 250: 566-572. allen d.a., austin b. and r.r. colwell. 1983. numerical taxonomy of bacterial isolates associated with of freshwater fishery. j. of general microbiology, 129, 2043 2062 33 vol 5 (1), january 2014; 30-35 figure 1: prevalence of isolated bacteria from infected fish samples. s. aureus s. pneumonia enterobactor bacillus pseudomonas nonhemolytic streptococci figure 2: infected nile tilapia fish (oreochoromis niloticus) table ii: cultural & biochemical characteristics of isolated gram negative bacteria from fish samples 34 vol 5 (1), january 2014; 30-35 aly, s.e.m. 1994. pathological studies on some fish in suez canal area. ph. d.thesis (pathology) fact. of vet. med. suez. canal university angka, s.l and k. g. lioe. 1982. control of bacterial hemorrhagic septicaemia in cultured fish. bul. perel inst. pertan bogor 3 (3) pp 13-14 (in bahasa indonesia). apun, k., yusof, a.m., and jugang, k. (1999) distribution of bacteria in tropical freshwater fish and ponds. int. j. environ. health res. 9, 285ñ292. attia, y. m. a. 2004. studies on problems affecting gills of cultured fishes ph.d. thesis vet. sci. fish disease department. fact. of vet. zagazig university austin, b. and austin, d.a. (1987) bacterial fish pathogens, disease of farmed and wild fish. ellis horwood, chichester. badran, a.f., and i. a. eissa. 1991. studies on bacterial diseases among cultured fresh water fish (oreochromis niloticus) in relation to the incidence of bacterial pathogens at ismailia governorate j. of egypt. vet med. association, 51 (4), pp 837 847. douglas, d. (2007). identifying fresh water aquarium fish disease. eissa, i.a.m., a.f. badran, a.s. diab and a.m. abdel-rahman. 1996. "some studies on prevailing bacterial diseases among cultured tilapias in abbassa fish farms". proceedings of the 3rd vet. med. congress, zag, 8-10 october 1996, zagazig university, egypt, pp. 185-202. el-altar, a.a and m. moustafa. 1996. some studies on tail and fin rot disease among cultured tilapia fishes. assiut vet. med. j, 35, 70, 155 – 159 el-refaee, m.e. 2005. streptococcus infection in fresh water fish ph.d. thesis submitted to faculty of vet. med., (microbiology). alex. university, egypt evelyn, t.p.t. and mcdermott, l.a. (1961) bacteriological studies of freshwater fish. 1. isolation of aerobic bacteria from several species of ontario fish. can. j. microbiol. 7, 357ñ382 gillespie, n.c. (1981) a numerical taxonomic study of pseudomonas-like bacteria isolated from fish in southeastern queensland and their association with s p o i l a g e . j . a p p l . b a c t e r i o l . 5 0 , 2 9 ñ 4 4 . goncalves, j.r., g. braum, a. fernandes, biscaia, i., m.j.s. and j. bastardo. 1992. aeromonas hydrophila fulminate pneumonia in a fit young man tborax 47, 482-483. kim, s.h., field, k.g., chang, d.s., wei, c.i., and an, h.j. (2001) identification of bacteria crucial to histamine accumulation in pacific mackerel during storage. j. food protect. 64, 1556-1564. ladipo, o; fabiyi and fatula, g.t. (1981), marketing and distribution of fish in nigeria. report submitted to the federal development of fisheries, lagos. pg 35. laila, a.m., f.r. el-seedy, m.a. abdelaziz, and w.s. soliman. 2004. "aerobic bacteria isolated from freshwater fishes in egypt". the first international conference of the veterinary research division, national research centre, 15-17 february 2004, pp. 117 leisner. j.j., vancanneyt, m., rusul, g., pot, b. lefebvre, k., fresi, a. and tee, l.t (2001). identification of lactic acid bacteria constituting the predom, nating microflora in an acid feamented condiment (tempoyak) popular in malaysia. international journal of food microbio 63: 147-157. malle, p. (1994) bacterial microflora in marine fish and evaluation of spoilage. recl. med. vet. 170, 147ñ157 micheal, w., johan, suen, f; carina, k and tor m. (2007). journal of clinical microbiology published & applied sciences similarity index of articles should be less than 19% all articles are checked for plagiarism through turnitin software rads journal of biological research 35 vol 5 (1), january 2014; 30-35 by the american society for microbiology. 45:1-7. nieto, t.p., toranzo, a.e., and barja, j.l. (1984) comparison between the bacterial flora associated with fingerling rainbow trout cultured in two different hatcheries in the north-west of spain. aquaculture 42, 193ñ206 plumb, j. a. (1994). health maintenance of cultured fishes: principal microbial diseases. crc press, boca raton, fl. roberts r.j. and m.t. horne. 1978. bacterial meningitis in farmed rainbow trout salmogarirdneri richrdson, affected with chronic pancreatic necrosis j. of fish diseases 1, 157-164 sugita, h., tokuyama, k. and deguchi, y. (1985). the intestinal microflora of carp, cyprinus carpio, grass carp, ctenopharyn godonidella, and tilapia sarotherodon niloticus. bulletin of the. japanese society of scientific fisheries, 51: 1325-1329. weinstein, m. r., m. litt, d. a. kertesz, p. wyper, d. rose, m. coulter, a. mcgeer, r. facklam, c. ostach, b. m. willey, a. borczyk, and d. e. low. 1997. invasive infections due to a fish pathogen, streptococcus iniae. n. engl. j. med. 337:589-594. zlotkin, a., s. chilmonczyk, m. eyngor, a. hurvitz, c. ghittino and a. 2003. trojan horse effect: phagocyte-mediated streptococcus iniae infection o f f i s h . i n f e c t i m m u n . 7 1 ( 5 ) : 2 3 1 8 2 3 2 5 . 1rabia ehsan, 2amna shafiq, 1sayyada ghufrana nadeem and 2shazia tabassum hakim 1medical mycology research and reference laboratory and 2virology and tissue culture laboratory, department of microbiology, jinnah university for women, karachi-74600, pakistan. 11 rads vol 2 (2), (2011); 11-17 abstract contamination of ready-to-eat foods and beverages sold by street vendors and hawkers rendering them unacceptable for human consumption has become a global health problem. the present study was done to compare contamination in tetra pack fruit juices and street vended fruit juices. a total of 25 fruit juice samples were analyzed for presence of bacterial pathogens. out of 25 samples, 07 were street vended fruit juices samples while 18 samples were tetra pack fruit juices. spread plate method is used for the isolation of these bacteria. the bacterial pathogens present in juices were escherichia coli (42%), staphylococcus (42%), enterococcus (28%), streptococcus (14%), bacillus (14%) & diptheroids (14%). only one sample of tetra pack fruit juice was contaminated and the organism identified was bacillus. it was found that street vended fruit juices were highly contaminated as compare to tetra pack fruit juices. the contamination is mainly due to poor quality of water used as well as unhygienic conditions related to washing of utensils. however, health education of the fruit juice vendors and implementation of standard hygienic practices should be enhanced to reduce contamination of fruit juices. key words: contamination, tetra pack, street vendors, bacteria, fruit juice introduction fruit juices are well recognized for their nutritive value, mineral and vitamin content. in many tropical countries they are common man’s beverages and are sold at all public places and roadside shops. however in view of their ready consumption, quick methods of cleaning, handling and extraction they could often prove to be a public health threat. there are reports of food borne illness associated with the consumption of fruit juices at several places in india and elsewhere (parish, 1997; sandeep, et al. 2001). improper washing of fruits will add these bacteria to the extracts leading to contamination. in addition, use of unhygienic water for dilution, dressing with ice, prolonged preservation without refrigeration, unhygienic surroundings often with swarming houseflies and fruit flies and airborne dust can also act as sources of contamination. such juices have shown to be potential sources of bacterial pathogens notably escherichia coli o157:h7, s p e c i e s o f s a l m o n e l l a , s h i g e l l a a n d staphylococcus aureus (splittstosser, 1979; harrigan, 1998; buchanana, et al.1999). practices used during their preparation, handling, cleaning, sorting and grading, packaging, storing and wrapping in low grade plastics are some of the factors that increase the risk of acquiring microbes (ryu and beuchat, 1998). the health risk of such products in particular is dependent about food safety and hygiene among vendors is also resulting in food contamination (uljas and ingham, 1998). contamination of fruit juices may be due to the injured or spoiled fruits used for making juice or improper sanitary conditions maintained by the vendors during the entire process of cutting to serving. the coliforms, and bacillus cereus and staphylococcus aureus growth may be due to improper handling or may be due to use of contaminated water for processing. moreover, punctures, cuts and splits bacterial contamination in tetra pack and street vended fruit juice samples corresponding author. e-mail: shaz2971@yahoo.com 12 rads vol 2 (2), (2011); 11-17 are a potential source of entry of pathogens into fruits and vegetables. these insults can occur during growing or harvesting, while processing and improper handling also contributes to the entry of bacterial pathogens into the product, especially in juices prepared from these fruits (sandeep, et al. 2001). a sugarcane juice is a popular refreshing drink in many parts of asian countries. it is extracted by crushing the sugarcane between rollers drums and served with or without ice. hygienic standard are usually not maintained during the transport of sugarcane from the field of point of extraction and preparation of juice. further the juice is consumed unpasteurized therefore it is possible that the sugarcane juice may be contaminated and pose health hazards. it is found to be contaminated with different bacteria e.g., escherichia coli, klebisella pneumonia, staphylococcus aureus, enterobacter spp, citrobacter spp, pseudomans, enterococcus faecalis, acinetobacter spp and bacillus (lewis, et al. 2006). though, fruit juices are stored at low temperature to retain their wholesome fruity character for long period of time such as the grape juice at 22 to280 f (-5.5 to 2.2c) in the bulk storage, the quality of product may naturally unchanged for a period of 1 year or more however yeast contamination of grape juice is often observed (wolford and berry, 1948). the ability of different pathogens to survive in low ph environments has also been documented at length (pederson, et al. 1959; lin, et al. 1995; gahan, et al.1996; eribo and ashenafi, 2003; subannayya, et al. 2007). therefore, it is not surprising that unpasteurized juices, such as orange and apple juices, have been identi?ed as the vehicle of food borne pathogens in several outbreaks (parish, 1997). in particular, unpasteurized orange juice has been linked to several outbreaks of disease caused by salmonella sp. (cdc, 1999; zaika, 2001; teeteh and beuchat, 2003), shigella sp. (cook, et al.1998), or viruses (fleet, et al. 2000; krause, et al.2001) in different countries. although foodborne pathogens can be destroyed by pasteurization, consumption of unpasteurized juice occurs frequently due to consumer preferences. freshly squeezed juice may be an important source of pathogens if the fruit is contaminated (thurston, et al.1998). high hydrostatic pressure can be used to inactivate microorganisms and quality-deteriorating enzymes in foods (martý´nez-gonzales, et al. 2003), and, at least in some foods, like fruit juices, this process allows a better retention of the original ?avour and taste than does thermal treatment (hoover, 1993; knorr, 1993). besides good retention of ?avour, an important reason for using this process is that the low ph of fruit products (ph 3 to 4) does not support growth of pathogenic bacteria which may eventually survive pressurization (ogawa, et al. 1990). this was particularly well documented for escherichia coli o157:h7 after this organism was implicated in a number of recent infectious o u t b r e a k s c a u s e d b y c o n s u m p t i o n o f unpasteurized apple juice and cider (besser, et al. 1993; weagant, et al. 1994; leyer, et al. 1995). clearly, the ef?cient inactivation of escherichia coli will be a primary and nonnegotiable requirement for hp processes for the production of high-quality and safe fruit juices (ogawa, et al. 1990). a combination of bacterial and yeast biocontrol strains may also be effective against pathogenic bacteria (laio, 1 9 9 9 ) . r e p o r t e d m e c h a n i s m s f o r microorganisms that control food-borne pathogens include the reduction of the ph (laio, 2001), as in the application of lactic acid bacteria or the competition for nutrients and/or space (brashears and durre; 1999; geisen; 1999; spadaro, et al. 2004). lactic acid bacteria also produce bacteriocins (ganzle, et al. 1999; spadaro, et al. 2002) and they may act as biocontrol agents (laukova, et al. 2000). the main objective of this comparative study is to assess bacterial contamination in tetra pack fruit juices and street vended fruit juices. materials and methods sample collection: 25 samples were collected from different places of karachi city out of which 07 are street vended fruit juices and 18 are tetra pack fruit juice. sample analysis: for the analysis 25 ml of the sample diluted as 1:10 with 250 ml distilled water and filtered if any solid particle present. 100ul filtrate was used for inoculation. bacterial enumeration: isolation and enumeration was made by using different media like nutrient agar, macconkey’s agar, mannitol salt agar, emb agar, xylose lysine deoxycholate agar, and blood agar. the inoculation was done by using spread plate method. spread plate method: the spread plate method consists of evenly spreading the diluted sample over an agar plate.ê when using this method, a volume of 0.1 ml of the diluted sample should not be used since the agar will not be able to absorb the excess.ê using this method yields colonies that form on the surface of the agar. results and discussions street vended fruit juices are well appreciated by the consumers because of their taste, low price, and availability at right time (ohiokpehai, 2003). in spite of the potential benefits offered by fruit juices, concerns over their safety and quality have been raised; as freshly prepared juices have no process or steps to minimize the microorganisms if they are contaminated (mahale et al. 2008). in the present study a total of 25 juice samples were analyzed for the presence of bacterial pathogens. microbiological analysis of fruit juice samples was carried out and morphological as well as biochemical characterization of isolated organisms was done. heavy microbial growth was seen in street vended fruit juices while only one sample of tetra pack fruit juice shows the growth of bacillus and no growth observed in rest of the tetra pack fruit juice samples. the dominant bacterial pathogen present in street vended fruit juices were escherichia coli (42%), and staphylococcus (42%) (figure). a study carried out in india also reported similar results and the food borne illness associated with the consumption of road side freshly squeezed fruit juices. the dominant pathogen accounted in their study was also escherichia coli (40%). while 4% samples found to be contaminated with staphylococcus in their study (tambekar, et al. 2009). overall the results of the present study indicate that, majority of the street vended fruit juices in many parts of the city showed contamination with bacterial pathogens. figure: prevalence rate of bacteria isolated from tetra pack and street vended fruit juice samples. faecal coliforms are often found in street vended 45 40 35 30 25 20 15 10 5 0 es ch er ic hi a co li st ap hy lo co cc us st re pt oc oc cu s ba ci llu s en te ro co cc us di ph th er oi ds tetra pack fruit juice samplesstreet vebded fruit juice samples bacterial strains p re ce n ta g e o f b ec te ri al s tr ai n s (% ) 13 rads vol 2 (2), (2011); 11-17 14 rads vol 2 (2), (2011); 11-17 fruit juices it may be because the water used in preparation of juices is highly contaminated with fecal coliforms. moreover, unhygienic physical practices of street vendors also contribute to contamination of fruit juices. the condition of street food preparation and vending rise many concern for consumers health. in most cases running water is not available at vending sites, hands and utensils washing are usually done one or more buckets and sometimes without soap, wastewater and garbage are discarded nearly, providing nutrient for insects and rodents. some of the juices are not efficiently protected against flies, which may carry food borne pathogens safe food storage temperature are rarely applied to street vended juices in addition there are potential health risk associated with initial contamination of food by pathogenic bacteria as well as subsequent contamination by venders during preparation handling and cross contamination (barro, et al. 2006). escherichia coli, the most common faecal coliform is found to be associated with serious diseases in human. it causes diarrhea, urinary infections, pyogenic infections and septicemia etc (samonis et al., 2009). conclusions the present study’s aim to establish the status of street vended juices and tetra pack fruit juices. based on the undertaken study, it is concluded that street vended fruit juices are highly contaminated with bacterial pathogens as compare to tetra pack fruit juices. therefore, tetra pack fruit juices found to be hygienic for human consumption as they are haccp (hazard analysis and critical control points) control. the practice of selling fruit juices by street vendors cannot be prohibited as such activities provide them with a source of livelihood but government & the health agencies must adopt measure to educate the vendors about food safety and hygienic practice. the occurrence of pathogenic micro organisms is alarming enough for an immediate action by the suitable agency. monitoring of the quality of fruit juices on regular basis should be done to avoid bacterial pathogen outbreaks in future. refrences: barro, n., bello a.r., aly, s., ouattara, c. a. t., ilboudo, a., traoré, a. s. 2006. hygienic status an ssessment of dishwashing waters, utensils, hands and pieces of money from treet food processing sites in ouagadougou (burkina faso). afr. j. biotech., 5 (11), 1107-1112. besser, r. e., lett, s. m., weber, j. t., doyle, m. p., barrett, t. j., wells, j. g. and grif?n, p. m. 1993. an outbreak of diarrhea and hemolytic uremic syndrome from escherichia coli o157:h7 i n f r e s h p r e s s e d a p p l e c i d e r. j a m a , 269:2264–2266. beuchat, l. r. 2002. ecological factors in?uencing survival and growth of human pathogens on raw fruits and vegetables. microbes infect., 4:413–423. brashears, m. m. and durre, w. a. 1999. antagonistic action of lactobacillus 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escherichia coli o157:h7 in apple cider with and without preservatives. appl. environ. microbiol., 59:2526–2530. microbial degradation of plastic: a short study vol 8 (1), jan 2017 issn: 2305 8722 32 r a d s j . b i o l . r e s . a p p l . s c i 32 op e n ac c e ss f u l l l e n g t h a r t i c l e microbial degradation of plastic: a short study huda ali1 and irsa wahab1* 1 department of microbiology, jinnah university for women a b s t r a c t plastic is also one of the keycauses of ecologicalcontamination. accumulation of plastic pollutants in the environment becoming an ecological threat. the aim of this study is to isolate plastic degrading organism. plastic is not broken down the plastic. absence or low activity of catabolic enzymes that can attack its components. polyesters containing a high ratio of chemically inert components, such as pet, shown resistance against microbial degradation. buried soil and shake flask methods were used in this study. plastic samples were buried and incubated in different soil samples for 3month intervals. pseudomonas species (15.2%), proteus species (26.4%) and micrococcus species (46.1%) were isolated by the soil buried method. these species were tested forconfirming degradation of plastic by using the shake flask method. keywords plastic, degradation, enviornment, pollutants address of correspondence girl_gemini@live.com article info. received: april 3, 2017 accepted: may 29, 2017 cite this article: ali h, wahab i. microbial degradation of plastic: a short study. rads j. biol. res. appl. sci 8(1):32-36. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n plastic is a polymer consist of carbon along with hydrogen, nitrogen, sulfur and other organic and inorganic elements and is manufactured from fossil fuel . plastic is a non-renewable source which is non-biodegradable, tough, sturdy, moisture resistant, and light in weight (1). the manufacture of plastic increased from 0.5 million tons in 1950 to 260 million tons now (3). plastic is being gradually used in agriculture field also to obstruct weeds and for other purposes (4). accumulation of plastic wastes created the thoughtful threat to the environment and wildlife (5). soil pollutions are mainly produced due to seven different types of human activities (6). synthetic polymers, like plastic waste and synthetic polymers that are soluble in water are the major cause environmental pollution (7). poly venyle chloride is used in common consumer commodities, containing adhesives, detergents, oiling, diluents, automotive plastics, plastic type clothing, building material and toys and soap, shampoo, deodorants, fragrances, hair spray, nail polish are personnel care products as well as toys and building materials. furans and dioxins deadly hazardous gases are produced due to the burning of the plastic. one of the causes of ozone depletion (8). plastic are of two types: one is thermoplastics and the other is thermosetting polymers. thermoplastic plastic can be hard or soft by repeating heating and cooling process and they can be shaped repeatedly. thermosets plastics have endless molecular weight as they are highly cross linked structures (1). light, heat or biological processes are the part of biodegradation process (9).biodegradation is the method in which living organisms decompose the organic substances. biodegradation is frequently used regarding environmentalism, management of waste, bioremediation and to the plastic materials, due to their extended life cycle. throughout aerobic biodegradation of plastic, co2 o r i g i n a l a r t i c l e microbial degradation of plastic: a short study vol 8 (1), jan 2017 issn: 2305 8722 33 r a d s j . b i o l . r e s . a p p l . s c i 33 and h2o are produce and throughout anaerobic biodegradation c2o, h2o and ch4 are produced (10). microorganisms are responsible for degradation of ordinary and artificial plastic (10). biodegradation of plastic continues under different soil circumstances because microbes that are accountable for the course of biological degradation they have their own ideal growth circumstances in the soil (1). initially the polymer mineralized into different monomers because the polymers of large size cannot permit through the cell membrane, so first they depolymerized to enter into the cell and then they are mineralized or biodegraded inside the cell. many fungi that grow on polymers can also responsible of enlargement, to assist polymer permeation into the fungal cell (11). two sets of enzymes are involved in the biological degradation of polymers: extracellular and intracellular depolymerase (12). during degradation exoenzymes from micro-organisms split complex polymers to yield short chains of smaller molecules, to permit the bacterial outer semi-permeable membranes, then make use of bacteria as carbon and energy sources. this whole process is called as depolymerization. the co2, h2o, or ch4 produced as an end products called mineralization (13). degradation is made in two steps. the aerobic degradation in which oxygen is used to change complex material to produce co2 and h2o and microbial biomass, as an end product. the second type is anaerobic degradation in which microorganisms do not use oxygen for degradation. the biomass of microbial origin, co2, ch4, and h2o are products under anaerobic settings (15). m a t e r i a l s a n d m e t h o d sample collection: the soil samples were collected from sea view soil from karachi, garden soil from near by area and sludge soil from the dump site near board office, nazimabad, karachi. isolation of microorganisms: multiple soil sample was collected and serially diluted:1 gm of soil sample was serially diluted in 9.0 ml or distilled water to make 1:10, 1:100, 1:1000, 1:10000, and 1:100000 and from the last dilution 0.1 ml was taken and streak in to nutrient agar plate and incubate the plates at 370 c for 24 hours. after 24 hours any visible colonies were observed and subjected to biochemical, serological identification for the conformation. identification of microorganism: identification of the isolates were performed according to their morphological, cultural and biochemical characteristics. all the isolates were subjected to gram stain for the conformation of morphology and subjected to specific biochemical tests and spot test such as coagulase test, oxidase test, imvic and motility test were performed. r e s u l t s plastic is also one of the key causes of ecological contamination. accumulation of plastic pollutants in the environment is becoming an ecological threat. the aim of this study is to isolate plastic degrading organism. three bacterial strains were used in this study are checked for their morphological, biochemical, and cultural characteristics. the organism isolated and inoculated with the sea view soil sample was proteus mirabilis are gram negative rods and scattered in arrangement and gives gray swarming colonies with a uniform film ad fishy odor. catalase positive, coagulase negative, oxidase negative, highly motile, indole negative. on tsi butt acidic and slant alkaline, with h2s and gas production. from buried soil method percentage of plastic degraded by proteus mirabilis was 7.1% in comparison from a shake flask method percentage of plastic degraded by proteus mirabilis was 26.4%.the microbe isolated and inoculated with garden soil sample was pseudomonas being short gram negative rods which are scattered in arrangement and gives large, opaque, flat colonies with irregular margins with green pigmentation and fruity odor. catalase positive, coagulase negative, oxidase positive, motility positive, indole negative.at tsi both butt and slant are acidic with no h2s and no gas production. from soil buried method percentage of plastic degraded by pseudomonas was 8.1% and in comparison shake flask method percentage of plastic degraded by pseudomonas was 15.2%.the microbe isolated and inoculated with the sludge soil sample was micrococcus lutes are gram positive cocci arranged in pairs or in tetrads and gives circular, pinhead, bright yellow colonies with entire margins. catalase positive, coagulase negative, oxidase microbial degradation of plastic: a short study vol 8 (1), jan 2017 issn: 2305 8722 34 r a d s j . b i o l . r e s . a p p l . s c i 34 negative, motility negative, indole negative. at tsi both butt and slant are acidic with no h2s and no gas production. from soil buried method percentage of plastic degraded by micrococcus luteus was 34% and from shake flask method percentage of plastic degraded by micrococcus luteus was 46.1%. d i s c u s s i o n this study was conducted to overcome the pollution caused by plastic and its polymers. this study deals about the isolation, identification and the plastic degradative ability of microorganism isolated from different soil samples. microorganisms play a vital role in plastic degradation. in this depolymerization process 2 types of enzymes are actively involved, i.e.extracellular and intracellular depolymerases (10). during degradation exoenzymes from microorganisms breaks complex polymers into smaller molecules of short chain, e.g., oligomers, dimmers, and monomers, and are smaller that can pass the semipermeable outer membranes of the microbes, and then utilized as carbon and energy sources (14). depolymerization results caused by physical strength or biological that produced aerobic metabolism those are carbon dioxide and water, whereas anaerobic metabolism produced carbon dioxide, water and methane as a final product. it is necessary to investigate the distribution and population of polymer-degrading microorganisms in various ecosystems. generally, the adherence of microorganisms on the surface of plastics followed by the colonization of the exposed surface is the major mechanisms involved in the microbial degradation of plastics (17). each microbe has different characters, so degradation ability possessed will be varied between one microbe to another microbe. different characteristic includes type of enzyme produced in degradation process that helped in polymer degradation. from the sea view soil sample isolated the microbe was proteus mirabilis which can degrade plastic about 7.1%. thomas (2011) reported that staphylococcus aureus had the lowest degradation power. our result agreed with thomas who also found from the garden soil sample isolated the microbe was pseudomonas sp. which can degrade plastic about 8.1% (17). kathiresan k. 2003 reported that the pseudomonas sp. was able to degrade plastic by 8.16%. we agree with katherisan results. reported in 2003 (2). from the sludge, soil sample the isolated microbe was micrococcus luteus which can degrade plastic about 34%. sivasankari et.al (2014) reported that the micrococcus luteus were found most active in degrade 46.1% of plastic. our results are closely related to that value so we agree with sivasankari’s statement. in the test flask of sludge, soil the degradation rate of plastic by micrococcus luteus was about 46.1%. sivasankari et. al (2014) reported that the micrococcus luteus were found most active in degrade 38% of plastic. our results are higher than sivasankari’s results (16). table i: colonial morphology after the collection of samples were serially diluted and plated for the observable colonies. sample dilution no. colonial morphology seaview soil 10ˉ5 gray swarming colonies with a uniform film ad fishy odor later identified as proteus mirabilis garden soil 10ˉ5 large, opaque, flat colonies with irregular margins with green pigmentation and fruity odor later identified as pseudomonas sludge soil 10ˉ5 circular, pinhead, bright yellow colonies with entire margins later identified as micrococcus lutues microbial degradation of plastic: a short study vol 8 (1), jan 2017 issn: 2305 8722 35 r a d s j . b i o l . r e s . a p p l . s c i 35 table ii: results of gram staining of different sample process sample shape and arrangement gram reaction identification sea view soil rods and scattered in the arrangement negative proteus mirabilis garden soil short rods and scattered in the arrangement negative pseudomonas sludge soil cocci in tetrads and pair positive micrococcus luteus table iii: results of biochemical and other confirmation tests. sample catalase coagulase oxidase tsi motility test indole test sea view soil +ve -ve -ve butt acidic and slant alkaline, with h2s and gas production motile -ve garden soil +ve -ve +ve -ve motile -ve sludge soil +ve -ve -ve -ve non motile -ve table iv: result of degradation of plastic sample by the soil buried method after 3 months. sample initial wt. (gm.) final wt. (gm.) difference weight loss/month in % sea view soil 11.02 10.23 0.79 7.1% garden soil 12.3 11.3 1 8.1% sludge soil 7.5 4.95 2.55 34% table v: results of degradation of plastic samples by shake flask method. sample initial wt. (gm.) final wt. (gm.) difference weight loss/month in % sea view soil 1.4 1.03 0.31 26.4% garden soil 2.5 2.12 0.38 15.2% sludge soil 1.3 0.7 0.6 46.1% microbial degradation of plastic: a short study vol 8 (1), jan 2017 issn: 2305 8722 36 r a d s j . b i o l . r e s . a p p l . s c i 36 figure 1. graphical representation of results of shake flask method b) graphical representation of results of buried soil method. c o n c l u s i o n microbes isolated are: pseudomonas spp., micrococcus luteus and proteus mirabilis, micrococcus luteus has ability to degrade plastic too much that of other bacteria. although other proteus mirabilis and pseudomonas spp has not as much of an ability to degrade plastic as compared micrococcus luteus. micrococcus luteus show much better degradation of plastic as compare to other org like pseudo, proteus in other study. r e f e r e n c e s 1. kumari n.a., kumari p. and murthy n.s. a novel mathematical approach for optimization of plastic degradation, int. j. engg. trends and tech. 2013; 4 (8), 35393542 2. kathiresan k. polythene and plastics-degrading microbes from the mangrove soil, rev. biol. trop. 2003; 51(3), 629634 3. thompson rc, moore cj, vom saal fs, swan sh. plastics, the environment and human health: current consensus and future trends. philosophical transactions of the royal society of london b: biological sciences. 2009 jul 27;364(1526):2153-66. 4. colin g, j. d. cooney, d. j. carlsson, d. m. wiles. j. appl. polym. sci.,1981;26, 509–519. 5. chua asm, takabatake h, satoh h and mino t. production of polyhydroxyalkanoates (pha) by activated sludge treating municipal waste water: effect of ph, sludge retention time (srt), and acetate concentration influent. water res., 2003;37(15):360-361. 6. ghosh m, singh sp. a review on phytoremediation of heavy metals and utilization of its byproducts. appl environ., 2005; 3:1-18. 7. premraj r, mukesh cd. biodegradation of polymer. indian j biotech. 2005; 4:186-193. 8. iarc (international agency for the research on cancer). agents classified by the iarc monographs. lyon, france: world health organization, 2011. 9. olayan, h.b., hamid, h.s. and owen, o.d. photochemical and thermal crosslinking of polymers.jmacromolsci rev macromol chem phys. 1996; 36:671–719. 10. gu, j.d., ford, t.e., mitton, d.b. and mitchell, r. microbial degradation and deterioration of polymeric materials. w. revie (ed.), the uhlig corrosion handbook (2nd edition), wiley, new york. 2000. 11. toncheva, v., bulcke, a.v.d., schacht, e., mergaert, j. and swings, j. synthesis and environmental degradation of polyesters based on poly (ε-caprolactone) j environ polym degrad. 1996; 4;71–83. 12. winursito, i. and matsumura, s. biodegradability, hydrolytic degradability, and builder performance in detergent formulations of partially dicarboxylatedalginic acid. j environ polym degrad. 1996;4:113–121. 13. hiltunen, k., seppälä, j.v., itävaara, m. and härkönen, m. the biodegradation of lactic acid-based poly (esterurethanes).j environ polym degrad. 1997;5:167–173. 14. hamilton, j.d., reinert, k.h., hogan, j.v. and lord, w.v. polymers as solid waste in municipal landfills. j air waste manage assoc. 1995;43: 247–251. 15. barlaz, m.a., ham, r.k. and schaefer, d.m. mass-balance analysis of anaerobically decomposed refuse. j environ eng. 1989; 115:1088–1102. 16. sivasankari s., and vinotha t. in vitro degradation of plastics (plastic cup) using micrococcus luteus and masoniella sp. sch. acad. j. biosci. 2014; 2(2): 85-89. 17. tokiwa y., calabia b.p., ugwu c.u. and aiba s. biodegradability of plastics. int. j. mol. sci. 2009; 10, 3722-3742. isolation and study of cellular compoments of aerobacillus polymyxa along with its comparision in soil layers vol 8 (1), jan 2017 issn: 2305 8722 18 r a d s j . b i o l . r e s . a p p l . s c i 18 op e n ac c e ss f u l l l e n g t h a r t i c l e isolation and study of cellular components of aerobacillus polymyxa along with its comparison in soil layers warda shahid1 and naheed afshan1* 1 department of microbiology, jinnah university for women a b s t r a c t objectives: the main objective of this research was to isolate and to study the cell morphology and biochemical reactions of aerobacillus polymyxa and bacillus megaterium along with its habitat either in deep soil or aerobic soil. background: aerobacillus is from the family of panebacillus polymxa and bacillus megaterium belongs from “bacillaceae” family. these two organisms are gram positive, non-pathogenic bacteria found in soil that helps in nitrogen fixation. they both are equally important today but the main aim of this research to isolate them from the soil due to the characteristic importance of a. polymyxa to produce antibiotic and helps to remove biofilm formation where as b. megaterium is a good a source of producing industrial proteins due to its larger size than any other organisms. methodology: total16 samples were collected from aerobic & anaerobic soil, water and milk. the soil samples were cultured on tgb media and na for four days. results: out of 16 samples 9 samples have shown positive results for the colonies of a. polymyxa and out of 16 samples 12 samples showed positive results for b. megaterium further confirmed by biochemical reactions. keywords aerobacillus polymyxa, bacillus megaterium, soil layers address of correspondence naheedafshan7@hotmail.com article info. received: april 3, 2017 accepted: may 29, 2017 cite this article: shahid w, afshan n. isolation and study of cellular components of aerobacillus polymyxa along with its comparison in soil layers. rads j. biol. res. appl. sci 8(1):18-22. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n microorganisms are the microscopic organisms which may be single cell or multi-cellular. the study of microorganisms became vast after the discovery made in 1674 by antonie van leeuwenhoek(1).they may be either prokaryotic or eukaryotic. microorganisms are the diverse groups that are divided into bacteria, fungi, viruses, protozoa and algae. in every part of biosphere microorganisms are found anywhere. for example, we naturally contain microorganisms in every part of the body as the normal flora to protect us from other harmful microorganisms and their pathogenicity.these microbes are also present in soil that helps in the better growth of plants and soil expansion by nitrogen fixation (2). aerobacillus polymyxa and bacillus megaterium both are equally the most important micro-organisms today in the fields of industry, agriculture and medicines. they both are gram positive, endospore forming rod shaped bacterium having peritrichous motility that is nonpathogenic found in plants roots that help in nitrogen fixation and increases root expansion and plant growth. they are also found in aerobic and anaerobic soil and in marine sediments. besides this, these species are also important in medical and in agriculture fields (3). some strains of a. polymyxa are capable of producing an antibiotic known as “polymixin, paenibacillin, and fusaricidin”effective against gram positive and gram negative organisms and helps in removing biofilm o r i g i n a l a r t i c l e isolation and study of cellular compoments of aerobacillus polymyxa along with its comparision in soil layers vol 8 (1), jan 2017 issn: 2305 8722 19 r a d s j . b i o l . r e s . a p p l . s c i 19 formation formed by escherichia coli, streptococcus pneumonia and staphylococcus aureus (4, 5). their wide range of applications in industries is due to the secondary metabolites produce by this specie (6). they are also used in bio preservation of foods due to their antimicrobial activity (2, 7). a. polymyxa became its own genus in 1993 as it was first classified under the genus bacillus. this classification was done when the 16s rrna of 3 different bacilli was analyzed comparatively which showed the phylogenetic difference enough to classify a. polymyxain its own genus(7)(8). a. polymyxa is a chemoorganoheterotrophs. it utilizes variety of carbon source for the energy i.e. glucose, mannitol, glycerol, xylan, xylose and arabinose and produces acetoin, lactate and ethanol as metabolites. besides this, a. polymyxaare also mesohphilic that grows around at 30˚c optimally maintaining the ph around 4-7. they also produce h2 gas as a by-product. there are total 13 strains of this species (9, 5). b. megaterium is the best cloning host. it is the largest eubacteria than any other micro-organisms about 60 micrometers cubed hence the name “mega” means “relatively big”. due to its large size b. megaterium is wellsuited for research on cell morphology, such as cell wall and cytoplasmic membrane biosynthesis, sporulation, spore structure and cellular organization, dna partitioning, and protein localization (3, 6). bacillus megaterium is one of the first bacteria’s genome to be coded completely (7). it can produce numerous plasmids remaining stable to its unique external proteases (3). this specie is able to produce variety of industrial proteins that due to the absence of alkaline proteases which allows for recombinant protein synthesis (8). besides this b. megaterium is a commercially available, non-pathogenic host for producing many biotechnological substances, including vitamin b12, penicillin acylase, and amylases (9). b. megaterium is known to produce poly-γglutamic acid (3, 9).using the penicillin amidase produced in this organism many synthethic penicillin have been derived (10). harvested glucose dehydrogenase is used in glucose blood tests; ß-amylases which are often used in the bread industry; and neutral proteases which are used by the leather industry (3, 10). b. megaterium is a cell factory towards the production of vitamin b-12.in addition to being a common soil bacterium and an endophyte, it can be found in various foods, including honey, in which most microorganisms do not grow and on a variety of surfaces, including clinical specimens, leather, paper and stone (23, 24). it is also able to survive in extreme conditions such as desert environments due to the spores it forms (11, 12). a. polymyxa and b. megaterium are fastidious organisms therefore they grow best on tgb media (15). they required 3 – 40oc to grow (3, 16). to isolate them from soil it requires 4 days of incubation period in an incubator (17). they appear as white, mucoid, opaque on cultural plates. they may appear as spreaded or pin-pointed colonies. microscopically they may appear as long, scattered or diploid or in chains as well (25). they may be vegetative cells microscopically (18). there many microorganisms found in soil i.e. bacteria, fungi, actinomyces, protozoa and algae. up to 10 billion bacterial cells inhabit each gram of soil in and around plant roots, a region known as the rhizosphere (20). in this research, i have studied about the isolation of a. polymyxa and b. megaterium from different sources of soil and other samples to study its cell morphology and biochemical reactions to differentiate it from other species (21). isolating this organism was difficult due to the high chances of growth of bacillus subtilis and other organism commonly found in soil and environment (22). m a t e r i a l s a n d m e t h o d samples collection: total 16 samples for a. polymyxa and 18 samples for b. megaterium were collected, for a. ploymyxa aerobic and 8 anaerobic soil samples from different sources were taken while for b. megaterium4 aerobic soil samples, 4 anaerobic soil samples, 4 milk powder samples and 4 distilled water sample were taken. isolation of cultures: samples were serially diluted upto 104 dilution and were poured in the tgb and na media. 8 samples were poured in tgb medium and 8 samples were poured in nutrient agar for a. polymyxa. 2 samples from each source were poured in tgb while 2 samples from each source were poured in na for b. megaterium. plates were then incubated at 37˚c and were allowed to isolation and study of cellular compoments of aerobacillus polymyxa along with its comparision in soil layers vol 8 (1), jan 2017 issn: 2305 8722 20 r a d s j . b i o l . r e s . a p p l . s c i 20 grow for 4 days. plates were checked daily in order to check the growth. identification of cultures: 5th day plates were observed for cultural morphology. the grams staining technique was applied on the cultures and observe the gram stained smear under the oil immersion lens (100x). the biochemical tests are also performed for the further identification of colonies. r e s u l t s 16 out of 9 samples have showed positive results for a. polymyxa. 8 samples appear to be white, thick, opaque and mucoid colonies on tgb medium while 1 plate of nutrient agar showed clonies of a. polymyxa along with bacillus subtilis. other 7 plates of na show colonies for bacillus subtilis. these results were further confirmed by biochemical reactions. 16 out of 8 samples showed positive results for b. megaterium. from the anaerobic samples 2 plates of tgb media and 1 plate of na showed positive results. the colonies appear to white mucoid thick colonies. aerobic soil samples give positive results on both tgb media. milk samples give positive results from 1 tgb media and 1 na media. water sample show no results for b. megaterium in any of the plates except for bacillus subtilis. the remaining negative plates have sown the results for bacillus subtilis, staphylococcus aureus and streptococcus. table i: the colony morphology of a. polymyxa on tgb and na table ii: the colony morphology of b. megaterium on tgb and na s. no. sample tgb na microscopy 1. anaerobic soil sample large, white, thick, opaque, mucoid and round colonies spreaded, mucoid, round white colonies thick chains of capsulated spore forming scattered rods. 2. aerobic soil sample white, thick and pin pointed, small, round colonies capsulated scattered spore forming rods 3. powdered milk spreaded large and small, white, hard and pin pointed colonies spreaded, large and pin pointed white round colonies capsulated spore forming cocci in chains 4. distilled water s. no. sample tgb na microscopy 1. anaerobic soil sample large, white, thick, opaque, mucoid and round colonies large, thick, opaque, mucoid round colonies. small scattered rods and diplococcic. 2. aerobic soil sample white, thick and pin pointed, small, round colonies long and short scattered rods isolation and study of cellular compoments of aerobacillus polymyxa along with its comparision in soil layers vol 8 (1), jan 2017 issn: 2305 8722 21 r a d s j . b i o l . r e s . a p p l . s c i 21 fig 1: colonies of a) a.polymyxab) b. megaterium chart: proportion of a) a. polymyxa b) b. megaterium fig 2: microscopic examination of a) b. megaterium b) a. polymyxa d i s c u s s i o n this article concludes two main points for both microorganisms. the ratio of a. polymyxa in anaerobic soil is greater (70%) as compared to aerobic soil i.e., the upper layer of soil (30%). these organisms are mainly involved in nitrogen fixation that also promotes the soil fertility. they protect plants from other harmful microorganisms by producing an antibiotic “polymixin” that is also used medicinally. second part of this research was to study the cell morphology and their arrangements on growth culture media as well as under microscope. they appeared as white, opaque, thick, mucoid, small spread, and pin pointed colonies on tgb media and na. under microscope these organisms appeared as long and small scattered rods. the ratio of b. megaterium in anaerobic soil is greater (60%) as compared to aerobic soil i.e., the upper layer of soil (40%). likewise, these organisms are also helpful in nitrogen fixation, promote soil fertility. they are the best producers of proteins and synthetic penicillin. isolating this specie can be useful in research for cell morphology and others, also being helpful in housing several plasmids. second part of this research was to study the cell morphology and their arrangements in media as well as under microscope. they appeared as large and small spread, white, hard, and pin pointed colonies on tgb media and na. under microscope these organisms appeared as thick chains of capsulated spore, forming scattered rods. c o n c l u s i o n this research provide the information about the isolation method of a. polymyxa and b. megaterium, their habitat and their cell morphology. it also showed that they are mostly present in deep roots where they play an important role in fixing nitrogen and protecting plants. the antibiotic polymixin produce by a. polymyxa is highly efficient in removing biofilm formation. these organisms can be widely use in industries due to the activity involved in bioflocculation and biopreservation of foods. b. megaterium on other hand is highly involved in protein synthesis which is widely used in industrial processes and is helpful in making synthetic penicillins. growing these two microorganisms in laboratory according to their growth conditions can be helpful for various purposes. they can be very helpful in today’s environment. r e f e r e n c e s 1. van der heijden, m. g., bardgett, r. d., & van straalen, n. m. the unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. ecol lett, 2008; 11(3), 296-310. 2. gest, h. the discovery of microorganisms by robert hooke and antoni van leeuwenhoek, fellows of the royal society. notes rec royal soc. 2004; 58(2), 187-201. 3. eppinger, m., bunk, b., johns, m. a., edirisinghe, j. n., kutumbaka, k. k., koenig, s. s., ... & martin, m. genome sequences of the biotechnologically important bacillus megaterium strains qm b1551 and dsm319. j bacterio, 2011; 193(16), 4199-4213. 4. zengguo, h.d. kisla et al. isolation and identification of paenibacillus polymyxa strain that coproduces a novel lantibiotic and polymixin. app environ microbiol. 2007; 73: 168-178. 5. biiniie v, pozsgi n, bacteriologie medicala, vol ii, ed, medicala, bucuresti. microbewiki.source 1985. 6. a. e. francis and joan e. rippon, bacillus polymyxa and its bacteriophages, wellcome research laboratoriest beckenham, kent. 1949. isolation and study of cellular compoments of aerobacillus polymyxa along with its comparision in soil layers vol 8 (1), jan 2017 issn: 2305 8722 22 r a d s j . b i o l . r e s . a p p l . s c i 22 7. ama am j dis child. antibiotics derived from bacillus polymyxa, 1995. 8. rudacunescu h., valeria bicaa-popii. bacteriology veterinara, ed. ceres , bucuresti, 1986. 9. gordan r.e., haynes w.c., pang c.h. – the genus bacillus. agriculture handbook no. 427, u.s.d.a., washington d.c. 1973. 10. ash c., farrow j. a. e., wallbanks s., collins m. d. phylogenetic heterogeneity of the genus bacillus revealed by comparative analysis of small subunit ribosomal rna sequences. lett appl microbiol. 1991; 13:202–206. 11. biedendieck r., et al. export, purification, and activities of affinity tagged lactobacillus reuteri levansucrase produced by bacillus megaterium. appl microbiol biotechnol. 2007; 74:1062–1073. 12. christie g., lazarevska m., lowe c. r. functional consequences of amino acid substitutions to gervb, a component of the bacillus megaterium spore germinant receptor. j bacteriol.,2008; 190:2014–2022. 13. chambon p, dupraw ej, kornberg a. biochemical studies of bacterial sporulation and germination. ix. ribonucleic acid and deoxyribonucleic acid polymerases in nuclear fractions of vegetative cells and spores of bacillus megaterium. j biol chem. 1968; 243(19):5101–5109. 14. setlow p. polyamine levels during growth, sporulation, and spore germination of bacillus megaterium. j bacteriol. 1974; 117 (3):1171–1177. 15. setlow p. identification and localization of the major proteins degraded during germination of bacillus megaterium spores. j biol chem., 1975; 250(20):8159– 8167. 16. de vos, p. et al. bergey's manual of systematic bacteriology: volume 3: the firmicutes. springer (2009). 17. shimizu, k., nakamura, h. & ashiuchi, m. salt-inducible bionylon polymer from bacillus megaterium. appl environ microbiol., 2007; 73:2378–2379. 18. claus, d. anreicherung und direktisolierung aerober sporenbildender bakterien. in schlegel, 1965. 19. ravi, a.v., k.s. musthafa, g. jegathammbal, k. kathiresan, and s.k. pandian. 2007. screening and evaluation of probiotics as a biocontrol agent against pathogenic vibrios in marine aquaculture. lett appl microbiol. 2007; 45(2): 219-223. 20. lal, s. and s. tabacchioni. ecology and biotechnological potential of paenibacillus polymyxa: a minireview. indian j microbiol. 2009; 49(1): 2-10. 21. kim, j.f., h. jeong, s.y park, s.b. kim, y.k. park, s.k. choi, c.m. ry, c.h. hur, s.y. ghim, t.k. oh, j.j. kim, c.s. park, and s.h. park. genome sequence of the polymyxin-producing plant-probiotic rhizobacterium paenibacillus polymyxa e681. j bacteriol. 2010; 192(22): 6103-6104. 22. eppinger, m., bunk, b., johns, m. a., edirisinghe, j. n., kutumbaka, k. k., koenig, s. s., ... & martin, m. genome sequences of the biotechnologically important bacillus megaterium strains qm b1551 and dsm319. j bacteriol. 2011; 193(16), 4199-4213. 23. celińska, e., & grajek, w. biotechnological production of 2, 3-butanediol—current state and prospects. biotechnol adv. 2009; 27(6), 715-725. 24. kim, j. f., jeong, h., park, s. y., kim, s. b., park, y. k., choi, s. k., ... & kim, j. j. genome sequence of the polymyxin-producing plant-probiotic rhizobacterium paenibacillus polymyxa e681. j bacteriol. 2010; 192(22), 6103-6104. 25. mishra, a. k., lagier, j. c., rivet, r., raoult, d., & fournier, p. e. non-contiguous finished genome sequence and description of paenibacillus senegalensis sp. nov. stand genomic sci. 2012; 7(1), 70. application of zno nanoparticles in biological treatment of municipal wastewater. vol 8 (1), jan 2017 issn: 2305 8722 42 r a d s j . b i o l . r e s . a p p l . s c i 42 op e n ac c e ss f u l l l e n g t h a r t i c l e application of zno nanoparticles in biological treatment of municipal wastewater wajeeha khan department of microbiology, jinnah university for women a b s t r a c t with the fast development of nanomaterials and nanotechnology, environmental nanotechnology has captivated increasing concerns in the past few decades. there are several nanomaterials in the earth, and a variety of modification held in future development. these zinc nanoparticles illustrate antibacterial, anti-corrosive, and uv filtering premises. in the study, we determine the antibacterial action of nanoparticles of zinc oxide for the curing of city wastewater. sample of wastewater were collected from sewage water, make three samples the sample i exposed to uv, and sample ii is treated with chlorine while other is control. samples were than cultured on na and emb agar, next day microscopy, biochemical test, spot test to identify the specie was performed accordingly. from control sample escherichia coli while from uv exposed and chlorine treated organism identified as staphylococcus. epidermidis respectively, nano particles of zinc were prepared by mechano-chemical method then these nanoparticle were characterize by transmission electron microscopy, then their antimicrobial action obtain to separated wastewater bacteria were calculated by determining the disk diffusion test results shows clear zone of inhibition against both escherichia coli and staphylococcus epidermidis on mha, the mic test was performed and we observed mic at concentration 32 and 64 concentration in s.epidermidis while in e.coli concentration 64 . wastewater obtain after chlorination and uv exposure are compared by the previous results. the resultant of experiment suggests that to teat wastewater, zno nanoparticles can act as antimicrobial agent. the class of bacterial strains is directly depended by effectiveness of antimicrobial action of nanoparticles of zno, in other words activity increase by increase in the concentration of nanoparticles. keywords antibacterial action, chlorinetreated,uv exposure, wastewater, minimum inhibitory concentration, zinc nanoparticle address of correspondence vj.smilestar@gmail.com article info. received: april 3, 2017 accepted: may 29, 2017 cite this article: khan w. application of zno nanoparticles in biological treatment of municipal wastewater. rads j. biol. res. appl. sci 8(1):42-7. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n nano sized particles of semiconductor materials have increased considerably more interest for later years because of their sensitive properties and applications in various ranges, for example, impetuses, sensors, photoelectron instruments, very functional and dominant instruments. these nanomaterials have novel electronic, basic, and warm properties which are of high research interests in essential and connected fields (1).in this research, we detailed the successful combination of nanoparticles of zno by a basic arrangement combusting strategy utilizing a blend of ethanol and ethyleneglycol (v/v = 60/40) powdered x-ray diffraction, transmission electron microscopy were utilized to reveal the stage and the structure of the end product. the ultraviolet–visible (uv–vis) absorption of the product was examined (2). chlorination is considered as a most usually conventional purification prepare for the treatment of both drinking and recycled water. chlorination has been found to have an important part in inactivating waterborne pathogens in numerous nations o r i g i n a l a r t i c l e application of zno nanoparticles in biological treatment of municipal wastewater. vol 8 (1), jan 2017 issn: 2305 8722 43 r a d s j . b i o l . r e s . a p p l . s c i 43 before discharge into accepting streams, waterways or seas (3). in this study, we also carried out the potential of zno nanoparticle against e. coli and s.epidermidis a using the zno plate forms and observed that zno nanoparticles have significant potential against the s.epidermidis and e. coli as compare to control. water is the most vital substance in our life. reliable and convenient access to clean, protected and moderate water is thought to be a prominent amongst the most basics for person. around, one-6th of the world’s population experiences access to clean drinking water (4). in the study, it widely considered thoughts behind the antimicrobial action of zno-nanoparticles covering procedures of assessing microorganism’s sustenance. in the ensuing segments, we have discussed about the elements influencing the antimicrobial action, including uv light, zno molecule estimate, focus, morphological, facial development by toughening, surface imperfections, and the base inhibitory fixation. a concise introduction of an exploratory contextual investigation, conveyed by creators on antibacterial activity to e. coli and s. epidermidis, was investigated (5). m a t e r i a l s a n d m e t h o d synthesis of zno nps: zinc acetate was dried at 60 ºc in air for 4 hours prior to its use. zinc acetate and oxalic acid were mixed in an agate mortar with molar ratio of 2:3 that means 2.5g zinc and 7.5g oxalic acid and were powdered for 45 min at room temperature. the white precursor was calcined at 160 ºc in air in a porcelain crucible for 30 min to prepare the zno nps. the transmission electron microscopy (tem) micrograph of the zno nps are shown in the study by alinezhad et al. waste water treatment: sewage waste water were collected in different areas of karachi, pakistan. the unfiltered and untreated samples were collected in 10ml syringes. through settling large particles were drain out. the samples were then separated into two portion and marked as 1 and 2. the chlorine treatment was given to one sample marked as 1, 1ml sewage sample was poured into petri plate, was treated with disinfectant, using the technique of treatment by liquid bleach commonly known as “chlorination”. 0.5ml chlorine solution was added by using sterile pipette to be treated with water. the solution was agitated thoroughly and left for 30 minutes. ultraviolet exposure was given to sample marked as 2, uv experiment was done by utilizing standard light emission illumination. the uv light was turned on for no less than 5 min before start of the trial to get a consistent uv force yield. 1 milliliters of sample was pipetted to a sterile glass petri dish, so that the uv light shaft was straightforwardly engaged onto the petri dish through source for 2 minutes. bacterial separation: the wastewater tests (previously, then after purified by chlorine or ultraviolet) were refined on supplement agar medium. after being incubated at 37 ˚c for 24–48 h. next day observe colonies of 3 nutrient agar plates. small and irregular cream mucoid colonies were observed on control plate. pinpointed small mucoid colonies were observed in uv plate. pinpointed mucoid opaque small colonies were observed in chlorine plate.the growth was then visualized by performing standards gram stain procedure given in the manual of clinical microbiology. antibacterial activity of zno nps: disk diffusion assay: prepared zno nps were disinfected through uv light for 1 minute. at that point, they were suspended in clean ordinary saline and stir until a clear suspension formed after which 1.5×108 cfu/ml of every bacterium (e. coli, and s. epidermidis isolated from each specimen) were splashed on mueller– hinton medium. after giving the microscopic organisms a chance to dry (inside 5–10min), twenty microliters of zinc oxide nanoparticles suspensions were added into the hole which was burrow by the assistance of borer. the immaculate dissolvable was likewise utilized for control. the zone of inhibition was measured after the brooding procedure at 37 wc for 24h. the test was tried three circumstances and the outcomes were found the middle value of. minimum inhibitory concentration: the minimum inhibitory concentration is the least fixation that may bring about entire development barrier. the soup microdilution strategy was connected to assess the minimum inhibitory concentration. 100ul of mueller–hinton stock, contain 105 cfu/ml of every microbe, was drop to each well. 100 application of zno nanoparticles in biological treatment of municipal wastewater. vol 8 (1), jan 2017 issn: 2305 8722 44 r a d s j . b i o l . r e s . a p p l . s c i 44 microliters of zinc oxide dilutions (fixations differed from 0.01 to 64 mm) was additionally filled 96 sanitized well microplates and after that incubated at 37 wc for 24 h. the first well, which had no development of bacteria, was considered for minimum inhibitory concentration. every focus was tried three circumstances and the outcomes were found the middle value. r e s u l t s the outcomes of uv radiations in most of the wastewaters samples have been reported to be almost the same as those in chlorine, indicating that it had very heavy or high impact on inactivation of bacteria. after incubation the percentage of growth of microorganism after exposure with uv dose as a disinfectant treatment was found to be very low. chlorine have inhibited 90% of microorganisms in all samples which is higher than that of uv.a chlorine concentration of 0.5 ml reduced the percentage of growth of microorganism to a detectable level. in e.coli and s.epidermidis both observe complete zone of inhibition. the presence of a large zone showed the antibacterial activity of zno nanoparticles. size of the zone was distinctive as indicated by the sort of microorganisms, the size and the properties of zno nanoparticles. zno nanoparticle inhibited e. coli high as compare to s. epidermidis. fig 1:inhibition of growth after uv disinfectant on na and emb agar fig 2: inhibition of growth after chlorine disinfectant on na and emb agar fig 3: zno nanoparticle inhibited e. coli high as compare to s. epidermidis figure 4: results of inhibition of e. coli and s. epidermidis by zno nanoparticles 0.1 10.1 20.1 30.1 40.1 50.1 60.1 c o n ce n tr a ti o n s o f z n o n a n o p a e rt ic le s growth of inhibition e.coli s.epidermidis fig 5: inhibition increases with the increases concentration of zno nanoparticles application of zno nanoparticles in biological treatment of municipal wastewater. vol 8 (1), jan 2017 issn: 2305 8722 45 r a d s j . b i o l . r e s . a p p l . s c i 45 d i s c u s s i o n for the extraordinary significance of wastewater checking extraordinarily the discovery of pathogenic microorganisms, there is focal requirement for inventive sensors. the natural impact and danger of a material are basic issues in materials choice and outline for wastewater sanitization. most likely nanotechnology is superior to anything other strategy utilized as a part of water treatment. although water assumes a critical part in each element of human action. in pakistan, the condition is not distinctive by any stretch of the imagination, the water accessible for residential and family unit utilize is generally tainted with sewage water or in a few ranges even the sewage water itself is utilized as the main water asset. it is perceived that sewage water treatment and their applications assume an imperative part in settling issues identifying with water deficiency and water quality. sterilization from pathogen organisms is one of the fundamental stride in the treatment of sewage water. in any case, the most widely recognized methods for sterilization i.e. chlorination and uv radiation, are presently observed to be unsuccessful keeping in mind the end goal to give 100% purification against pathogenic microorganism. to beat these lacks uv and chlorine disinfectant medicines, we researched the antibacterial movement of zno nanoparticles. the essential sizes of zinc oxide nanoparticles are one of the critical variables for the antibacterial movement. in our review, zinc oxide nanoparticles were described by amrita vish was method. the sizes of zinc oxide nanoparticles were 75±20 nm as dictated by this technique. in this review, the portrayal of the nanoparticles was inspected. antibacterial exercises against e. coli and s. epidermidis were assessed by good dissemination agar techniques. the nearness of a restraint zone unmistakably showed the antibacterial impact of zno nanoparticles. as it was additionally appeared in the investigation of razvan et al. (2010c) it has been found in this review by expanding the convergence of zno nanoparticles in wells, the development restraint has additionally been expanded. the measure of restraint zone was diverse as indicated by the kind of microscopic organisms, the size and the convergences of zno nanoparticles. number of state shaping unit (cfu) of e. coli and s. epidermidis after overnight hatching at the nearness of various groupings of zno nanoparticles was appeared. the base convergence of zno nanoparticles which hindered the development of microorganisms was 64 mg/ml for e. coli and 32 mg/ml and 64 mg/ml for s. epidermidis. this is in concurrence with beforehand distributed reports on the antibacterial properties of zno nanoparticles which demonstrated that the base fixation at which the development of e. coli and s. epidermidis was restrained was also to this fixation individually. considering the outcomes acquired from mic, and good agar dissemination strategies, it can be recommended that in correlation with gram-positive microbes, the development of gram-negative microorganisms is restrained at higher centralizations of zno nanoparticles (6). reddy et al. have announced similar outcomes, accentuating on the higher powerlessness of grampositive microorganisms in examination with gramnegative microbes. in the review done by selahattin et al., it has been suggested that the higher weakness of grampositive microbes could be identified with contrasts in cell divider structure, cell physiology, digestion system or level of contact (8). our investigation looked to investigate the antibacterial properties of both zno nps towards essential waterborne pathogens, e. coli, and s. epidermidis. the outcomes demonstrated that the inhibitory impacts expanded as the groupings of zno nps expanded. our review does not, be that as it may, demonstrate the imperative relationship between the extent of zno nps and antibacterial properties. these outcomes demonstrate that zno nps can be possibly viewed as a successful antibacterial operator for waterborne pathogens and can be considered as an integral strategy for water and wastewater treatment (7). an objective of this audit is to set a well-manufactured reference for researchers inspired by antibacterial exercises alongside their useful applications by considering nanotechnology standards as it identifies with the nano biological lethality of zno-nps. the honorable properties and appealing qualities of zno-nps present significant poisonous quality to creatures, which have made zno-nps fruitful applicant among other metal oxides. other specific properties are anticipated to extend zno-nps applications in a few ranges, especially in application of zno nanoparticles in biological treatment of municipal wastewater. vol 8 (1), jan 2017 issn: 2305 8722 46 r a d s j . b i o l . r e s . a p p l . s c i 46 catalysis and biomedicine. a conceivable research road is the blends with different classes of antibacterial operators, for example, the utilization of zno-nps as supporter of silver nps, which are antibacterial specialists that contain silver as antecedent. this point is viewed as an effective application figure and attractive significance. more accentuation ought to be given to the connection between’s zno-nps auxiliary, optical, electrical, compound properties, and their bacterial harmfulness. zno-nps can go about as keen weapon toward multidrug-safe microorganisms and a gifted substitute way to deal with anti-infection agents. in future, these nanoparticles may supplant routine anti-infection agents in people and creatures. be that as it may, antibacterial impacts, security, and nitty gritty instruments of zinc oxide nanoparticles ought to be further considered in vitro and in vivo. r e f e r e n c e s 1. world health organization (who). emerging issues in water and infectious disease; who: geneva, switzerland, 2003. available from: http://www.who.int/water_sanitation_health/emerging/emer ging.pdf 2. world health organization (who). drinking water, fact sheet; who: geneva, switzerland, 2015. available from: http://www.who.int/mediacentre/factsheets/fs391/en/ 3. shang, c.; cheung, l.m.; liu, w. ms2 coliphage inactivation with uv-light and free chlorine/monochloramine. environ eng sci. 2007;24, 1321–1332. 4. liu, y., he, l., mustapha, a., li, h., hu, z. & lin, m. antibacterial activities of zinc oxide nanoparticles against escherichia coli o157: h7. j appl microbiol. 2009; 107:1193–1201 5. zhang z, couture a, luo a. an investigation of the properties of mg-zn-al alloys. scripta materialia. 1998; 39(1):45-53. 6. elmi f et al. the use of antibacterial activity of zno nanoparticles in the treatment of municipal wastewater. water sci technol. 2014; 70:763–770 7. emami-karvani z, chehrazi p. antibacterial activity of zno nanoparticle on gram-positive and gram-negative bacteria. afr j microbiol res. 2011; 5(12):1368-73. 8. ansari ma, khan hm, khan aa, sultan a, azam a. synthesis and characterization of the antibacterial potential of zno nanoparticles against extended-spectrum β-lactamases-producing escherichia coli and klebsiella pneumoniae isolated from a tertiary care hospital of north india. appl microbiol biotechnol. 2012; 94(2):467-77. lactic acid bacteria from fresh fruits and vegetables as biocontrol agent of foodborne bacterial pathogens syesa anum zehra1, sumaira javed2, sayyada ghufrana nadeem2, shazia tabassum hakim3 1department of microbiology, jinnah university for women, karachi-74600, pakistan 2medical mycology research and reference laboratory, department of microbiology, jinnah university for women, karachi -74600, pakistan. 3virology and tissue culture laboratory, department of microbiology, jinnah university for women, karachi -74600, pakistan. abstract the presence of lactic acid bacteria (lab) in dairy products has long been established but the occurrence of the particular group of bacteria in fruits has rarely been focused. therefore, different lactic acid bacteria generally found in fruits, cultivated in pakistan were studied. following the culture enrichment method randomly selected fruit samples were analyzed. about 12 different isolates of lactobacillus spp. were obtained from 25 different fruits and vegetables. the identification was based on conventional morphological and biochemical analysis. prior to all these manipulations the growth conditions were carefully optimized for the respective strains. this study evaluated the efficacy of lactic acid bacteria (lab) isolated from fresh fruits and vegetables as biocontrol agents against the foodborne human bacterial pathogens (escherichia coli, enterococcus faecalis, proteus sp. and pseudomonas aeruginosa). the antagonistic activity of lab strains were tested in vitro and all tested microorganisms except few were inhibited by at least one isolate. cell-free supernatants of selected antagonistic bacteria were studied to determine the nature of the antimicrobial compounds produced. k e y w o r d s : a n t a g o n i s t i c a c t i v i t y, l a c t i c a c i d b a c t e r i a , m i r c o o rg a n i s m s , p r o b i o t i c s . introduction lactobacillus is a genus of gram positive facultative anaerobic or microaerophilic rod-shaped bacteria, occurring in chains. they are non-sporulating, facultatively heterofermentative, catalase negative, cytochrome oxidase negative and non motile. the lactobacilli are strictly fermentative and have complex nutritional requirements (madigan et al., 1997). they are a major part of the lactic acid bacteria (lab) group, named as such because most of its members convert lactose and other sugars to lactic acid (makarova et al., 2006). in humans they are present in the vagina and the gastrointestinal tract, where they make up a small portion of the gut flora. lactic acid bacteria may isolate from fermented foods, dairy products, grain products, meat and fish products, pickles, sour dough, fruits & vegetables. in nature, all plant surfaces contain lactobacilli in low numbers (mundt and hammer, 1968) and also grow luxuriantly with other lactic acid bacteria in manure decaying plant material, especially decayed fruits. hence, lactobacilli are also important for the production and spoilage of fermented vegetable feed and food (e.g. silage, mixed pickles) and beverages (e.g. beer, wine, fruit juices). these organisms have been used for many years for production of fermented food. lactic acid bacteria are chiefly responsible for the raw material through the production of organic acids, mainly lactic acid. in addition, their production of acetic acid, ethanol, aroma compounds, bacteriocins,*corresponding author: samira__javed@hotmail.com 36 vol 5 (1), january 2014; 36-45 exopolysaccharides, and several enzymes is of importance. lactic acid bacteria produced different antimicrobial molecules such as lactic acid, acetic acid, hydrogen peroxide, co2 and bacteriocin. bacteriocin is widely known to inhibit spoilage microorganism and food borne pathogens, thereby extending shelf life of food products. lactic acid bacteria exert strong antagonistic activity against many microorganisms, including food spoilage organisms and pathogens. in addition, some strains may contribute to the preservation of fermented foods by producing bacteriocins (adeniyi et al., 2006). research on bacteriocins from lactic acid bacteria has expanded during the last decades, to include the use of bactericocin or producer organisms as natural food preservatives. adeniyi et al. (2006) reported varied inhinbitory acitivities of lactic acid bacteria isolated from indigenous fermaented diary foods against commonly encounted bacteria implicated in urinary tract infections (coconnier et al., 1993). the killing activity of antisalmonella enterica serovar typhimurium produced by lactobacillus and bifidobacterium strains in the persence of luria broth (lb) has also beed reported (graver and wade, 2011). a small number of clinical studies suggest that eating yogurt with l. acidophilus cultures may also help. some people also use l. acidophilus to treat or prevent vaginal yeast infections. some clinical research suggest lactobacillus acidophilus may be effective when used to prevent diarrhea (caused by eating contaminated food). the health benefits derived by the consumption of foods containing probiotic bacteria are well documented and more than 90 probiotic products are available worldwide. to provide health benefits, the suggested concentration for probiotic bacteria is suggested to be 106 cfu/g of a product (fao/who, 2010). in the present investigation the microflora of different fruit samples of karachi, pakistan were studied with special focus on lactic acid bacteria. we isolated lactic acid bacteria (lab) from fresh vegetables and fruit and then tested their potential as bioprotective agents against food-borne human bacterial pathogens. this study was focused in two different areas. first part of the study aimed to isolate lactobacillus and second part was focused on inhibition properties. material and methods sample collection: fruit markets in different areas of karachi, pakistan were visited and the seasonal fruits (apple, banana, cheeko, custard apple, sugarcane, peach, grape, papaya, plum, grape fruit, mango, melon, pomegranate, pear, lettuce, cabbage, tomato and cucumber) of reasonable quality were purchased. the samples were collected in sterile plastic seal bags and brought to the laboratory within 24 hours of purchase for analysis (table i). isolation of lactic acid bacteria: to isolate lab, the fruit samples were brought to the laboratory and thoroughly washed with tap water following a final rinse with sterile saline (0.9%). each fruit was processed separately and diced in a sterile petri plate. small portion of the solid fruits sample was chopped manually with sterile cutter while the citrus fruits (grapefruit) were squeezed manually till the considerable amount of extract was obtained. following the culture enrichment method 1 ml of fruit extract so obtained was inoculated into 9ml of sterile mrs broth and incubated overnight at 37oc for 48 hours in shaking water bath at 120 rpm. after incubation samples were appropriately diluted and spread on mrs-agar plates. the plates were incubated an aerobically at 35oc for 24-48 hr. identification of isolates based on phenotypic characteristics: the isolates were characterized for gram reaction and catalase reaction and fermentative catabolism of various carbohydrates. the strains were also tested for their motility by sim (sulphide, indole, motility) test. the fermentative behavior for various carbohydrates was determined in bromothymol blue lactose broth supplemented with 1.5% of carbohydrate to be tested (arabinose, fructose, galactose, glucose, lactose, maltose, mannitol, raffinose, sucrose, xylose). 37 vol 5 (1), january 2014; 36-45 assay of antagonistic activity: for this assay, 48 hrs broth cultures of the lab were centrifuged at 10,000 rpm for 30 min. the supernatnants were membrane filtered (millipore, 0.22um) and the ph was measured using ph meter. the ph of sterile mrs broth treated the same way as the lab culture. the cell free supernantant 100 ul was transferred in to 6mm diameter well bored in mueller hinton agar previously seeded with 0.5 macfarland of clinical isolates of pseudomonas aeruginosa, enterococcus faecalis, escherichia coli and proteus sp., equivalent to 108 cfu/ml. the culture plates were incubated at 37oc for 24hrs and the zones of inhibition measured in millimeter. results a total of 12 lactic acid bacterial strains were isolated from 25 fruit samples collected and analyzed during present course of work. all isolates were identified on the basis of colony/cell morphology and biochemical tests (figure 1-7). majority of the colonies on mrs agar plates were whitish to offwhite in color, gram reaction showed gram positive rods, catalase test gives negative result and motility was not observed in sim media (table i). small number of the analyzed fruit sample gave colonies of bacillus with positive catalase reaction. all gram positive and catalase negative isolates were selected for further analysis. the 12 isolated lactic acid bacterial strains were tested for their antagonistic activity against e. coli, enterococcus fecalis, pseudomonas aeruginosa and proteus. all except few strains show moderate to high inhibition activity against these organisms (table ii). vol 5 (1), january 2014; 36-45 table i: identification test for lactobacilli. 38 -ve -ve -ve -ve -ve +ve -ve -ve -ve -ve -ve ----ve -ve -ve -ve sim test -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve +ve -ve -ve -ve -ve catalasefruit source grape sugarcane banana apple peach cucumber plum grape fruit tomato papaya cheeko pomegranate mango melon lettuce cabbage bromothymolblue test +ve +ve +ve +ve +ve -ve +ve +ve +ve -ve -ve ----ve -ve +ve +ve colonial morphology large, gummy colonies flat, gummy colonies small colonies flat, gummy colonies large, gummy colonies pin pointed transparent colonies large, gummy colonies gummy colonies gummy colonies small colonies pin pointed flat colonies spreaded colonies pin pointed flat colonies pin pointed flat colonies gummy colonies gummy colonies +ve rods +ve rods in chains +ve rods +ve rods +ve rods gram +ve diplococci gram-ve rods +ve cocci +ve rods +ve rods +ve rods in chains +ve rods +ve rods in chains +ve rods in chains +ve rods +ve rods microscopy 39 vol 5 (1), january 2014; 36-45 figure 3: catalase test. figure 4: oxidase test. figure 2: colonial morphology of lactobacillus. figure 1: microscopy of lactobacillus. figure 5: bromothymol blue lactose broth. figure 6: sim test. figure 7: zone of inhibition of lab against bacterial pathogens. 40 vol 5 (1), january 2014; 36-45 discussion this study evaluated the efficacy of lab isolated from fresh fruits and vegetables as biocontrol agents against the food borne pathogens. the densities of lab in fruit and vegetable products usually range from 102 to 106 cfu/gm (ongeng et al., 2006). lactic acid bacteria are generally considered as microorganisms with no pathogenic activities (sexline et al., 1996). in the present study, the approaches in preliminary identification of isolated bacterial strains were morphological and biochemical characterization like positive growth on mrs agar medium, negative catalase activity, colony color, gram positive staining reaction, outcomes of microscopic analysis and carbohydrate fermentation pattern. all were to suggest the presence of lactic acid bacteria in the samples under consideration. these identification approaches were in agreement with the previous studies on lactic acid bacteria (sudi et al., 2008). the present study had little success in isolating lactic acid bacteria from fruit samples by agar plate culture method. instead a predominance of bacillus by this method was found. this correlated the different bacillus species as widespread pollutants in the environment. consequently, the present investigation focused on enrichment culture as an approach to determine the diversity of lactic acid bacteria associated with fruit samples. enrichment substantially increased the isolation frequency of lactic acid bacteria from the above stated samples. nevertheless many of the fruit samples in present study did not give detectable lactic acid bacteria. present investigation deals not only with the distribution of lactic acid bacteria in fruits, but also with antagonistic activity. several clinical applications of lab against human diseases have attracted the attention of many research groups. therefore, in vitro antagonistic assays performed revealed that lab strains isolated from fresh fruit and vegetable products had antagonistic table ii: antagonistic activity of lactic acid bacteria. 2mm 0 mm 8mm 0 mm 0 mm 3mm 0 mm 0 mm 0 mm 2mm 0 mm 0 mm pseudomonas 11mm 10mm 9mm 7mm 0 mm 0 mm 11mm 0 mm 0 mm 0 mm 0 mm 7mm proteus 0 mm 0 mm 0 mm 4mm 6mm 0 mm 4mm 0 mm 0 mm 3mm 0 mm 0 mm enterococcus 0 mm 0 mm 3mm 0 mm 5mm 6mm 7mm 0 mm 0 mm 5mm 0 mm 0 mm e. coli 1 2 3 4 5 6 7 8 9 10 11 12 organism code avtivity against common foodborne pathogens like e. coli, proteus sp., enterococcus sp. and pseudomonas sp. a previous work using lab showed that most of the selected strains had good antagonistic activity against food borne pathogens, including listeria monocytogenes, salmonella typhimurium, and escherichia coli (trias et al., 2008). a major advantage of using lab as biocontrol agents is that they are considered as gras (generally recognized as safe) and usually comply with all recommendations for food products. moreover, lab is natural colonizer of fresh fruit and has been previously described ad good antagonists of several bacteria and fungi in different food products (stiles and holzapfel, 1997). studies on the antifungal properties of lab are relatively scare, when this effect was reported; it was attributed to the production of proteinaceous compounds (rouse et al., 2008) or organic acids (goueama, 1998). the preferred antimicrobial substances produced by the strains described in this study are organic acids, this was the case for all the bacteria and fungi tested. the combination of different organic acids, such as lactic and propionic, has been reported to have a synergistic fungistatic effect (batish et al., 1997). conclusion in conclusion, fruits and vegetables have been found in this study to be carriers of some lab and present potential sources of the organisms. since lab strains have inhibitory effect against number of bacterial pathogens, they can be used in food industry to ensure the safety of different food products. references adams mr, hall cj. (1988). growth inhibition of foodborne pathogens by lactic and acetic acids and their mixtures. int j food sci technol, 23: 287-291. adeniyi ba, ayeni fa, and ogunbanwo st (2006). antagonistic activities of lactic acid bacteria isolated from nigerian fermented diary food against organisms implicated in urinary tract infection. biotech, 5: 183-188. batish vk, roy u, lal r, grover s. (1997). antifungal attributes of lacticacid bacteria--a review. crit rev biotechnol, 17: 209-225. brink ten, b., m. minekns, j.m.b.m. vander vossen, r.j. leer and j.h.j. huis in’t veld, 1994. anti microbial activity of lactobacilli. j. appl. bacteriol., 77:140-148. carr, f.j., chill, d., maida, n., 2002. the lactic acid bacteria: a literature survey. critical reviews in microbiology, 28: 281-370. coconnier mh, bernet mf, kerne is s, chauvie’re g, fourniat j, and serving al (1993). inhibition of adhesion of enteroinvasive pathogens to human intestinal caco-2 cells by lactobacillus acidophilus strain lb decreases bacterial invasion. fems microbiol lett., 100: 299-306 fao/who. (2010). codex standard for fermented m i l k s ( 2 n d e d . ) . . c o d e x s t a n 2 4 3 2 0 0 3 . gourama h (1997) inhibition of growth and mycotoxin production of penicillium by lactobacillus species. lebensm-wiss u-technol, 30: 279-283. graver ma and wade j (2011). the role of acidification in the inhibition of neisseria gonorrhoeae by vaginal lactobacilli during anaerobic growth. annals clin microbiol antimicrob. 10:8-12. madigan m.t, martinko j. m., parker j. biology of microorganisms, 8th ed.,, prentiee hall internaional, inc., 1997. makarova, k., slesarev, a., wolf, y., sorokin, a. mirkin, b., koonin, r., pavlov, a., pavlova, n. et al, (2006). comparative genomics of the lactic acid bacteria proc natl acad sci usa 103 (42): 156116. doi: 10.1073/pnas.0607117103. pmc 1 6 2 2 8 7 0 p m i d 1 7 0 3 0 7 9 3 . 41 vol 5 (1), january 2014; 36-45 mundt, j.o. and j.l. hammer (1968). lactobacilli on plants. applied microbiology, 16, 1326-1330. ongeng d, devlieghere f, debevere j, coosemans j, ryckeboer j. (2006). the efficacy of electrolysed oxidizing water for inactivating spoilage microorganisms in process water and on minimally processed vegetables. int j food microbiol, 109: 187-197. ph. makela, h. salminen and s.l. gorbach. 1996. lactobacilli and bacteremia in southern finland, 1989-1992. clin. infec. dis., 22:564-566. rodondo lopez v., cook r.l., sobel j.d. (1990) rev infect dis 12, 856-872. rouse s, harnett d, vaughan a, van sinderen d. (2008). lactic acid bacteria with potential to eliminate fungal spoilage in foods. j appl microbiol, 104: 915923. sathe sj, nawani nn, dhakephalkar pk, kapadnis bp. (2007). antifungal lactic acid bacteria with potential to prolong shelf-life of fresh vegetables. j appl microbiol, 103: 2622-2628. schillinger u, lucke fk. (1989). antibacterial activity of lactobacillus sake isolated from meat. a p p l e n v i r o n m i c r o b i o l , 5 5 : 1 9 0 1 1 9 0 6 . sexline, m., n.h. chuand, b. chassy, h. rautelin, sharpe, m.e. (1962). lactobacilli in meat products. foodmanufa. 37:582-582. stiles m, hozapfel w.(1997). lactic acid bacteria of foods and their current taconomy. int j food microbiol, 36: 1-29. sudi, i.y., n. de and u. ali-dunkara. 2008. mutagenesis and for potential use as starter culture. j. amr. sci., 4(3): 8087. trias r, baneras l, badosa e, montesinos e. (2008). bioprotection of golden delicios apples and iceberg lettuce against foodbrone bacterial pathogens by lactic acid bacteria. int j food microbiol, 123:5060. 42 vol 5 (1), january 2014; 36-45 “rads” publishes articles within the whole field of physical science. generally these articles will be in or related to need of the country. manuscripts or data will be considered that have not been previously published or submitted elsewhere for publication. however, re-analysis of previously published data can be accepted. 1. all articles submitted for publication will be reviewed by referees. submit three copies of the article / manuscripts along with a floppy d i s k e t t e ( i b m c o m p u t e r ) c o n t a i n i n g t h e manuscript in duplicate. while submitting diskette it should only the latest version of the manuscript. name the file clearly, label the diskette with the format of the file and the file name. provide information of the hardware and software used. 2. manuscript 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vesicular arbuscular mycorrhizal fungi on growth and yields of soybeans. soil sci. soc. amer. j., 44: 528-532. 5. the manuscript should be accompanied by a covering letter signed by corresponding authors. instruction to authors a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 61 r a d s j . b i o l . r e s . a p p l . s c i . 61 op e n ac c e s s f u l l l e n g t h a r t i c l e a preliminary up-to-date review on pakistani medicinal plants with potential antioxidant activity adil hussain department of biotechnology, faculty of life sciences, university of okara, okara 56130 pakistan a b s t r a c t background: there exist natural antioxidants in plants that scavenge harmful free radicals from the body. free radicals are species of chemical origin with an unpaired electron and play a pivotal role in combating health-related problems like lung damage, inflammation, and cardiovascular ailments etc. antioxidants halt the development of these free radicals called the reactive oxygen species either by chelating the trace elements or by enzymes inhibition. objectives: this present review aimed to collect information about pakistani medicinally important plants with the exploration of their antioxidant potential. methodology: a total of 206 papers were looked over, which were obtained from numerous sources like; google scholar, medline, pubmed, research gate, science direct, scopus and web of science. results: overall, 95 plants representing 44 families with potential antioxidant activity reported from pakistan have been presented in this review. the maximum number of species from asteraceae, poaceae and rutaceae families were scrutinized for their potential antioxidant activity from pakistan. conclusion: the present review clearly authenticates that the presence of phytochemicals and antioxidant activity of medicinal plants from pakistan vary with the species of the plants and material/extracts used. from this review, it is recommended to perform comprehensive experimental investigations based on toxicology and ethnopharmacology on these precious plants from pakistan. it will be advantageous in the provision of trustworthy information to patients and determine further innovative compounds for safer and new drugs development with fewer side effects. keywords antioxidant activity, flavonoid compounds, phenolic compounds, medicinal plants, pakistan *address of correspondence adil@uo.edu.pk article info. received: february 13, 2020 accepted: august 21, 2020 cite this article: hussain a. a preliminary up-to-date review on pakistani medicinal plants with potential antioxidant activity. rads j biol res appl sci. 2020; 11(1):61-88. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. funding source: nil conflict of interest: nil i n t r o d u c t i o n antioxidants are the compounds that halt the initiation rate of lipid oxidation reactions in the food system. in living organisms, the primary source of health problems is oxidative damage due to the free radicals, and reactive oxygen species (ros) attack on key macromolecules of the cell. antioxidants counteract free radicals and are significant in terms of cell prevention from injuries caused by ros1. a group of phytochemicals like amines and phenols are antioxidants, with radical scavenging ability and break down radical chain reactions. these are the substances with significant inhibition of adverse effects caused by the ros on a human being, and their physiological function. some antioxidants like reactive oxygen or nitrogen scavengers vitamin c and e, transition metal chelators and oxidative enzyme (cyclooxygenase) inhibitors are non-enzymatic in nature2. the induced ros r e v i e w a r t i c l e a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 62 r a d s j . b i o l . r e s . a p p l . s c i . 62 oxidation disintegrates the cell membrane, damages the membrane protein, and causes mutations in living organism’s dna. plants generally contain numerous phytochemicals in their extracts, and their utilization could be fruitful for new therapeutic methodologies development to treat diseases. it could be accomplished only if the antioxidant and antimicrobial profile of the plant is well understood3. antioxidants from plants origin guard the cell by offsetting ros through scavenging them4. antioxidants from plants are structurally diverse and their activity and extraction dependents on their structure5. total flavonoids and phenols are herbal compounds showing free radical scavenging potential, which are active against neurological disorders6. these compounds also act as cardio-protective, antimutagenic and anti-cancerous representatives due to their free radical scavenging ability7. ferreira and stade8 proposed that polyphenolics compounds are chemo-preventive agents because they decrease the level of cholesterol. because of these unique discoveries, researchers around the world attempts to replace the synthetic antioxidants used in medicinal/foods industries with those of natural healthfriendly antioxidants6. according to a survey in pakistan, 6,000 different types of flowering plants are present, out of which nearly 400-600 species are medicinally important9. additionally, in pakistan, most of the flora is still unexplored medicinally and their utilization through the unani medicine system is still not well documented10. in this review, efforts made by the researchers to evaluate the antioxidant efficacy of different plant species from pakistan have been compiled. m a t e r i a l s a n d m e t h o d s the data on different pakistani plants with antioxidant activity were assembled from search engines like google scholar, scopus, science direct, pubmed, web of science, research gate and medline. numerous indicators including “antioxidant activity”, “phytochemistry” and “pakistani medicinal plants” were used to search the data. the correct species names and taxonomic description with synonyms were confirmed from databases (http://www.theplantlist.org/) and (http://www.eflora.org/). about 206 research articles published on the antioxidant activity were studied, and information on the pakistani medicinal herbs with potential antioxidant activity has been provided. pakistani medicinal plants with antioxidant activity data on 95 plants representing 44 families with potential antioxidant activity reported from pakistan have been compiled here (table 1 & fig. 1). the assembled reported studies concerning antioxidant activities of plants from pakistan are discussed in detail below: acacia leucophloea acacia leucophloea is a species from the leguminosae family with ~650 genera and more than 18,000 species11. it is one of the major plant families with traditionally used plant species12 having medicinal significance13. a study from multan, pakistan confirmed phenolic compounds existence in a. leucophloea bark methanolic extracts. antioxidant capacity measured by ferric reducing antioxidant power (frap), trolex equivalent antioxidant capacity (teac) and total peroxyl radical trapping antioxidant parameter (trap) assays indicated that the methanolic extracts of a. leucophloea stem bark are rich sources of natural antioxidants with acceptable antioxidant activity14. acacia nilotica acacia nilotica belongs to the plant family leguminosae. in bahawalpur, pakistan, an in vitro antioxidant and antibacterial profile of a. nilotica methanolic leaves extract through 2,2-diphenyl, 1-picrylhydrazyl hydrate (dpph) inhibition assay showed maximum antioxidant potential with antibacterial activity among tested plant extracts of leguminosae family15. adenium obesum (forssk.) adenium obesum is a plant species from the family apocynaceae16 which is indigenous to south african regions17. this plant contains flavonoids, carbohydrates, cardiac glycosides, triterpenes, pregnanes and steroids18. in a study from karachi, pakistan, the methanolic extracts of a. obesum leaves have been evaluated for antioxidant activity with dpph free radical scavenging assay using standard antioxidants like ascorbic acid and butylated hydroxytoulene (bht). the tested extracts of a. obseum leaves displayed inhibition and better antioxidant activity19. a. obesum methanol extract was assessed by http://www.theplantlist.org/ http://www.eflora.org/ a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 63 r a d s j . b i o l . r e s . a p p l . s c i . 63 ahmed et al.,18 and described 42 compounds including cardiac glycosides, steroids, and triterpenoids. aegle marmelos l. aegle marmelos belongs to the rutaceae family20. this plant is sub-tropical and commonly found in regions of bangladesh, bhutan, burma, ceylon, india and pakistan21. rahman et al.,22 and khatoon et al.,19 studied antioxidant efficacy of a. marmelos from pakistan. the antioxidant and lipoxygenase inhibitory potential of some aqueous extracts and organic fractions of mature fruit of a. marmelos have been reported from karachi, pakistan22. the aqueous and chloroform fractions were supported with greater ability to inhibit soya bean lipoxygenase activity (89-69%), reduction of free radicals (88-65%) and the minimizing of ferric chloride. the methanol extracts of a. marmelos were tested from karachi, pakistan, with a dpph assay for assessing free radical scavenging action19. all tested extracts of a. marmelos validated better radical inhibition action followed by a. obseum. albizia lebbeck (l.) benth. albizia lebbeck is a vital species from mimosaceae family. in many regions of pakistan, it is grown as an ornamental plant due to pleasant morphology23. the antioxidant activity with phytochemistry of a. lebbeck extracts was investigated from pakistan24, 25. in an inquiry from multan city of pakistan, the antioxidant activity of a. lebbeck (seed, flower and bark) methanol extracts validated greater phenols. antioxidant activity estimated by trap, teac and fraps assays, substantiated that the investigated of a. lebbeck methanolic extracts are worthy natural antioxidants sources24. another investigation from multan, pakistan displayed the antioxidant potential of aqueous methanol pods, stems, and roots extracts of a. lebbeck. studies based on composition specified carbohydrates as the main constituents and saponins as chief antinutrient in both seeds and pods extracts of a. lebbeck. in the pods extract and seed oil of a. lebbeck, a major fatty acid linoleic acid is present. α-tocopherol is a major tocopherol component in oil. in vitro frap, trap and teac substantiates the aqueous methanol pods, stems and roots extracts of a. lebbeck with potent antioxidant activity25. aloe vera l. the aloe vera plant belongs to the plant family liliaceae. this plant possesses almost 200 phytochemicals and 7075 diverse nutrients like anthraquinones, amino acids, lignin, enzymes, minerals, saponins, sugars, salicylic acid and vitamins26. antioxidant activity and phytochemistry of a. vera extracts from pakistan have been described by malik et al.,27 and waris et al.,28. a study from bhimber ajk of pakistan27 exposed the presence of phytochemicals and nutrients like amino acid, carbohydrates, flavonoids, glycosides, phenolic compounds, steroids, reducing sugar, tannins, saponin and terpenoids in the ethanol, aqueous and methanol and extracts of a. vera leaves. waris et al.,28 from peshawar, pakistan documented the presence of flavonoids, alkaloids, glycosides, saponins and tannins in the a. vera plant extracts with antioxidant activity. artemisia l. species artemisia genus belongs to the family asteraceae. many artemisia species are significant economically due to their antitumor, antispasmodic, antiseptic, antirheumatic*and hepato-protective86, antioxidant, antihelmintic, anticancerous, antimicrobial and antimalarial possessions87. a lot of artemisia species have been discovered from pakistan and scrutinized for their antioxidant activity namely artemisia annua33, artemisia persica30, artemisia roxburghiana31 and artemisia rutifolia34. an enquiry from the islamabad region of pakistan has been performed for antioxidant activity assessed with trolox equivalent, dpph, frap and lipid peroxidation assay (lpd). as a result, the maximum total flavonoid contents (tfc), total phenolic contents (tpc), dpph and teac and minimum peroxidation of lipid for the methanol extracts were recorded, however, the aqueous extract showed maximum ferric reducing antioxidant power33. akhtar et al.,31 checked the presence of phytochemicals with potential antioxidant activity of aqueous and methanol/chloroform extracts of artemisia roxburghiana from nathiagali region of abbotabad, pakistan. a study from the northern pakistan displayed that the methanol extracts from leaves of a. persica contains phenolics and flavonoids with a startling antioxidant activity30. in a study from chitral kpk, pakistan, total phenolic and flavonoid contents, antimicrobial activity, dpph activity and reducing power http://www.efloras.org/florataxon.aspx?flora_id=5&taxon_id=10781 a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 64 r a d s j . b i o l . r e s . a p p l . s c i . 64 of a. rutifolia leaves methanolic extracts showed antioxidant activity34. berberis lycium (royle.) the plant berberis lycium is from the genus berberis of the berberidaceae family. studies of ahmed and shakeel36, sabir et al.,37, akhtar et al.,31 and shan et al.,88 validated the potential antioxidant action of b. lycium from pakistan. a noteworthy association amongst phenolic compounds and the antioxidant dimensions substantiated that these are the major factors that provides antioxidant efficacy to the b. lycium (ahktar et al., 2015). from the north himalayan region of pakistan, b. lycium was collected and the total antioxidant activity was scrutinized in its acetone fruit extract. the presence of polyphenolics and the antioxidant activity caused by these compounds discovered a synergistic part of secondary metabolites in inhibiting diseases88. akhtar et al.,31 from nathialgali abbottabad, pakistan scrutinized the methanol/chloroform and aqueous extracts of b. lyceum for its antioxidant activity and corroborated great antioxidant activities in the testes extracts due to better reduction capacity and 2,2-dpph radical scavenging action. sabir et al.,37 extracted six phytochemicals from petroleum ether and methanol extracts of the roots of b. lycium in tehsil rawalakot, pakistan and characterized them structurally with the help of low-resolution mass, infrared, 13c and 1hnmr (nuclear magnetic resonance) spectroscopy. all the extracted compounds from this plant exhibited prospective antioxidant activity. another inquiry from rehar mansehra kpk, pakistan inspected the stem and fruit extracts of b. lycium and the presence of potential antioxidant compounds with superoxide anion radical scavenging activity has been validated36. figure 1. family-wise distribution of medicinally important plants with potential antioxidant activity reported from pakistan. the numbers in brackets specify species from the corresponding family. a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 65 r a d s j . b i o l . r e s . a p p l . s c i . 65 table 1. reported plants with potential antioxidant activity from different localities of pakistan. s.no. family no. of plants plant name locality parts used extraction solvent reference 1 anacardiaceae 1 pistacia integerrima (stew.) malakand kpk fruit methanol 29 2 apiaceae 1 centella asiatica (linn.) urban sys. northern areas leaves & stem methanol 30 3 apocynaceae 2 adenium obesum (forssk.) pakistan fruit methanol 18* karachi leaves methanol 19 catharanthus roseus (g. don) attock whole plant methanol/chloroform & aqueous 31 4 acanthaceae 1 justicia adhatoda l. islamabad leaves methanol, ethanol, butanol, chloroform & n-hexane 40 islamabad leaves methanol/chloroform & aqueous 31 5 asclepiadaceae 1 periploca aphylla toormang, dir kpk aerial part methanol 32 6 asteraceae 9 artemisia annua l. islamabad leaves hexane, chloroform, ethyl acetate, methanol & water 33 artemisia persica northern areas leaves & stem methanol 30 artemisia roxburghiana (wall. ex besser) nathiagali abbotabad whole plant methanol/chloroform & aqueous 31 artemisia rutifolia (spreng.) chitral, kpk leaves methanol, chloroform & hexane 34 cichorium intybus l. gujrat whole plant methanol 35 cousinia minuta boiss kalabagh leaves methanol/chloroform & aqueous 31 cirsium arvense (l.) scop. carthamus oxyacantha (m.bieb) mianwali whole plant pentanema vestitum (linn.) malakand, kpk whole plant methanol 29 7 berberidaceae 1 berbaris lycium (royle). mansehra, kpk fruit dmso 36 nathiagali abbottabad whole plant methanol/chloroform & aqueous 31 rawalakot roots methanol & petroleum ether 37 8 brassicaceae 2 brassica campestris l. shahdra whole plant methanol/chloroform & aqueous 31 nasturtium officinale (w. t. aiton) north kpk whole plant crude ethanol 38 salgira park whole plant methanol/chloroform & aqueous 31 tanawal area whole plant mthanol 39 9 cannabaceae 1 cannabis sativa l. lahore leaves & stem methanol and ethanol 41 bajaur, kpk leaves methanol, ethanol & chloroform 42* peshawar, kpk whole plant acetone 28 10 capparidaceae 1 capparis decidua (forsk.) edgew multan leaves, flowers & fruit methanol 43 multan roots methanol 24 mianwali bark methanol/chloroform & aqueous 31 continue… a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 66 r a d s j . b i o l . r e s . a p p l . s c i . 66 s.no. family no. of plants plant name locality parts used extraction solvent reference 11 caryophyllaceae 1 stellaria media l. north kpk whole plant crude ethanol 38 12 chenopodiaceae 2 chenopodium ambrosioides kotli, ajk bark & fruit aqueous, petroleum ether, chloroform & methanol 44 haloxylon griffithii hanna, quetta leaves, stem & roots methanol 45 13 combretaceae 2 conocarpus lancifolius pattoki & lahore whole plant dichloromethane & methanol 46 terminalia arjuna l. faisalabad leaves & stem bark aqueous ethanol & aqueous methanol 47 14 convolvulaceae 1 convolvulus leiocalycinus hanna, quetta leaves, stem & roots methanol 45 15 cucurbitaceae 1 momordica charantia lahore seed, peel & flakes aqueous 48 16 dennstaedtiacea e 1 pteridium aquilinum north kpk whole plant crude ethanol 38 17 elaeagnaceae 3 elaeagnus angustifolia chitral valley fruit crude ethanol 49 elaeagnus umbellate thunb. rawlakot, ajk fruit methanol 50 hippophae rhmnoides l. skardu berries methanol 51 skardu, gilgitbaltistan leaves aqueous, acetone, ethanol, chloroform, ethyl acetate, methanol & n-hexane 52 18 euphorbeaceae 1 euphorbia royleana faisalabad whole plant methanol, hexane, and aqueous 34 19 fabaceae 1 cicer arietinum l. mianwali leaves & stem methanol/chloroform & aqueous 31 multan seed methanol 24 faisalabad seed acetone 53 20 fagaceae 1 quercus incana (roxb.) abbottabad whole plant ethyl acetate & n-butanol 54 pakistan bark ethyl acetate & methanol 55 21 gentianaceae 1 swertia chirata (roxb.) galyat, kpk aerial part methanol 56 22 juglandaceae 1 juglans regia chitral valley fruit crude ethanol 49 23 lamiaceae 6 colebrookea oppositifolia (sm). islamabad leaves & flower methanol/chloroform & aqueous 31 mentha piperita l. rawalpindi whole plant mentha longifolia l. peshawar whole plant acetone 28 mentha spicata muzaffarabad, ajk leaves diethyl ether, chloroform, ethanol & methanol 57 origanum majorana l. faisalabad aerial part essential oil 58 origanum vulgare l. 24 leguminosae 3 acacia leucophloea multan stem bark methanol 24 acacia nilotica bahawalpur leaves methanol 15 trifolium repens north khyber pakhtunkhwa whole plant ethanol 38 25 liliaceae 1 aloe vera l. bhimber, ajk leaves ethanol & methanol 27* peshawar, kpk leaves acetone 28 continue… https://www.google.com/search?rlz=1c1chbd_enpk804pk804&q=elaeagnaceae&stick=h4siaaaaaaaaaongvuluz9u3mezkstnaxmrjmpoympiel5icmpgkaafjdrucaaaa&sa=x&ved=2ahukewjwqoajisjjahvhofwkhy96c_qqmxmoatadegqidrak a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 67 r a d s j . b i o l . r e s . a p p l . s c i . 67 s.no. family no. of plants plant name locality parts used extraction solvent reference 26 meliaceae 2 melia azedarach faisalabad leaves, fruit & stem bark aqueous methanol 59 azadirachta indica a. juss. mianwali leaves methanol/chloroform & aqueous 31 27 malvaceae 1 grewia asiatica l. mianwali leaves methanol/chloroform & aqueous 31 multan leaves methanol 24 28 mimosaceae 1 albizia lebbeck (l.) benth. multan pod, stem & roots aqueous methanol 25 multan bark, flower & seed methanol 24 29 moraceae 5 fiscus carica bannu, kpk fruit aqueous methanol 60 ficus sarmentosa toormang, dir kpk aerial part methanol 32 ficus microcarpaa l. (fil) islamabad whole plant methanol/chloroform & aqueous 31 morus alba mardan leaves aqueous extract 61* chitral valley fruit crude ethanol 49 morus nigra islamabad whole plant methanol/chloroform & aqueous 31 30 moringaceae 1 moringa oleifera (lam.) faisalabad leaves ethanol 62 kundian mianwali whole plant methanol/chloroform & aqueous 31 31 myrtaceae 1 psidium guajava mardan leaves aqueous extract 61* chitral valley fruit crude ethanol 49 32 oleaceae 3 jasminum humile abbottabad flower ethyl acetate, n-butanol & n-hexane 35 jasminum officinale l. mianwali whole plant methanol/chloroform & aqueous 31 jasminum sambac l. (aiton) 33 pinaceae 01 cedrus deodara (roxb. ex d. don) rawalakot ajk stem & leaves aqueous extracts & oil 63 northern areas stem & leaves methanol 30 continue… a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 68 r a d s j . b i o l . r e s . a p p l . s c i . 68 s.no. family no. of plants plant name locality parts used extraction solvent reference 34 poaceae 9 dactyloctenium aegyptium l. bahawalpur aerial part ethanol & acetone 64 dichanthium annulatum (forsk.) eleusine indica l. imperata cylindrica l. oryza sativa l. gujranwala rice bran methanol 65 karachi rice bran methanol 66 kalashahkako lahore seed aqueous, methanol, ethanol & isopropanol 67 poa annua l. bahawalpur aerial part ethanol and acetone 64 saccharum spontaneum l. vetiveria zizanioides (l.) zea mays multan corn silk powder hexane, ethyl acetate, chloroform, methanol & water 68 sahiwal seed methanol 69 faisalabad ear tissue extraction buffer 70 35 rhamnaceae 1 zizyphus jujube (mill.) chitral fruit ethanol 49 kohat kpk fruit aqueous methanol 60 faisalabad shoot methanol 71,149 36 rosaceae 5 cydonia oblonga poonch aj&k fruit ethanol 72 chitral valley fruit crude ethanol 49 rosa indica l. islamabad leaves & stem methanol/chloroform & aqueous 31 pakistan leaves ethanol 73 rosa hybrida rawalakot, ajk petals aqueous 74 rosa damascene (mill.) rosa moschata 37 rubiaceae 1 ixora coccinea l. karachi leaves & flower methanol 19 continue… a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 69 r a d s j . b i o l . r e s . a p p l . s c i . 69 s.no. family no. of plants plant name locality parts used extraction solvent reference 38 rutaceae 9 aegle marmelos l. karachi fruit crude aqueous 22 karachi fruit methanol 19 citrus sinensis (var. valencia), citrus sinensis (var. sungin), citrus sinensis (var. washington navel), citrus reticulata (var. page), citrus aurantium and citrus paradise sargodha peel & fruit methanol 75 citrus sinensis islamabad powder methanol 76 citrus limon l. (burm.f.) malakwal whole plant methanol/chloroform & aqueous 31 39 saxifragaceae 1 bergenia ciliata (haw.) sternb nathiagali, kpk rhizome methanol 77 kaghan valley rhizome methanol, chloroform, ethyl acetate & n-hexane 56 40 solanaceae 4 datura innoxia (mill.) islamabad leaves & stem distilled water chloroform, n-hexane, ethyl acetate, acetone, ethanol & methanol 78 bahawalpur fruit & leaves ethanol 79 mianwali whole plant methanol/chloroform & aqueous 31 solanum nigrum l. mirpur bhimber fruit, leaves and stem chloroform, distilled water ethanol & petroleum ether 80* bahalwalpur fruit & leaves ethanol 79 withna somnifera malakand, kpk fruit methanol 29 bahalwalpur fruit & leaves ethanol 79 withna coagulans (dunal.) mianwali whole plant methanol/chloroform & aqueous 31 malakand kpk fruit methanol 29 41 typhaceae 1 typha domigensis (pers.) lahore pollen chloroform, methanol, ethyl acetate n-hexane & water 81 42 urticaceae 1 urtica dioica l. islamabad aerial part ethanol 82* north khyber pakhtunkhwa whole plant crude ethanol, n-hexane, ethyl acetate & chloroform 38 continue… a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 70 r a d s j . b i o l . r e s . a p p l . s c i . 70 s.no. family no. of plants plant name locality parts used extraction solvent reference 43 verbenaceae 1 lantana camara l. soon sakesar punjab flower & fruit methanol and ethanol 83 karachi aerial part methanol, aqueous, petroleum ether & ethyl acetate 84 44 zygophyllaceae 1 fagonia cretica l. hazara stem & spines aqueous & methanol/chloroform 31 *represents studies concerning only phytochemical investigations of the plants bergenia ciliata (haw.) sternb bergenia ciliata is a species of the genus bergenia from saxifragaceae family. in pakistan, two species of the genus bergenia are locally distributed like b. strachey and b. ciliata89. inquiries of khan et al.,56 and ahmed et al.,77 acknowledged the phytochemistry and antioxidant profile of b. ciliate from pakistan. investigation of methanol crude extracts and chloroform, n-hexane and ethyl acetate fractions of rhizome of b. ciliata from kaghan valley (kpk) pakistan proposed that the chloroform fraction is highly anti-oxidative with the ic50 value of 4.15 ± 0.82 as compared to ethyl acetate56. in an inquiry from nathiagali kpk, pakistan, the improved free radical inhibition percentage with dpph assay with high flavonoid and phenolic contents validated the antioxidant potential of b. ciliate. the aqueous and ethyl acetate extracts have been corroborated with better activity against h157 cell line77. according to rauf et al.,32 flavonoids such as quercetin from b. ciliata remarkably holds antiradical, antioxidant, and iron-chelating properties. brassica campestris l. brassica campestris belongs to the family brassicaceae. the samples of b. campestris from shahdra, pakistan showed promising antioxidant efficacy in the tested extracts with better reduction and total antioxidant activities due to phenolics and flavonoids presence31. cannabis sativa l. cannabis sativa is an important dioicous species from the cannabis genus of the family cannabaceae. this plant is extensively distributed globaly90 including pakistan91. many researchers28, 41, 42, 91 documented the antioxidant activity and phytochemical profile of c. sativa from pakistan. an antioxidant investigation from lahore, pakistan using dpph scavenging assay showed that the ethanol extracts of c. sativa leaves retain great antimicrobial activity and contains natural antioxidants41. in bajaur agency of pakistan, the ethanol, methanol and chloroform leaf extracts of c. sativa were scrutinized that validated the presence of important phytochemicals. the presence of alkaloids, cardiac glycosides, carbohydrates, flavonoids, phlobatannins, proteins, phenols, saponin, terpenoids and tannins was confirmed in c. sativa leaf extracts42. another phytochemical inquiry authenticated the presence of alkaloids, glycosides and saponins in extracts of c. sativa. antioxidant activity with dpph assay specified that this plant holds a promising antioxidant activity28. capparis decidua (forsk.) edgew capparis decidua from capparidaceae family is a drought resistant plant grown as dense tufts in dry areas of pakistan92. in pakistan, akhtar et al.,31, imran et al.,24 and haq et al.,43 documented the antioxidant activity and phytochemistry c. decidua extracts. an in vitro antioxidant investigation of roots methanolic extracts of c. decidua from multan, pakistan corroborated the phenolic compounds presence. antioxidant capacity measured by frap, teac and trap assays indicated that the root extracts of c. decidua are good sources of natural antioxidants24. another inquiry from multan, pakistan exposed presence of amino acids, proximate chemicals, fatty acids, sterols and tocopherols in seeds oil and tpc and total glucosinolates (tg) in methanol extracts of c. decidua. the antioxidant activity validated remarkable antioxidant activity of c. desidua extracts due to the presence of phenolics and glucosinates with potent antidiabetic and antihemolytic activity43. akhtar et al.,31 a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 71 r a d s j . b i o l . r e s . a p p l . s c i . 71 validated bark extracts of c. decidua with antioxidant action from mianwali city of pakistan. carthamus oxyacantha (m.bieb) carthamus oxyacantha belongs to the family asteraceae. in pakistan, akhter et al.,31 have described the antioxidant potential of c. oxyacantha. they collected samples of c. oxyacantha from mianwali city of pakistan and checked the presence of phenols and flavonols in methanol/chloroform and aqueous extract with potential antioxidant activity. they confirmed presence of phenols and flavonoids in the c. oxyacantha plant with antioxidant activities in its methanol/chloroform and aqueous extracts due to reduction and total antioxidant capacity. catharanthus roseus (g. don) catharanthus roseus belongs to the genus catharanthus of the family apocynaceae. it has wide-ranging biological activities like antibacterial, antifungal, antiviral and antioxidantal71. akhtar et al.,31 reported the antioxidant activity of this important plant from attock city of pakistan. the extracts (methanol/chloroform and aqueous extract) demonstrated potential antioxidant actions due to their great reduction, total antioxidant capacity and scavenging of 2,2-diphenyl-2-picrylhydrazyl radicals. cedrus deodara (roxb. ex d. don) cedrus deodara is a species of pinaceae family. flavonoid like quercetin, taxifolin and saponins are the major phytochemicals of c. deodara. phytosterols present in this plant are very operative against hyperlipidemia93. c. deodara ethanol wood extracts also holds a significant anti-spasmodic activity94. the antioxidant activity and phytochemistry of c. deodara extracts have was studied by zaman et al.,95 and raza et al.,30 from pakistan. an inquiry from district poonch, rawalakot azad kashmir, pakistan evaluated the biological activities of c. deodara aqueous stem, leaves extracts and essential oil. the crude leaves and stem extracts and essential oil are authenticated with phenolics and flavonoids exhibiting metal chelating and antioxidant activity95. samples of c. deodara collected from northern areas of pakistan showed flavonoids and phenolics presence in stems and leaves methanol extracts retaining antioxidant activity. centella asiatica (linn.) the plant centella asiatica is from the centella genus of the apiaceae family. the phytochemicals present in this plant encouraged many researchers with the aim of exploring its antioxidant potential96. a study from northern pakistan suggests that c. asiatica extracts are good antioxidants source in food preservation because the leaves and stem methanol extracts hold antioxidant activity in foods rich with fats and oil30. chenopodium ambrosioides chenopodium ambrosioides is a species from chenopodiaceae family containing ~103 genera and ~1300 species found all over the world. in pakistan, 35 genera and 106 species of this family are present. they are mainly found along sea shores and in the desert or semi desert areas97. c. ambrosioides fruit and bark collected from kotli, ajk, pakistan were evaluated for antioxidant and antimicrobial potential using aqueous, chloroform, petroleum ether and methanol extracts. the fruit and bark aqueous extracts with greater antioxidant activity was documented44. cicer arietinum l. cicer arietinum belongs to the leguminosae family. legumes including c. arietinum possess polyphenolic compounds like flavone, glycosides, flavonols, polymeric and oligomeric proanthocyanidins98. in pakistan, c. arietinum (chickpeas) are grown as major legume crop99 and several chickpea varieties are prevalently consumed as a dietary protein source. methanol/chloroform and aqueous leaves and stem extracts of this plant assessed from mianwali, pakistan corroborated the presence of antioxidant inducing flavonols and phenolic with great antioxidant and reduction abilities31. in an inquiry from multan, pakistan, an in vitro antioxidant activity of c. arietinum seeds methanol extracts corroborated the presence of phenols. studies validated that c. arietinum extracts possesess great antioxidant potential24. haq et al.,53 studied the antioxidant action of seed acetone extracts of four indigenous desi chickpeas varieties from faisalabad, pakistan showing total phenolic, condensed tannin and flavonoid content with antioxidant activity. a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 72 r a d s j . b i o l . r e s . a p p l . s c i . 72 cichorium intybus l. cichorium intybus is a species from the asteraceae family. this plant holds great biological significance because of having flavonoids, alkaloids and vitamin a, e & c which are antioxidants in nature35,100. more specifically, the hydro-alcoholic extracts of c. intybus legitimate the presence of phytochemicals with potential antioxidant activity100. the antioxidant prospective of crude methanolic extracts of whole plant of c. intybus from gujrat, pakistan was evaluated through dpph on tlc assay for their antioxidant actions. the total phenolic contents (tpc) of c. intybus calculated and acknowledged as the rich source of innocuous natural antioxidants35. cirsium arvense (l.) scop. cirsium arvense plant belongs to the asteraceae family also called creeping thistle. samples obtained from kalabagh, pakistan revealed that the presence of flavonoids and phenolics in aqueous methanol extracts are crucil features providing antioxidant activity to this plant with great reduction potential31. citrus l. species the citrus species belongs to the family rutaceae. it is used as an anti-diabetic, antifungal, anti-microbial, antioxidant, hypotensive, insect repellent and anti-yeast agent. investigations of akhtar et al.,31, asjad et al.,75, shah et al.,76 and mehmood et al.,101 validated the antioxidant activity of citrus species from pakistan. the azinobis-3-ethylbenzothiazoline-6-sulfonate (abts) and dpph free radical scavenging approaches with citrus species unveiled their antioxidant and phytochemicals profile that could be fruitful in the prevention fatal diseases like cancer, cardiovascular disorder and diseases related to stress caused by radicals101. in pakistan, c. sinensis species collected from islamabad, pakistan substantiated the presence of flavonoids in its crude methanol extracts from plants powder and their scrutiny revealed a potential antioxidant activity76. samples of c. limo collected from malakwal city were analyzed for antioxidant potential of aqueous and methanol-chloroform extracts where the presence of flavonoids and phenols validated antioxidant activities due to the reduction and total antioxidant abilities31. in sargodha, pakistan, the fruits and peels of different citrus species like c. sinensis var. washington navel, c. sinensis var. valencia, c. reticulata var. page, c. paradise, c. aurantium and c. sinensis var. sungin scrutinized with methanol proved promising antioxidant activity of the extracts75. colebrookea oppositifolia (sm). colebrookia oppositifolia belongs to lamiaceae family. c. oppositifolia root extracts contains flavones and are employed for epilepsy treatment102. in pakistan, c. oppositifolia whole plant evaluated from kotli, ajk confirmed cardiac glycosides, flavonoids and phenolics in ethyl acetate, n-butanol and chloroform fraction. the soluble ethyl acetate fractions showwed greater percentage inhibition of frap, dpph value and inhibition of lipids peroxidation. the chloroform fraction also holds highest total phenolic contents with highest total antioxidant activity103. this species from islamabad, pakistan scrutinized for the antioxidant activity of its aqueous and methanol/chloroform leaves and flower extracts displayed phenolic and flavonoid compounds with antioxidant efficacy31. conocarpus lancifolius the conocarpus genus is from the combretaceae family having only two species104. in a study from pakistan, c. lancifolius collected from pattoki and lahore, elucidated the antioxidant potential of whole plant’s methanol and dichloromethane extracts in correlation with total flavonoid and phenolic content. presence of highest phenolic contents and flavonoid with greater hydroxyl radical and dpph scavenging actions of the methanolic extracts has been validated105. convolvulus leiocalycinus convolvulus leiocalycinus is member of the family convolvulaceae. convolvulus is a genus consisting of nearly 200-250 species of flowering plants. a study from hanna quetta, pakistan, assessed the antioxidant potentials of methanol extracts of c. leiocalycinus through the dpph, frap and phosphomolybdenum assay. the dpph assay showed c. leiocalycinus with activity of 97.16% at concentration of 1.0mg/ml. however, the increased frap value for c. leiocalycinus extract proved to have greater antioxidant activity45. a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 73 r a d s j . b i o l . r e s . a p p l . s c i . 73 cousinia minuta (boiss.) cousinia minuta is a vital member of the family asteraceae. a study from kalabagh, pakistan confirmed phenolic compounds and flavonoids presence in its aqueous and methanol/chloroform leaves extracts with potential antioxidant actions31. cydonia oblonga (quince) cydonia oblonga belongs to the rosaceae family. it is a significant dietary source of essential phytochemicals including phenolic acids and flavonoids106. in pakistan, rahman et al.,49 and rasheed et al.,72 investigated the antioxidant activity of c. oblonga. an attempt from the chitral valley of pakistan performed a dpph scavenging assay of c. oblonga crude ethanol extract that validated maximum activity of 27.18% at 500µg/ml and minimum activity 9.70% at 100µg/ml. it has been elucidated that c. oblonga plant possesses great free radical scavenging properties49. the mature fruits of c. oblonga collected from different locations of poonch ajk, pakistan, evaluated for physicochemical analysis revealed the presence of certain compounds and essential nutrients. the phenolic content (gallic acid) present in fruit of this plant possesses potential antioxidant72. dactyloctenium aegyptium l. dactyloctenium aegyptium belongs to the poaceae family. it has antipyretic, anti-inflammatory, anticancer, antioxidant and antimicrobial properties. d. aegyptium is employed as a traditional medicine against smallpox and ulcers107. in an investigation from bahawalpur, pakistan, fatima et al.,64 evaluated the aerial parts of d. aegyptium plants with ethanol and acetone to assess phytochemicals with antioxidant activity. secondary metabolites with elevated antioxidant potential have been documented in ethanol and acetone extracts of d. aegyptium64. datura innoxia (mill.) datura innoxia is a member of the solanaceae family108. studies of akhtar et al.,31, fatima et al.,78 and fatima et al.,64 acknowledged the antioxidant activity of d. innoxia from different regions of pakistan. in pakistan, samples of d. innoxia from mianwali, pakistan were checked for antioxidant activity showing better antioxidant potential due to its great reduction capabilies31. different leaves and stem extracts of d. innoxia were screened for phytochemical and assessed for their antioxidant potential from islamabad, pakistan. the combined acetone and ethyl acetate stem extracts of d. innoxia proved maximum dpph radical scavenging activity with ic50 values of 16.14μg/ml. moreover, the ethyl acetate stem and aqueous leaf extracts of d. innoxia are considered to possess maximum reducing power and antioxidant actions78. in bahawalpur, pakistan, the antioxidant activity of d. innoxia leaves and fruits ethanolic extracts with 1, 1-diphenyl-2-picrylhydrazyl assay was measured where maximum activity was validated in its ethanol fruits and leaves extracts79. dichanthium annulatum (forsk.) dichanthium annulatum belongs to the family poaceae. in an inquiry from bahawalpur, pakistan, the aerial parts of d. annulatum extracted with ethanol and acetone showed secondary metabolites in the extracts of plant. greater level of antioxidant activity with dpph, abts, nbt and reducing power assay has been validated for ethanol and acetone extracts of d. annulatum64. elaeagnus species elaeagnus species are members of the elaeagnaceae family. the fruits of many species of the genus elaeagnus are sweet and contain vitamins c and vitamin e. flavonoids and alkaloids are also present in it. rahman et al.,49 documented the antioxidant action of crude ethanol extract of e. angustifolia from chitral valley of pakistan. their results showed great antioxidant activity of this plant extracts. in one study, the radical scavenging activity and presence of phytochemicals from berries extract of elaeagnus umbellate with methanol was validated from rawlakot, ajk, pakistan. a conspicuous dpph scavenging activity and presence of alkaloid, flavonoid, phenolic, carotenoid, saponin and tannin contents made this plant a worthy natural asset with potential antioxidant activity50. eleusine indica l. the plant eleusine indica is from poaceae family. fatima et al.,64 screened and evaluated the aerial parts of e. indica plants form bahawalpur, pakistan, extracted with ethanol and aceton for phytochemical and antioxidant activity. secondary metabolites presence has been https://www.google.com/search?rlz=1c1chbd_enpk804pk804&q=elaeagnaceae&stick=h4siaaaaaaaaaongvuluz9u3mezkstnaxmrjmpoympiel5icmpgkaafjdrucaaaa&sa=x&ved=2ahukewjwqoajisjjahvhofwkhy96c_qqmxmoatadegqidrak a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 74 r a d s j . b i o l . r e s . a p p l . s c i . 74 confirmed in extracts of plant. an acceptable range of antioxidant activity and reducing power assay has been authenticated in the acetone and ethanol extracts of e. indica. euphorbia royleana euphorbia royleana from the family euphorbeaceae has been utilized as traditional medicine against anemia, asthma, cough, and constipation, jaundice and skin problems109. an evaluation of antioxidant, antitumor and antimicrobial activities of hexane, methanol and aqueous extracts of e. royleana whole plant has been performed from faisalabad, pakistan. antioxidant activity assessed by dpph and ferric ions reduction assay, validated a linear increase in inhibition values with increase in extract concentration. the ferric reducing assay of hexane extract substantiated with supreme ferric reducing power of 12.70 ± 0.49mg equivalents to gallic acid. maximum flavonoid content of 63.68 ± 0.43 mg equivalents of querectin and phenolic content of 47.47 ± 0.71 mg as equivalents of gallic acid were confirmed in the hexane extract110. fagonia cretica l. fagonia cretica is a vital member of the zygophyllaceae family. in one study85 from district layyah, pakistan, samples of f. cretica collected were assessed for antioxidant potentials of its methanol extracts of roots, fruit and upper parts. the total flavonoid and phenolic content values for methanol extracts and numerous fractions of roots and aerial part displayed prodigious total flavonoid and phenolic contents. all the tested extracts and fractions of f. cretica particularly, the root extracts have extraordinary antioxidant and radical scavenging capabilities85. akhtar et al.,31 collected samples of f. cretica from hazara, pakistan and determined the antioxidant activity of the stem and spines methanol/chloroform extracts. they confirmed that the plant extracts contains flavonoids and phenols that gives antioxidant activity due to the reduction and total antioxidant abilities. ficus l. species ficus species belongs to the moracea family. species of this genus possess a material that provides self healing tendency and protection to the plant from foreign attacks111. rauf et al.,32 and akhtar et al.,31 have documented the antioxidant activity of ficus species from pakistan. in a study from razagram toormang dir, kpk, pakistan, the chemical test of aerial portion of ficus sarmentosa plant showed presence of amino acids, anthraquinones, beta cyanin, cardiac glycoside, flavonoids, phlobatanins, reducing sugars, steroids, terpenoids, and tannins in methanol extracts and ethyl acetate fraction with antioxidant activity32. ficus carica is another vital species of the moraceae family commonly found in the temperate parts of the world. leaves and fruits of this plant possess great antioxidant and antimicrobial potentials112. samples of f. carica, collected from bannu, pakistan confirmed the presence of phytochemicals and antioxidant action assessed with dpph assay revealed promising outcomes. results corroborated that the methanol extract of f. carica fruit is good source of flavonoids, polyphenols, terpenoids, alkaloids and saponins with potential antioxidant activity. fruit of this plant is also a worthy source of chromium iron, copper, selenium, manganese and zinc60. fiscus microcarpaa from islamabad, pakistan has been scrutinized for the free radical scavenging activity of its aqueous and methanol/chloroform extracts. extracts of this plant validated the presence of phenolics and flavonols with greater antioxidant activities due to antioxidant and reduction possessions31. grewia asiatica l. grewia asiatica belongs to the family malvaceae. species of the malvaceae family are significant medicinally because of the presence of effective antioxidant compounds in their extracts including melvin, phenols and vitamins a and c35. akhtar et al.,31 pooled samples of g. asiatica from mianwali, pakistan and the antioxidant activity of its methanol/chloroform and aqueous extracts assessed ascertained significant antioxidant outcomes because of its wonderful reduction capacity. an investigation of methanol leaves extracts of g. asiatica from multan, pakistan evident the presence of phenolic contents. the antioxidant potential designated the leaves extracts of g. asiatica as beneficial assets with natural antioxidants24. haloxylon griffithii haloxylon griffithii plant belongs to the chenopodiaceae family. it consists of ~100 genera with 1200 species113. a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 75 r a d s j . b i o l . r e s . a p p l . s c i . 75 baki et al.,45 informed the antioxidant activity of h. griffithii from balochistan, pakistan. they determined the antioxidant potentials of methanol extracts of h. griffithii spectro photometrically through the frap, dpph and tac of phosphor molybdenum assay. the values for dpph assay of h. griffithii recorded were 95.03% at the concentration of 1.0 mg/ml. furthermore, the frap value of the h. griffithii extract of extract proved a high and persuasive antioxidant activity. hippophae rhmnoides l. (sea buckthorn) hippophae rhmnoides is a bush species that belongs to the family eleagneaceae. in pakistan, hippophae rhamnoides berries obtained from the skardo district and their methanol extracts were scrutinized for potential antioxidant activity. the extract containing emulsions and polyphenol-rich plant-derived extracts from this plant have proved promising antioxidant possibilities102. leaves samples of h. rhamnoides collected from skardu, pakistan were inspected for the antioxidant activity of their methanol, acetone, n-hexane, chloroform, aqueous, ethanol, ethyl acetate and extracts where methanolic extracts displayed the highest activity. the leaves extracts of h. rhamnoides exhibited improved dose-dependent antioxidant activity and thus designated as precious natural antioxidant52. imperata cylindrica l. imperata cylindrica belongs to the family poaceae. it is also called cogon or thatch grass. it possesses antioxidant, anticancer, immunomodulatory and neuroprotective activities114. investigations of fatima et al.,64 form bahawalpur, pakistan validated the ethanol and acetone extracts of i. cylindrica aerial parts with important phytochemicals having antioxidant activity. secondary metabolites were confirmed with greater antioxidant action in acetone and ethanol extracts of i. cylindrical. ixora coccinea l. ixora coccinea belongs to the family rubiaceae. this plant is a great source of essential compounds including flavonoids, alkaloids, steroids tannins and terpenoids115 which gives antioxidant, hepatoprotective, antimicrobial, anti-mitotic, antinociceptive, anti-inflammatory and cardiovascular activities116. various researchers19,115,116 have acknowledged the antioxidant activity of this essential plant from pakistan. in karachi, pakistan, the methanolic extracts, fractions and sub-fractions of i. coccinea were assessed for free radical scavenging activity based on dpph assay. i. coccinea leaves methanol extracts showed greater antioxidant action among the tested extracts with values ic50= 0.00315 ± 0.0001mg/ml. twenty-six pure chemical compounds were obtained after further purification of the active fractions of i. coccinea where 5-o-caffeoyl quinic acid was obtained from flower fraction showed better antioxidant activity with ic50= 0.0467 ± 0.0018mg/ml than standard antioxidants (bht and ascorbic acid)19. jasminum l. species jasminum l. is from the oleaceae family. about ~28 genera with ~900 species have been assigned to this family. the genus jasminum contains ~200 species117. the methanol/chloroform and aqueous extracts of two jasminum species like jasminum sambac (l.) aiton and jasminum officinale l. showed flavonols and phenolic compounds with antioxidant potencies because of the great antioxidant and reduction capacities31. another jasminum species, j. humile was collected from abbottabad, pakistan and assessed for total phenolic content, antioxidant activity, no scavenging and antibacterial profile of its flower extracts (n-hexane, nbutanol and ethyl acetate). these plant extracts have been designated with vital antioxidant action with better ic50 values. furthermore, phenolic contents present in the extracts of j. humile flower with antioxidant activity are a natural asset to impede microbial growth and inhibition of oxidative degradation, particularly in the food industry148. juglans regia juglans regia is a member of juglandaceae family. its fruit used in the diet reduces the chances of coronary heart diseases118. linoleic acid among the fatty acids is the most important one, followed by palmitic acid, stearic acid and oleic acid119. the antioxidant activity of j. regia from the chitral valley of pakistan was validated by rahman et al.,49 where they confirmed that j. regia holds greater antioxidant potential. the dpph scavenging possessions of methanol crude extract of j. regia revealed activities of 72.98% at a concentration of 100µg/ml and 95.42% at a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 76 r a d s j . b i o l . r e s . a p p l . s c i . 76 500µg/ml concentration. nevertheless, the lowest activity of 90% at 100µg/ml and highest activity of 98% at 500μg/ml was presented by ascorbic acid. justicia adhatoda l. justicia adhatoda of the family acanthaceae grows wild in india, nepal and pakistan120. it is commonly called malabar nut or vasaka with bitter taste and unpleasant smell121. the medicinal significance and antioxidant activity of j. adhatoda from pakistan have been reported by various researchers19,31,40,83. in a study from soon sakesar, punjab, pakistan, the phytochemical, antioxidant and antibacterial evaluation exposed a very good range of phenolic compounds in ethanol or methanol fruit andflower extracts of j. adhatoda. antioxidant potential of fruit and flower extracts authenticated moderate to good ability to dpph free radicals scavangeing, linoleic acid peroxidation inhibition with better antibacterial activity83. in vitro study from islamabad, pakistan also validated leaf extracts of j. adhatoda with total alkaloid, crude oil, dietary fiber, flavonoid, protein, phenols, saponins, tannins and essential and non-essential metal ions. the ethanol extract of j. adhaotda have been designated as a better antioxidant candidate than butanol, chloroform, methanol and water extracts40. akhtar et al.,31 collected sample of j. adhatoda from islamabad, pakistan and the antioxidant activity of aqueous and methanol/chloroform leaves extract demonstrated antioxidant potential due to its reduction capacity. another study from mardan, kpk, pakistan focused on the pharmacological activities of justicia adhatoda with antioxidant activity. the dpph activities and concentration of fractions were proportional because when the concentration of fractions increases the percent scavenging activity also increases19. lantana camara l. lanata camara is a member of the verbenaceae family. it is utilized as a traditional medicine in pakistan and grows at in altitude of 1500m58. in one study, a phytochemical, antibacterial and antioxidant evaluation from soon sakesar, punjab pakistan exposed a very good range of phenolic compounds in ethanol or methanol fruit and flower extract of l. camara. the flower and fruit extracts were authenticated to scavenge dpph free radicals and inhibit linoleic acid peroxidation with better antibacterial activity83. in another inquiry from karachi, pakistan, the antioxidant activity of methanol extracts and its fractions with lantadene-a, lantanilic acid and oleanolic acid, from upper portion of l. camara have been endorsed with good dpph activity. the methanolic extract, its aqueous, petroleum ether and ethyl acetate fractions possess active antioxidant agents. one pure compound (oleanolic acid) was authenticated better antioxidant activity in contrast to ascorbic acid84. melia azedarach melia azedarach is a deciduous tree from the meliaceae mahogany family. an investigation from faisalabad, pakistan m. azedarach leaves, stem bark and fruit aqueous and methanol extracts showed total flavonoid and phenols. a greater linoleic inhibition capacity and dpph scavenging activity for sun-dried methanol extracts as compared to ambient dried extracts has been acknowledged and substantiated the potent antioxidant activity of m. azedarach59. azadirachta indica (a. juss.) is another significant species of the family meliaceae. methanol/chloroform and aqueous leaves extracts of this plant assessed from mianwali pakistan corroborated the presence of antioxidants like flavonols and phenolic compounds with great antioxidant possessions and reduction abilities31. mentha l. species the genus mentha l. is from the lameaceae family. species of this genus are commonly found in central asia, europe, and non-tropical regions of africa. extracts from mentha species are used against headache, cold, cough, indigestion and stomach cramps122. investigations of andleeb et al.,57, akhtar et al.,31 and waris et al.,28 acknowledged the antioxidant action of mentha species from pakistan. mentha longifolia is another vital species of the genus mentha. the phytochemical analysis of whole plant acetonic extracts of m. longifolia leaves from peshawar, pakistan corroborated the presence of flavonoids, alkaloids, saponins, triterpenoids and steroids. moreover, the greatest scavenging of free radicals (%) of the acetonic extract of this plant measured was 59% at many concentrations (100-500μg/ml)28. mentha piperita is a crucial species of the mentha genus. its methanol/chloroform and aqueous extracts were a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 77 r a d s j . b i o l . r e s . a p p l . s c i . 77 assessed from rawalpindi, pakistan where presence of antioxidants like flavonols and phenolic compounds provided great antioxidant capabilities in the tested extracts due to reduction abilities31. a study on mentha spicata plant, collected from muzaffarabad, ajk pakistan revealed that its diethyl ether, chloroform, ethanol and methanol extracts from leaves possess significant antioxidant activity57. momordica charantia (bitter gourd) momordica charantia is from the cucurbitaceae plant family. it is cultivated and eaten as a vegetable worldwide123. a study from lahore, pakistan assessed the antioxidant activity of this significant plant which proven to be a potent antioxidant candidate. the flakes extract retain potent free radical scavenging action of 63.20% followed by seed with 33.05%, dpph % inhibition at 2mg/ml concentration related to bht as a standard antioxidant48. moringa oleifera l. moringa oleifera is a member of moringaceae family. m. oleifera leaves are rich in vitamins a and c124 and other phytochemicals like flavonoid pigments such as rhamnetin, kaempferol, isoquercitrin and kaempferitrin125. a lot of researchers31,62,124 have conveyed the phytochemical analysis and antioxidant potentials of this important plant from pakistan. akhtar et al.,31 collected a sample of m. oleifera from kundian, mianwali, pakistan and checked the antioxidant action of its aqueous and chloroform/methanol extracts. the antioxidant activity of m. oleifera extracts verified extraordinary antioxidant activities because of better reduction capacity. lemo, a phytochemical extracted from the ethanol leaves extracts of m. oleifera in faisalabad, pakistan has been verified as a better stabilizer of butter at refrigeration temperature. lemo attained from ethanol leaves extracts of m. oleifera to possess prospective antioxidant activity at a concentration of 600ppm and is convenient in retaining organoleptic features of butter in the course of storage62. morus alba (white mulberry) morus alba is a member of the moraceae family. m. alba is used as a traditional medicine against bronchitis, digestive disorder, headache, smallpox, scabies and ulcers etc. several researchers31,49,61 have described the antioxidant efficacy of morus species from different regions of pakistan. a phytochemicals screening from mardan, pakistan confirmed the presence of flavonoids, alkaloids, reducing sugar, terpenoids and phloba-tannins in aqueous leaf extracts of m. alba61 which give antioxidant activity to this plant. akhtar et al.31 studied the antioxidant activity of aqueous and methanol/chloroform extracts of morus nigra collected from islamabad, pakistan. the tested extracts of this plant validated better antioxidant actions with great reduction capability. the dpph activity of crude ethanolic extracts of m. alba from the chitral valley of pakistan showed acceptable antioxidant activity of crude ethanol extracts49. nasturtium officinale (w. t. aiton) nasturtium officinale belongs to the family brassicaceae. its extracts contain phenolic compounds, mainly in β-carotene and flavonoids in noe126. studies of akhtar et al.,31, khan et al.,128 and bibi et al.,127 have documented the antioxidant activity of this plant from pakistan. akhtar et al.,31 collected samples of n. officinalen from salgira park pakistan and scrutinized the methanol/chloroform and aqueous extracts for potential antioxidant activity. their results confirmed phenols and flavonols in the extracts with potential antioxidant action. bibi et al.,127 acquired samples of n. officinale from tanawal areas of pakistan and found the maximum antioxidant activity of its methanol extracts. wild plants of pakistan were evaluated for nutritional, antioxidant and antimicrobial assessment including the n. officinale and its antioxidant activity was authenticated in correlation with the presence of phenolic compounds128. from the north kpk region of pakistan, the n. officinale ethanol crude extracts and fractions were tested to check antioxidant action with dpph assay. their results validated ethanol crude extract with an acceptable range of antioxidant activity38. origanum species origanum is a member of the lamiaceae family with ~38 shrubby, perennial and annual plants species. the majority of species of this genus are native to east asia, europe, north africa and the mediterranean area129. in a study58 from faisalabad, pakistan determine 39 and 43 components from two origanum species. the major constituents like α-terpineol, terpinene-4-ol, linalool, https://en.wikipedia.org/wiki/moraceae https://www.sciencedirect.com/topics/medicine-and-dentistry/phenol-derivative https://www.sciencedirect.com/topics/medicine-and-dentistry/beta-carotene a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 78 r a d s j . b i o l . r e s . a p p l . s c i . 78 linalyl-acetate and limonene have been documented from o. majorana extract; however, α-terpineol, carvacrol, thymol and o-cymene have been validated in o. vulgare extract. the resazurin microtitre and disc diffusion assays corroborated o. majorana extract with better antibacterial activity than o. vulgare extract against bacteria. the revelations of dpph assay displayed o. vulgare extract with better activity than o. majorana extract and both inhibited better oxidation of linoleic acid. nevertheless, in another assay (bleaching β-carotene) o. vulgare extract substantiated improved antioxidant action as compared to o. majorana extract58. oryza sativa l. (rice bran) it belongs to the family poaceae. rice bran comprises 100 different antioxidants including oryzanol, tocotrienol and tocopherol. studies of iqbal et al.,65, anwar et al.,66 and zubair et al.,67 documented the antioxidant activity of oryza sativa from pakistan. the antioxidant potential of methanol extracts of indigenous varieties of rice bran was evaluated from gujranwala, pakistan. the presence of phenolic compounds with strong antioxidant activity was observed. rice bran variety (rb-kr) displayed the longest growth period with remarkable antioxidant activity65. another assessment from karachi, pakistan revealed the antioxidant action of rice bran methanol extracts. higher activity among the rice bran extracts was authenticated. moreover, the activity of some extracts in substrate oils stabilization under ambient was significantly different during storage66. from kalashahkako lahore, pakistan, 10 different varieties of rice screened with orbital shaker technique and the effect of pure and aqueous ethanol, methanol and isopropanol on phenolics was evaluated with antioxidant activity. the aqueous methanol and aqueous isopropanol as an effective solvent promoted higher content of total phenolics and a remarkable antioxidant activity through dpph assay, ferrous ionchelating activity and reducing power, particularly in three rice varieties67. pentanema vestitum l. pentanema vestitum is a plant from asteraceae family and sub family inuleae130. the antioxidant activity of whole plant methanolic extracts of p. vestitum from malakand division khyber pukhukhwa, pakistan on the basis of dpph assay was acknowledged29. a nonsignificant difference in the inhibition percent at different concentrations of methanol extracts of p. vestitum and ascorbic acid at p<0.05 with ic50 value of 13.00ppm in p. vestitum and 15.09ppm of ascorbic acid demonstrated potential antioxidant efficiency of this plant29. periploca aphylla periploca aphylla belongs to the family asclepiadaceae. p. aphylla is medicinally employed for the treatment of swellings and tumors and its bark is specifically used in contradiction of fever131. rauf et al.,32 acknowledged antioxidant potential of p. aphylla from razagram, toormang, dir khyber pakhtunkhwa, pakistan, where the photochemical of screening of aerial part of p. aphylla revealed the presence of amino acid, beta cyanin, flavonoids, cardiac glycoside, comurrine, steroids, terpenoids and tannins. the dpph assay validated methanol extracts and fraction of ethyl acetate of p. aphylla with significant scavenging activity among the entire fractions32. pistacia integerrima (stew.) pistacia integerrima is a tree from anacardiaceae family. it possesses promising biological activities like antioxidant, antimicrobial and hepatoprotective activities132. ilahi et al.,29 documented the antioxidant potential of p. integerrima from malakand division, khyber pukhukhwa, pakistan, based on dpph assay. methanol extract from fruit of p. integerrima corroborated considerably maximum activity at different concentrations with ic50 value of 5.75ppm for p. integerrima and 15.09ppm for ascorbic acid. poa annua l. poa annua belongs to the poaceae family. p. annua is cosmopolitan annual bluegrass with high phenotypic and genotypic variations. in pakistan, an inquiry from bahawalpur, appraised the aerial parts of p. annua plants with acetone and ethanol for the presence of phytochemical and antioxidant activity. secondary metabolites in majority of plant extracts has been recorded with minimum abts, dpph, nbt and reducing power assay in ethanol and acetone extracts of p. annua64. a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 79 r a d s j . b i o l . r e s . a p p l . s c i . 79 psidium guajava (guava) psidium guajava belongs to the myrtaceae family. p. guajava is utilized as a remedy to treat skin problems and healing wounds133. in pakistan, wadood et al.,61 and rahman et al.,49 documented the presence of phytochemicals and assessed potential antioxidant activity of this plant. a study from the chitral valley of pakistan revealed dpph scavenging possessions of p. guajava crude ethanolic extract. the maximum inhibition percent for the ethanol extracts noticed was 12.28% at a concentration of 500μg/ml and minimum was 4.91% at 100μg/ml. on the other hand, minimum activity of 90% at 100μg/ml concentrations and maximum activity of 98% at 500μg/ml concentration of ascorbic acid was recorded49. a similar study from mardan, pakistan reported phytochemical constituents like alkaloids terpenoids, flavonoids, phlobatannins and reducing sugars in the aqueous extracts of p. guajava leaves61 that gives potential antioxidant action. pteridium aquilinum pteridium aquilinum belongs to dennstaedtiaceae amily and utilized as wild vegetable in few countries of the world. it is employed as a traditional medicine contrary to headache, high fever and rheumatism126. from north khyber pakhtunkhwa, pakistan, the crude ethanolic extracts and various fractions of p. aquilinum were evaluated for antioxidant action with dpph assay that revealed crude ethanol extract with a remarkable highest range (93.97%) of antioxidant activity38. quercus incana (roxb.) quercus incana plant is from the fagaceae family. this family is characterized by 8 genera containing ~900 plant species distributed in world temperate region. in pakistan, species of two genera like castana and quercus represents this family. six wild species of the genus quercus are distributed in the northern pakistan54. an inquiry from abbottabad district of pakistan examined q. incana extracts for their potential antioxidant activity along with antimicrobial activity. the n-butanol fraction of this plant is considered as relatively good option for antioxidant activity with great ic50 value (55.4 + 0.21μg/ml). the ethyl acetate fraction has no scavenging activity (ic50 = 23.21 + 0.31μg/ml) and is considered to be fairly good than other fractions. moreover, q. incana extracts (n-butanol) possess substantial antifungal and antibacterial actions against certain microorganism54. likewise, the antioxidant activity of a compound (quercuschin) extracted from ethyl acetate fraction of q. incana bark holds a remarkable antioxidant activity (51.2μg/10μl) as compared to standard butylated hydroxyanisole (45.9μg/10μl)55. rosa indica l. rose is from the genus rosa of the rosacea family. species of the rosa genus holds promising phytochemicals with biological activities73. investigations of akhtar et al.,31, khurshid et al.,74 and zahid et al.,73 recognized the antioxidant activity of species of rosa from pakistan. in a study from islamabad, pakistan, the presence of phenolic compounds with antioxidant activity of the methanol/chloroform and aqueous extracts of rosa indica validated antioxidant and reduction capacities31. in rawalakot ajk, pakistan, the antioxidant activities of rosa species aqueous extracts revealed inhibition against lipid peroxidation, in mice liver homogenate induced by prooxidants. extracts of rosa species validated high antioxidant and metal chelating activities in the phosphomolybdenum assay. it has been corroborated that the presence of extraordinary contents of favonoids and phenolics in aqueous extracts gives antioxidant potential to rosa species74. in the ethanol leaves extracts of rosa indica varieties, the presence of phytochemicals with potential antioxidant activity was confirmed. r. indica var cardinal red variety contains highest tpc (gallic acid) and tfc (quercetin) in its leaves extracts. additionally, the phytochemicals present in the ethanol leaves extracts of r. indica var pink have highest percentage inhibition potency against free radicals73. saccharum spontaneum l. saccharum spontaneum is a member of the family poaceae. a. spontaneum has a folk traditional medicinal uses against abdominal disorders, anaemia, obesity and vomiting. roots of this plant are employed as diuretic, astringent, tonic, emollient and utilized against burning sensation, dyspepsia, respiratory ailments and piles134. in an inquiry form bahawalpur, pakistan, the aerial parts of s. spontaneum plants extracted with ethanol and aceton has been screened for phytochemicals and evaluated for antioxidant activity. phytochemicals inquiry corroborated a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 80 r a d s j . b i o l . r e s . a p p l . s c i . 80 that secondary metabolites are present in the extracts of plant with high dpph, abts, nbt activity and reducing power in ethanol and acetone extracts of s. spontaneum64. solanum nigrum l. solanum is a prevalent genus of the solanaceae family. in pakistan, 15 species of solanum are commonly found and 11 of them are acknowledged as medicinally important135. a crucial water insoluble steroidal alkaloid called solasodine is used to synthesize steroid drugs like corticosteroids136. solanum nigrum, an important species encompasses many important compounds like essential oils in different parts 137. in mirpur bhimber, pakistan, mazher et al.,80 unveiled the phytochemical profile of fruit, stem, and leaf extracts of s. nigrum prepared in distilled water, chloroform, ethanol and petroleum ether. phytochemicals like alkaloids, flavonoids, saponins, steroids, tannins, terpenoids were observed in fruits extracts of s. nigrum. in bahawalpur, pakistan, the antioxidant activity of the fruits and leaves ethanolic extracts of s. nigrum with dpph assay validated improved percentage of antioxidant activity. hence, it could be substantiated that s. nigrum is a potential source of antioxidants79. stellaria media l. stellaria media of the family caryophyllaceae is a small shrub with 20-30cm height. s. media is employed against inflammation, kidney problems, wounds, rheumatic joints and ulcers. s. media is also against bronchitis, skin diseases, rheumatic pains and period pain138. study of usman et al.,38 from north khyber pakhtunkhwa, pakistan evaluated the crude ethanolic extracts and various fractions of s. media for potential antioxidant activity with dpph assay. it has been revealed that the ethanol crude extracts of s. media possess an acceptable range of antioxidant activity. swertia chirata (roxb.) swertia chirata is a member of the gentianaceae family. it is being employed as anthelmintic, antipyretic139 and used for the treatment arthritis140. it is also used for disorders assossiated with urogenital or gastrointestinal tract disorders139 and for the treatment of cancer141. in a study from galyat region of kpk, pakistan, methanolic extract fractions of swertia chirata aerial parts confirmed flavonoid and phenolic compounds with significantly high dpph scavenging action. moreover, these fractions have active antileishmanial and antimicrobial activity56. terminalia arjuna l. terminalia arjuna from the family combretaceae is commonly called as arjuna. it is an extraordinary tree for having significant phytochemicals in its extracts142. in an inquiry from faisalabad, pakistan, the antioxidant activity of stem bark and leaves extracts of t. arjuna with aqueous methanol and ethanol were evaluated. the t. arjuna extracts contained considerable amount of total flavonoids as catechin and total phenolics as gallic acid equivalent. t. arjuna extracts also possesses a good dpph activity with ic50 values of 2.71-7.68μg/ml. t. arjuna extracts also showed inhibition of lipid peroxidation (64.79-71.43%) and reducing power (0.0011.584mg/ml)47. trifolium repens trifolium repens is a member of the family leguminosae. there are 103 species commonly found in turkey. trakya having 67 trifolium taxa and is a centre of diversity143. in north khyber pakhtunkhwa, pakistan, the crude ethanolic extracts and various fractions of t. repens were evaluated for antioxidant action with dpph assay that revealed crude ethanol extract with an acceptable range of antioxidant activity38. typha domigensis (pers.) typha domigensis is a member of the typhaceae family. pollen grains of typha genus are utilized in diet to cure fever and injuries and to prevent wound bleedings. sardar et al.,81 reported the inhibition of free radicals in various solvents with abts and dpph radical scavenging assays from pakistan. t. domigensis pollen extracts have better antioxidant action because of phenolic and flavonoid compounds which are are better candidates in the formulation of drugs against cancer and aging disorders. urtica dioica (l.) urtica dioica is a a plant species from the urticaceae family. leaf extracts of u. diocia are employed as an antihaemorrhagic to control nose bleedings and extreme menstrual flow. roots of this plant are used against benign prostate hyperplasia. it is also utilized against rheumatic conditions and urinary tract disorders and a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 81 r a d s j . b i o l . r e s . a p p l . s c i . 81 allergies144. the distribution of phytochemicals from the aerial parts ethanol extracts of u. diocia from islamabad, pakistan authenticated the presence of important flavonoids like orientin and luteolin82. in the north khyber pakhtunkhwa, pakistan, the crude ethanolic extracts and various fractions of u. dioica screened for antioxidant potential using dpph assay showed ethanol crude extract with superior antioxidant activity38. vetiveria zizanioides (l.) vetiveria zizanioides of the poaceae family has thick adventitious roots. it is vetiver grass traditionally used as stimulant and stomachic. it is used to treat inflammations and fevers. this grass has applications in perfume industry and also grown for oil production145. in an inquiry form bahawalpur, pakistan, the aerial parts of v. zizanioides evaluated for phytochemicals and antioxidant activity confirmed the secondary metabolites present in most of the plant extracts. the higher antioxidant activity with dpph, nbt, abts and reducing power assay has also been documented in acetone and ethanol extracts of v. zizanioides64. withna species withna species belongs to the family solanaceae. species of the withna genus have medicinal value and has been reported for their antioxidant and antihyperglycemic activity146. in pakistan, the antioxidant activity of two vital species of the withna genus namely w. somnifera29,79 and w. coagulans29,31 have been reported. in a study from malakand division of kpk, pakistan, ilahi et al.,29, estimated antioxidant action of w. somniferra with dpph assay. the percent inhibition (ic50 = 46.85ppm) caused by methanolic extract w. somniferra fruits was worth noticing with great antioxidant potential. akhter et al.,31 collected samples of w. coagulans from mianwali, pakistan and assessed the antioxidant activity of aqueous and methanol/chloroform extracts. the tested extracts validated promising antioxidant actions due to its total antioxidant and reduction dimensions. in an inquiry from bahawalpur, pakistan, the antioxidant activity with dpph assay corroborated enhanced percentage of antioxidant activity in the ethanol leaves and fruit extracts of w. somnifera79. zea mays (corn) zea mays belong to the family poaceae. its extracts contain steroids, alkaloids and flavonoids which are significant source of natural antioxidants. phenolic compounds can also be extracted from corn silk extract of zea mays and these constituents possess great antioxidant activity in nature147. various researchers68,69,70 documented the antioxidant potential of z. mays from pakistan. an inquiry on corn silk from multan, pakistan showed worthy phytochemical composition with a robust antioxidant activity. a polarity dependent rise in extracts yield, free radical scavenging action and phytochemical content of extracts reinforced with regression analysis of experimental data68. z. mays seeds collected from sahiwal, pakistan were screened for phytochemicals that showed coumarins, saponins, alkaloids, terpenoids and tannins. an increased tpc under lead stress and greater scavenging rate of maize seeds methanol extract sanctioned maize extracts with potential antioxidant activity69. a study from faisalabad, pakistan exposed three varieties of z. mays including golden, sultan and agatti: 85 containing phenolic compounds with effective inhibition in the oxidation process present in its ear tissues extracts. maize hinders the oxidation reaction and decreases diabetes mellitus complications. the total phenolics content, sod, peroxidase and catalase are the indicators for higher antioxidant activity in decreasing obstructions in diabetic patients70. ziziphus jujube (mill.) ziziphus jujube belongs to the family rhamnaceae. it contains simple carbohydrates (9%), protein (2%) and polysaccharides (2%)144. investigations of rasool et al.,149, shad et al.,60 and rahman et al.,49 validated antioxidant activity of species of z. jujube from pakistan. the dpph assay of crude ethanolic extract of this plant examined from chitral valley of pakistan exposed better antioxidant activity of 19.52% at concentration of 500μg/ml and 2.00% at 100μg/ml concentration). lowest activity of 90% at concentration of 100μg/ml and maximum activity of 98% at 500μg/ml concentration was showed by ascorbic acid49. a study from kohat, pakistan corroborated that the methanol extracts of fruit of z. jujube are good source of alkaloids, flavonoids, polyphenols, terpenoids and saponins. fruit of this plant has shown to a review on pakistani medicinal plants with potential antioxidant activity vol. 11 (1), june 2020 issn (print): 2305 – 8722 issn (online): 2521 – 8573 82 r a d s j . b i o l . r e s . a p p l . s c i . 82 be a good source of copper, iron, chromium, selenium, manganese and zinc. the phytochemicals present are potential source of antioxidant agents60. in faisalabad, pakistan, the antioxidant effectiveness of z. jujube showed the total flavonoid and phenolic contents in shoot extracts substantiated good dpph inhibition (ic50) and inhibition of linoleic acid peroxidation. methanolic extract and chloroform fraction of the z. jujuba shoots extracts have shown with highest antioxidant activity149. c o n c l u s i o n since the last decade, phytochemical investigations with potential antioxidant action of plant extracts from different families have got substantial consideration. antioxidants from plants origin, with free radical scavenging activity, have great applicability in the treatment and prevention of health-related complications caused by free radicals. asteracea, poaceae and rutaceae are the prominent plant families whose species are maximally scrutinized for their potential antioxidant activity from pakistan. 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https://bmccomplementalternmed.biomedcentral.com/articles/10.1186/1472-6882-11-65#auth-6 crohn's disease: retrospective study in algerian patients vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 134 op e n ac c e s s f u l l l e n g t h a r t i c l e crohn's disease: retrospective study in algerian patients hassina guetarni1,2*, meriem aissou1 1faculty of natural sciences, university bounaama djilali of khemis miliana, ain defla, algeria. 2laboratory of natural substances valorization, university bounaama djilali of khemis miliana, ain defla, algeria. a b s t r a c t background: inflammatory disease of crohn affects the entire digestive tract, with extra-intestinal manifestations and immune disorders. objectives: this work aims to represent the histopathological aspects of crohn‘s disease and the establishment of pathogenic bacteria as causal agents. methodology: the histopathological aspects of the disease were studied on a colonic resection specimen and on intestinal biopsies with colorations topographic staining. pathogenic bacteria responsible for crohn‘s disease have also been isolated and identified. the study is continued to establish a correlation between the disease and exposure to infections by unusual bacteria, particularly pathogens (salmonella, shigella, streptococcus pyogenes, klebsiella). results : the macroscopic appearance of the disease presented transmural involvement and can be complicated by abscesses and fistulas, microscopic appearance indicated the infiltrate of inflammatory cells (lymphocytes, plasma and cells) and lymphoid follicles after topographic staining. crohn’s disease is an idiopathic disease, it is assumed that it is a deregulation of the immune system due to an infectious agent in genetically predisposed people. in our work, we studied the microbiota of intestine and stool of crohn’s patients in which we found certain bacteria including proteus mirabilis with a predominance of e. coli. other pathogenic bacteria were found like salmonella spp., shigella spp., klebsiella spp. and streptococcus pyogenes. two cases which tested positive on ziehl neelsen stain represented mycobacterium avium paratuberculosis. conclusion: the histopathological aspect of cd can be better visualized and identified on surgical specimens than on endoscopic biopsies, which helps to monitor the evolution of the disease and must be accompanied by clinical, serological and radiological exploration. keywords crohn’s disease, inflammatory granulomas, lymphoid follicle, ziehl neelsen staining, hematoxylin eosin staining, mycobacterium avium paratuberculosis. *address of correspondence kmhg2009@yahoo.fr article info. received: september 03, 2022 accepted: december 02, 2022 cite this article: guetarni h, aissou m. crohn’s disease: retrospective study in algerian patients. 2022; 13(2):134-147. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. i n t r o d u c t i o n crohn’s disease is ibd1, affects the entire digestive tract, most often ileum and colon2. it’s involvement is transmural that is mucosal involvement extending to all layers of the wall. it’s diagnosis is based on a range of clinical, biological, endoscopic and histological arguments3. it develops in flare-ups, interspersed with remissions, and o r i g i n a l a r t i c l e crohn's disease: retrospective study in algerian patients vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 135 can be complicated by structures, abscesses and fistulas. the goal of treatment is to heal the lesions before the occurrence of irreversible complications3. crohn’s disease has two main peaks of occurrence in humans, the first around the age of 30 and second peak around the age of 60-70. however, it can occur at any age, especially in children and the elderly. crohn’s disease mainly affects women with a sex ratio of 1.2. every 6 to 8 patients per 100,000 inhabitants in france are affected with crohn’s disease which corresponds to a prevalence of 80,000 cases in france. this incidence is highly variable depending on the region with a north-south gradient found between the countries of northern europe and those of the south4. theories of cd etiology with many factors were proposed: alimentation, environment also dysregulation against the microbiota, all of the work have been viewed regarding genetic predisposition of patients5. the objectives of this work are to present the histopathological aspects of cd on a colon resection specimen and on intestinal biopsies with he topographic staining, to identify the pathogenic bacteria as causative agent (mycobacterium avium paratuberculosis, e. coli) and to determine the correlation between the disease and exposure to infections by unusual bacteria, particularly pathogens (salmonella, shigella, streptococcus pyogenes, klebsiella). m a t e r i a l s a n d m e t h o d s workplace our experimentation was performed in several units lasting four months from february to may 2015: • gastroenterology departmentmustapha bacha (algiers); internal medicine department-uhc kouba (algiers); internal medicine departmentuhc beni messouse (algiers) for the collection of intestinal biopsies (two fragments in 10% formalin and two others in physiological water). • anatomopathological and cytological department uhc (medea) for the anatomo-pathological examination of intestinal biopsies and of the colonic resection specimen. • ain defla medical analysis laboratory of dr zibouche for the bacteriological examination of intestinal biopsies. biological material intestinal biopsies were obtained from the 15 patients representing ileal and colonic cd (diagnosed) who underwent low endoscopy (control colonoscopy), for the search for map and bacteriological examination; two fragments were placed in physiological saline to store at 4°c. biopsies should be taken from pathological and healthy mucosa for bacteriological examination; and two biopsies fragments were taken from pathological mucosa embedded in 10% formalin for pathological examination (table 1). table 1. medical characteristics of patients. number sex age (years) duration of illness (years) diagnosis/ localization cd surgery 01 male 32 4 ileo-colic no 01 female 41 18 diffuse, several locations partial colectomy (distal part of right colon) 02 male 39 6 ileal no 02 male 25 3 colic no 02 female 17 0.8 ileal no 02 female 22 3 ileocecal no 02 female 53 14 ilea no 01 female 23 3 colic no 01 female 23 2 colic no 01 female 23 4 colic no crohn's disease: retrospective study in algerian patients vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 136 stools from the same patients (5 out of 15 patients) were collected in sterile plastic pots for comparison of bacteriological results. an operating specimen of an old woman of 39 years. undergoing a colectomy in the medea (internal medicine department), presented a treatment failure for a month with severe colitis and stenoses explored by colonoscopy. the anatomo-pathological examination of the specimen aims to compare the histological aspects of the colonic biopsies and of the surgical specimen. anatomo-pathological examination intestinal biopsies taken from patients by lower digestive endoscopy (colonoscopy) and an operating specimen were placed in 10% formalin for anatomo-pathological examination6. the samples were stained by ziehl neelsen and he fast. bacteriological examination of intestinal biopsies the biopsies were ground using a mortar in 0.5ml of bhib7. a drop of suspension was observed between slide and coverslip to visualize the possible presence of bacteria and their mobility. using the loop, a drop of suspension was placed on the slide. after fixation, the smear was stained with gram and read with the 100 immersion oil objective. stained smear was examined to observe the bacterial diversity in the biopsy suspension. the slides stained by the ziehl neelsen method from the biopsy suspension were observed under an optical microscope (bacterioscopy)8. culture a drop of the previously enriched medium was inoculated onto the hektoen and macconkey media using the streak method. another drop was placed in the bgb. incubation was done at 37°c for 24 h. each bacterium had a type of colony characterized by color, outline, smell, size and appearance. cloudiness present in the bgb tube indicated bacterial growth of streptococci. the suspected colonies were isolated using a pasteur pipette and transferred to a sterile tube filled with 5ml of physiological water, then inoculated on hektoen for 24 hours at 37°c. for bgb, a drop was taken using the loop and inoculated onto the blood agar medium to detect the type of haemolysis. identification macroscopic identification was performed according to the type of colonies one can suspect the bacterial species. for microscopic identification, a smear was prepared and stained according to gram technique for each colony. isolates were identified by macroscopic, microscopic and biochemical characters at the api 20 e. table 2. antibiotics to be tested for enterobacteriaceae and their mode of action. families mode of action antibiotic aminosides/ aminoglycosides inhibition of protein synthesis ak (amikacin) cn (gentamicin) ᵦ-lactam + ᵦ-lactamase inhibitor act on the synthesis of peptidoglycan (inhibition of bacterial wall synthesis) amc (amoxicillin + clavulanic acid) ax (amoxicillin) cephalosporin inhibition of bacterial wall synthesis ctx (cefotaxim) kz (cefazolin) cfm (cefixime) quinolones dna synthesis inhibitor cip (ciprofloxacin) na (nalidixic acid) sulfonamides and their associates act on the synthesis of folate, puric acid and nucleic acid (dna synthesis inhibitor) stx (trimethoprim + sulfamethoxazole) nitrofurans act directly on dna causing various lesions f300 (nitrofurantoin) crohn's disease: retrospective study in algerian patients vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 137 antibiogramme antibiotics to test for enterobacteriaceae are presented in the table29. the diameter of the zones of inhibition was measured, and classified the bacteria into different categories including susceptible, intermediate or resistant. coproculture the stools were collected from the emissions in a clean container. the samples were immediately stored at 4°c in order to avoid desiccation and the proliferation of commensal bacteria and yeasts. any coproculture must systematically implement the search for salmonella and shigella. in addition to a selective isolation medium (hektoen), an enrichment medium for salmonella is essential. the identification of pathogenic bacteria was done according to their macroscopic appearance on the agar, and biochemical identification. r e s u l t s histological examination of intestinal biopsies a. after hematoxylin eosin staining the figures of the histological sections of the intestinal biopsies are presented in fig.1, in which we observe: healthy mucosa with inflamed areas. -sub mucosa sometimes edematous. reorganization of the glands can be important. presence of inflammatory granuloma. presence of clear centered lymphoid follicles. -type of ulceration: cobblestone/pebble. figue 1. histological sections of intestinal biopsies. a: he stained biopsy section seen at low magnification (40). the fragment presents fissure ulcerations in the rotation, replacing the entire thickness of the mucosa by a polymorphic inflammatory granuloma (red arrow) arriving at the depth of the chorion with chronic infiltrate producing hyperplastic nodules presenting the lymphoid follicle with a clear center (blue arrow). b: he stained ileal biopsy section seen at low magnification (40). the layer of the glands are thick and of variable disposition and sometimes nibbled by inflammatory infiltrate in the acute phase (green arrow) in the center we note the presence of lymphoid follicles (blue arrow). c: he stained ileal biopsy section seen at low magnification (40). the fragment shows ulcerations of the epithelium with chronic inflammatory infiltrate; the glands are moderately inflammatory with a preserved appearance (green arrow) the polymorphic inflammatory granuloma and lymphoid follicle with a clear center are still present (blue arrow). d: at the magnification 100, the level of the follicle represents a richness in polymorphonuclear (gray arrow). crohn's disease: retrospective study in algerian patients vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 138 b. ziehl neelsen stain on histological sections all slides are negative for ziehl neelsen stain. colonic resection macroscopic study to colonic resection piece (colectomy) showed a colonic rigid segment of 12cm in length, opening large areas of ulceration in cobblestones (gridling by cracks of a swollen mucosa). the wall was thick with a firm consistency (stenosing), in addition to more or less deep ulcerations, the mesos were the seat of sclero-lipomatosis and the lymph nodes were enlarged. the mesentery was usually thick, retracted and fibrous (fig. 2). the resection specimen showed on microscopic study, a colonic mucosa with large trans-parietal ulcerations accompanied by a dense and diffuse, acute pyogenic and subacute polymorphic inflammatory granuloma rich in plasma cells and lymphocytes. noting pronounced vascular congestion, streaks and lymphoid follicles in the mucosa. absence of epitheloid follicles within the boundaries of the room; the serosa is the site of fibro-lipomatosis. this lesional aspect was focal, it contained areas of differentiated and edematous local mucosa ( fig. 3). figure 2. macroscopy of the colonic resection piece and the selected sections. a: photograph of a 12cm rigid surgical specimen representing sclero-lipomatosis on its macroscopic appearance. b and c: macroscopic sections of the colonic resection specimen obtained by dissection of the stenotic parts. figure 3. histological sections of the colonic resection piece. a: he stained section seen at low magnification (40). the extensive inflammatory granuloma represents large ulceration rich in polymorphonuclear. b:the section shows a zone rich in polymorphonuclear cells accompanied by pronounced vascular congestion (× 40) (yellow arrow). crohn's disease: retrospective study in algerian patients vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 139 c: he stained section seen at low magnification (40), depicts perivascular fibrosis (white arrow), inflammatory granuloma (light red arrow) and slightly inflamed muscle layer (black arrow). d: he-stained section seen at low magnification (40), shows a layer of fat on the serosa (dark red arrow) and an aspect of fibro-lipomatosis (light red arrow) with the presence of lipid droplets. figure 4. bacteriological examination before culture. a and b: smear of the biopsy suspension before culture stained by the gram method, seen under an optical microscope at 1000, shows a bacterial diversity of gram-negative bacilli (black arrow) and gram-positive (blue arrow) as well as gram cocci positive (red arrow). c: biopsy smear stained by hot ziehl neelsen method seen under light microscope at 1000. with different lighting, map is present inside macrophages appear as small curved bacillus stained red (black arrow). bacteriological examination of intestinal biopsies in the fresh state, scattered epithelial cells were observed with numerous red blood cells, leukocytes and macrophages. in some cases, germs (cocci and motile bacilli) were observed. the gram was verified, of which we observed gram+ and grambacilli, and gram+ cocci arranged in a chain. among the 15 biopsies taken, 2 cases were found to be positive for ziehl stain, afb present in the smear as small curved bacilli stained red. they were few in number (fig. 4). in all the cases studied, after 24 hours of incubation at 37°c, pure colonies of similar salmon color were observed on hektoen agar over the entire surface of the agar. blue-green colonies with a black center were isolated on hektoen agar, gram-bacilli had been observed, these bacilli could be salmonella spp. or proteus spp. in macconkey medium, lactose fermentation changed medium color to red. the strains were lac+ where the colonies were mucous in appearance, gelatinous in consistency, larger in size, with a tendency to confluence (klebsiella or e. coli). among the biopsies taken, 5 out of 15 cases gave a positive result in bgb (fig. 5). on blood agar medium, small, translucent colonies with total haemolysis were observed. these colonies were gram+ cocci arranged in a chain (streptococci) (fig. 6). crohn's disease: retrospective study in algerian patients vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 140 figure 5. results of the culture obtained after incubation at 37oc for 24h. a and b: culture of the biopsy suspension on hektoen agar; lac+ strains (salmon) and h2sstrains (green) were observed. other h2s+ strains (black center). c: culture obtained from macconkey medium. d: bacterial growth on bgb. figure 6. macroscopic and microscopic identification of isolated bacteria a: isolation of e. coli on hektoen medium, salmon colonies and mucous appearance. b: gram-stained smear observed under light microscope (100) represents gram-negative coccobacilli. c: isolation of h2s+ colonies (green with black center). d: smear represents gram-negative bacilli after gram staining, seen under an optical microscope (100). e: isolated colonies on macconkey medium. f: smear represents gram-negative bacilli after gram staining seen under an optical microscope (100). g: colony isolated on blood agar medium. h: smear showed gram positive chain sections after gram staining, viewed under light microscope (100). crohn's disease: retrospective study in algerian patients vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 141 figure 7. results of identification by the api20e gallery. a: api20 identification gallery after 24h/37°c incubation, followed by the code sheet which presented the biochemical characters of salmonella spp, b: api20 identification gallery, reader after incubation at 37°c /24h showed the biochemical characteristics of e. coli. figure 8. antibiogram of e. coli after incubated at 37°c for 24 hours on mh agar. a: presents 6 discs of antibiotics of amc; ctx; cfm; ax; ak and kz b: presents 5 discs of antibiotics of stx; nc; cip; f300; n / a. the results obtained by the api20 galleries showed in fig. 7; after reading the results by the apiwebtm software; we had identified the two species. antibiogram e. coli showed sensitivity for third generation cephalosporins (ctx; cfm), aminoglycosides (ak; cn) and also for quinolones (cip; na); and resistance for first generation cephalosporins (kz), b-lactams (amc; ax) signified the presence of b-lactamases, for sulfonamides (stx) and intermediate for nitrofurans (f300) (fig. 8) coproculture in stool analysis results were found similar to that of intestinal biopsies except that the ziehl neelsen stain was negative. we identified the same bacterial species determined by culture of intestinal biopsies, these species were: escherichia coli, salmonella spp., proteus spp. and klebsiella spp. the count of isolated bacteria showed a decrease in stool (salmonella spp., proteus spp. and klebsiella spp.). on the other hand, e. coli was uncountable that indicated e. coli dominancy in cd. d i s c u s s i o n crohn’s disease is inflammatory bowel disease affecting the digestive tract. this ibd was examined in association with clinical symptoms followed by serological exploration which showed the signs of inflammation (high esr and crp). we also noted the radiological exploration which was very important, during the endoscopy the doctor took intestinal biopsies for the purpose of the anatomopathological analysis which helped in the orientation of the diagnosis. crohn's disease: retrospective study in algerian patients vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 142 in our study, we analyzed endoscopic biopsies to understand cd pathophysiology association with the intestinal microbiota, followed by histopathology of the disease and bacteriological analysis. crohn’s disease is recognized by macroscopic bowel which reveals thickened and stiff mesentery with wat in retrospective study of bowel resections10; mesenteric fat is defined as an extension of the wat that covers a small and large intestinal circumference11, the mesentery is generally retracted and fibrous and the intestine, with its surrounding, is thickened and enlarged with fatty lobules, and the mesenteric lymph nodes are inflamed12. all of these characters were presented in our study piece it is the appearance of sclero-lipomatosis, packing fat has been correlated in change of structures11. in the present work, we were able to compare between the results of the histological aspects of the intestinal biopsies and those of the resection piece, and by coming to the conclusion that the mucous biopsies showed only one tunic of intestine unlike the operating piece which represented the different coats of mucous membrane down to the serosa; therefore the mucosal biopsies obtained by endoscopy had not shown all the characteristics of cd that made possible to evaluate the disease (activity and identification of precancerous lesions), but not an exact diagnosis of the disease. the results must be associated with radiological, serological and clinical exploration 12,3; because cd lesions is recognized "recognition of dysplasia". when surgical specimens are available, the disease can be much more easily identified 11. histological criteria are known in cd, including a diffuse transmucosal lymphoplasmacytic infiltrate associated with large and fissured ulcerations 3,12. cryptitis and crypt abscesses 13, lymphangiectasias 14 and perivascular inflammation 3 due to abnormalities in mwat of cd patients which also included marked macrophages in the infiltrate and fibrosis also thickening of the vessels 11, visceral adipocytes are smaller in adipocytes through mesentery cd patients not in group control 15. the hypothesis for the association of fat packaging and cd is that local production of mediators by mwat could promote intestinal mucosa damage 11; the relationship between mesenteric fat and cd needs to be investigated. although cd is believe has an infectious cause autoimmune in origin16. many works have been conducted to identifying the microbial diversity of healthy individuals and cd patients, in which a decline in bacterial diversity is found17. cd occurs in subjects genetically predisposed to inappropriate intestinal mucosal immune system activation and its stimulus is the intestinal bacterial content "microbiota"18. the intestinal microbiota contains approximately 1014 bacteria of various species, gram staining used on fresh biopsy smears showed a great bacterial diversity. a growing interest centered on the study of bacterial communities as an antigenic source fueling chronic inflammation in cd 18. the intestinal microbiota was identified by culture. 70% of this microbiota was not cultivable, it would remain unexplored 19. the study of several bacteria associated with intestinal biopsies taken from cd patients shows that cd is a bacteriologically distinct disease 18. the gut microbiota membrane favors sources of fa with which the immune system is dysfunctional 20. fecal microbial populations differ significantly from mucosal associated microbiota (mam), and each individual harbors a unique flora pattern 21,22. the analysis of the faecal microbiota remains informative, most often colonic and ileal biopsies are necessary for identifying mam 18. biopsies used from healthy and pathological mucosa did not show any differences. in this work, a difference in bacterial composition was not due to the condition of the inflamed tissue, as bacterial collections associated with biopsies did not differ in composition. according to vasquez et al. 23, there was no difference in injured and healthy areas of mam. the gut microbiota could carry pathogenicity in two ways: through pro-inflammatory or through anti-inflammatory; two complementary strategies were followed: the search for a candidate pathogenic microorganism and the analysis of the intestinal microbiota "search for dysbiosis" 18. many researchers have demonstrated that bacteria concentration in mucosa is higher in cd patients, in distal ileum and colon, precisely the concentrations of enterobacteriaceae 24,25. one study showed that when the disease is active, the number of coliforms increased in faeces of cd patients and lowered in subjects recovering26, and an increase in faecal enterobacteriaceae in the mam crohn's disease: retrospective study in algerian patients vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 143 24,25 such as e. coli25. identification of ileal mam of cd patients has indicated that e. coli was widespread, representing approximately half of total aerobic anaerobic bacteria27, escherichia coli charge has increased in mamin patients compared to symptomatic subjects controls 28. the strains were able to colonize the intestinal mucosa by adhesion to intestinal epithelial cells26. immunocytochemistry demonstrated that escherichia coli ag was found in resections of most intestinal cd patients26. high levels of anti-omp escherichia coli of cd patients appeared in bowel surgery such as stenosing and perforating18. certain e. coli strains containing particular adhesion have been found in ileal mucosal samples from cd patients 27. this strain of e. coli isolate did not contain the virulence factors of other aiec pathogenic e. coli (reference strain lf82), they are generally strongly infected immune cells secreting high levels of tnf-α 26. aiec (lf82) adhere to enterocytes of cd patients 29 and colonize the ileal mucosa, and phagocytosed and multiply in lamina propria, induce strong secretion pro-inflammatory cytokines tnf-α 18. the greater aiec richness was due to the stability of bacteria in cd patients mucosa 30,31,32. aiec was capable of inducing the formation of granuloma in vitro26,29. researchers suggested that optional pathogens cause disease in susceptible individuals 33. in addition to the high concentration, we noted the presence of unusual bacteria 34,35 such as those found in our study, including salmonella, shigella, streptococcus, proteusmirabilis, klebsiella, mycobacterium avium paratuberculosis. autophagy which is a process of degradation the damaged organelles by lysosomal eukaryotic 36. it limited growth intracellular bacteria 37,38. it’s dysfunction conducted to infection like s.typhimirium, s. pyogenes also mycobacteria39,38,40, lesions seen in this disease are mostly present at the peyer's patches. for which many bacteria have a particular tropism such as salmonella, shigella, vibrio, campylobacter, yersina, lesteria, klebsiella41. aphthous ulcerations resulting from m cell necrosis, which are lesions in cd patients. these ulcerations occur in many infections by shigella, salmonella also yersina, invasion is an element of virulence26. most possible infectious cause is mycobacterium avium paratuberculosis16, it may be there is association between johne's and crohn’s diseases42. map is increased in adults crohn’s disease43 and newly diagnosed children 44, the mycobacterial theory and the selfimmune system in cd are complementary, the first concerns the etiology of the disease and the second concerns its pathogenesis5. map has been studied in tissue of many cd patients 45. unlike other environmental mycobacteria, map can causing chronic inflammation of the intestine 45. maps are difficult to diagnose by ziehlneelsen staining, sometimes culture or dna or rna detection of maps is doing in laboratory16. mucosal biopsies were obtained from cd patients by endoscopy used for smear and examined by zn. smears of all patients were negative by zn, after 14 weeks cultures mgit shown in zn of the characteristic cells and afb 43, if the shape of cell wall is deficient in map, the zn will be negative. in prolonged period of culture (weeks to years) map can produce a protoplasmic 46,16, and zn staining will be positive 16 . important role, moreover the lifestyle and the diet of the people affected little appear modifications in the cell wall of the map. while geographical and environmental conditions can give resistance to this strain, it is therefore proposed that strains of map can differentiate according to environmental conditions. the use of zn staining on histological sections gave negative results, while their use on biopsy smears in the same patients appeared positive in two cases. it is concluded that this method is more effective on smears than on histological sections. in animals, tissues stained with zn for the direct visualization of mycobacteria in the study by wells et al. 47 who identified afb in 9% and 5.6% of cases respectively in the ileal mucosa and the lymph nodes. it is a method that requires a lot of specific manipulations and expertise in anatomo-pathology. for the study by weber et al.48 which was based on the zn technique on a matrix smear on a "smear" glass slide, a sensitivity of 48% and a specificity of 98% for animals in the clinical phase was established. nod2 an intracellular sensor of bacterial peptidoglycan 18, in mutations nod2/card15 gene of cd patientsinnate immune response a disease by invasive bacteria 49, nod2 intervenes in clearance of salmonella spp., streptococci 50,51 also enterococcus52. monocytes and cells epithelialexpress nod253, intracellular receptor for muramyl dipeptide53. nod2 is also expressed in cells paneth 54 synthesized and secreted several antimicrobial crohn's disease: retrospective study in algerian patients vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 144 peptides, including lysosomes, phospholipase and defensin. these peptides are sensitive to oral administration of pathogenic bacteria, likewise provide protection against salmonella55. studies showed biological effects of the peptides on pathogenic lumen microbiota gut 56, defensin is essential element cd pathophysiology57. defensins, in deficiency in innate immune defense, inducing adherence of bacteria in mucosa conducted to inflammatory response in cd58. c o n c l u s i o n in our study, the common histopathological appearance of cd is defined by the presence of inflammatory granuloma with clear-centered lymphoid follicles (rich in lymphoplasmocytes). the use of ziehl neelsen staining, for detection of map, is effective on smears as well as on histological sections, it must be followed by culture with meta-analysis and pcr for consistent results. the results obtained on the intestinal microbiota in patients with cd are different, on one side of increased concentrations of certain bacteria such as e. coli and proteus mirabilis; and on the other hand the appearance of certain unusual pathogenic bacteria such as salmonella, shigella, klebsiella and streptococcus pyogenes. the results are due to the deficit in immune regulation caused by the nod2/card15 gene mutation. over the last few years, ibd has become very frequent in algeria. our perspective is to provide research groups for the study of the pathophysiology and especially the etiology of the disease which is different according to the geographical areas. we also want to conduct more research on maps which we assume are more resistant and different from the reference strains and to introduce new animal and human vaccines against map. e t h i c a l a p r o v a l ethical approval for the study was obtained from khemis miliana university, algeria. c o n f l i c t s o f i n t e r s t no conflict of interest. f u n d i n g s o u r c e no funding source a c k n o w l e d g e m e n t s we would like to appreciate the health officials below for their welcome and their precious help at the level of their establishments: the head of the gastroenterology department of uhc mustapha bacha pr. berkane saadi, pr. bounab nassima and pr. kissili (endoscopy unit). the head of the internal medicine department, uhc kouba endoscopy unit, mr. athmane omar, mr. slinani farid and dr. fissah. the head of the internal medicine department uhc beni messouse pr. boumedienne and the head of the endoscopy unit dr. benkanoune. the head of service of anapath uhc medea mr. bailiche mohamed and head of medicine dr. hamadou and also dr. sounna. the director of the medical analysis laboratory ain defla dr. zibouche and all the team of his work. l i s t o f a b b r e v i a t i o n s cd: crohn’s disease bgb: buffered glucose broth. map: mycobacterium avium paratuberculosis ibd: inflammatory bowel disease nod2: nucleotide-binding oligomerization domain 2. card/15: caspase recruitment domain-containing protein 15. pcr: polymerase chain reaction zn: ziehl neelsen he: hematoxylin eosin bhib: brain heart infusion broth. dna: deoxyribonucleic acid mgit: mycobacteria growth indicator tube rna: ribonucleic acid afb: acid fast bacillus lac: lactose aiec: adherent invasive escherichia coli tnf-α: tumor necrosis factors-alpha. omp: outer membrane protein ag: antigen crohn's disease: retrospective study in algerian patients vol. 13 (2), december 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 145 mam: mucosal associated microbiota wat: white adipose tissue api20 e: application programming interface 20test enterobacteriaceae. uhc: university hospitalier center crp: c-reactive protein. e. coli: escherichia coli. esr: erythrocyte sedimentation rate. s.typhimirium: salmonella typhimirium s.pyogenes: streptococcus pyogenes r e f e r e n c e s 1. nahon s. prise en charge de la maladie de crohn iléale localisée: chirurgie ou traitement médical? post’u.2022; 37-44. 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crohn’s disease: a defensin deficiency syndrome? eur. j. gastroenterol. hepatol. 2003; 15: 627-634. 58. wehkamp j, harder j, weichenthal m, schwab m, schaffeler e, schlee m, et al. nod2 (card15) mutation in crohn’s disease are associated with diminished mucosal αdefensin expression. national library of medicine, gut. 2004; 53: 1658-1664. global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 83 op e n ac c e s s f u l l l e n g t h a r t i c l e global overview of sars-cov-2 induced covid-19 in 2020: biological characterization, epidemiology with social, economic and environmental implications ayesha batool1, ayesha kashif2, muhammad haq nawaz3, ashfaq ahmad khan4, nafees iqbal5, muhammad kashif shahid6,* 1department of chemistry, quaid-e-azam university, islamabad, pakistan. 2department of senior health care, eulji university, daejeon, republic of korea. 3department of physics, university of gujrat, hafiz hayat campus, gujrat, pakistan. 4department of chemistry, government post graduate college, haripur, kpk, pakistan. 5h.e.j. research institute of chemistry, international center for chemical and biological sciences, university of karachi, karachi, pakistan. 6research institute of environment & biosystems, chungnam national university, daejeon, republic of korea. a b s t r a c t background: covid-19 is a global pandemic initiated in january 2020 that caused 79 million cases and more than 1.7 million deaths worldwide. the causative agent of covid-19 is severe acute respiratory syndrome coronavirus-2, a member of betacoronvirus. covid-19 patients are classified into asymptomatic, mild symptomatic, and severe symptomatic cases. objectives: to review the prevalence, therapeutic interventions for the treatment, vaccination, and containment of covid-19 in four quarters of 2020, emphasizing the advancements in biological studies, and the social, economic, and environmental impact of the pandemic. methodology: data of covid-19 spread, identification, prevention, and control measures was analyzed. the impacts of pandemic on society, economy, and the environment were assessed. results: owing to distinct genome of covid-19, de novo diagnostic tests have been designed, optimized, and carried out in individuals. the specimen for viral detection can be selected from sputum, nasal, and pharyngeal swabs, anal swabs, blood, bronchoalveolar lavage fluid (blf), and secretions of lower respiratory tract. primary treatment includes antiviral therapeutic agents, whereas, supplementary treatment includes corticosteroid therapy, antibiotic treatment, and oxygen therapy with the help of noninvasive and invasive mechanical ventilation. the lack of targeted therapeutics failed to induce a 100% mortality rate as recovered patients’ immune system produces cd4+ and cd8+ t cell responses and antibodies against the spike protein of the virus. in order to contain the virus spread and build herd immunity in the masses, protein subunit vaccines, rna-based vaccines, and vlps were developed. conclusion: the social, economic, and environmental impact of covid-19 has threatened the global community. the novel prevention and control measures offered significant benefits however, an effective treatment will possibly always be required even with the end of pandemic. keywords covid-19, diagnosis, mutants, pathology, sars-cov-2, vaccination. *address of correspondence mkbutt2000@gmail.com article info. received: march 09, 2021 accepted: february 04, 2022 cite this article batool a, kashif a, nawaz mh, khan aa, iqbal n, shahid mk. global overview of sars-cov-2 induced covid-19 in 2020: biological characterization, epidemiology with social, economic and environmental implications. 2022; 13(1):83-122. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. r e v i e w a r t i c l e global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 84 i n t r o d u c t i o n coronavirus disease-2019 is a respiratory infection caused by sars-cov-2. the first case was reported in december 2019 in wuhan. its emergence was associated with the huanan seafood market, the largest wholesale market for live animals and seafood in the jiangshan district of wuhan, china1. the first epicenter of the covid19 pandemic was also identified in wuhan, hubei province, china2. in a period of one month, the respiratory disease and pneumonia of unknown origin and vague diagnostic symptoms significantly spread to other parts of china and rest of the world, and the world health organization (who) declared the “global public health emergency” on january 30, 20203,4. the widespread of the pandemic has been fluctuated across the globe, from country to country, and within regions of the same country5. on february 11, 2020, the guidelines of the international committee for the classification of viruses, scientifically named the virus “severe acute respiratory syndrome-related coronavirus2”, abbreviated as sars-cov-26. on december 29, 2020, the who reported 79,231,893 cumulative cases of covid-19, 1,754,574 cumulative deaths, and a mortality rate of 2.2%7. the possible identification methods as well as timeline of infection spread has been generated in fig. 1. figure 1. an overview of covid-19. global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 85 covid-19 cases are classified as asymptomatic, mild symptomatic, and severe symptomatic cases. the signs and symptoms of sars-cov-2 are established after 7-14 days of infection, including fever, anorexia, dysplasia, muscular pain, and fatigue8,9. the progression of infection severely damages the lower respiratory tract (trachea, bronchoalveolar surfaces, and lungs). the disease is transmitted through both direct and indirect contact with infected individuals. covid-19 can propagate through sporadic spread, clustered transmission, and community transmission. in order to contain the virus, public safety policies were implemented throughout the world to contain the horizon of sars-cov-2 virus; however, most of them compromised mental health and caused social distress among the masses. the lockdown of industries and transnational borders caused an economic downfall in advanced, developing, and underdeveloped nations. the social, economic, and environmental aspects of the covid-19 pandemic have threatened the survival of the global community. this review summarizes the source, spread, treatment, and development of vaccines in four quarters of 2020, with special attention given to the advancements in biological studies to combat the viral threat and the social, economic, and environmental impact of the covid-19 pandemic. b i o l o g i c a l c h a r a c t e r i z a t i o n o f n o v e l c o r o n a v i r u s 2 0 1 9 taxonomical classification the phylogenetic evidences from genomic studies of the initially unidentified agent revealed sequence homology to the rdrp region of betacoronaviruses, particularly sarscov. hence, it was named as “2019 novel-coronavirus” (2019-ncov) by zhu et al.10 and later termed as sarscov-2 by the coronaviridae study group (csg) of the international committee on taxonomy of viruses (table 1)6. taxonomically, sars-cov-2 has been assigned to the genus betacoronaviruses of the family coronaviridae11. the coronaviridae family consists of three additional genera, alphacoronaviruses, gammacoronaviruses, and deltacoronaviruses, which are responsible for viral ailments in birds, animals and humans12,13. alphacoronaviruses and betacoronaviruses primarily infect mammals, whereas gammacoronaviruses and deltacoronaviruses infect birds14. the two distinct members of alphacoronaviruses (hcovnl63 and hcov-229e) and four distinct members of betacoronaviruses (hcov-oc43, hcov-hku1, sarscov, and mers-cov) have been responsible for coronaviruse-induced pathologies in the human population15. hcov-nl63, hcov-229e, hcov-oc43, and hcov-hku1 account for 15-30% cases of seasonal common cold or mild respiratory infections16 . however, sars-cov causes life-threatening lower respiratory infection and is responsible for severe acute respiratory syndrome epidemic during 2002-2003 with 8,096 reported viral infections and a 9.6% mortality rate. the middle eastern respiratory syndrome (mers) outbreak 2012 in saudi arabia infected 2,494 humans in 27 countries and reported a mortality rate of 34%, corresponding to 858 deaths17. sars-cov-2 marked the first “pandemic” of the 21st century. table 1. taxonomical classification of 2019-novel coronavirus. scientific serial classification order nidoviralis family coronoviridae subfamily orthocoronovirinae genus betacoronavirus sub genus sarbecovirus species severe acute respiratory syndrome coronavirus-2 genomic and proteomic characterization of novel coronavirus-2019 the whole genome sequencing, multiple sequence alignment, and phylogenetic analysis of sars-cov-2 exhibited striking sequence homology to bat severe acute respiratory syndrome-related coronaviruses and human coronaviruses6. the genetic analysis of sars-cov-2 has indicated 75% sequence homology of viral nucleotides to sars-cov18,19. the whole genome sequencing of sarscov-2 has also revealed 87.9% and 87.3% sequence homology with sars-like bat coronaviruses, sarscovzc45 and sars-covzxc21, respectively20. moreover, sars-cov-2 has also been more closely related to the bat cov ratg13 extracted from rhinolophus global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 86 affinis. these similarity indices strongly suggest the zoonotic transfer of sars-cov-2 and bats as primary hosts of the 2019-novel coronavirus21,22. another candidate for the zoonotic transfer of sars-cov-2 is suspected to be pangolins, as phylogenetic studies conducted by lam et al. highlighted 99.9% sequence homology of sars-cov-2 with the coronavirus gx/p2v present in manis javanica23. however, sars-cov-2 has shown the least homology to mers, with 50% sequence resemblance24. genomic studies of sars-cov-2 indicate that it is an enveloped rna virus, characterized by the presence of positive-sense single stranded rna molecule10. instead of requiring rna transcriptase enzyme, positive-sense rna viruses are capable of using genomic rna as mrna for direct protein synthesis. the coronavirus genomic rna is structurally similar to eukaryotic mrna with a 5' cap structure and 3' poly-a tail, which are essential for durability and the translation of rna molecules in the cytosolic component of eukaryotic host cells25. during the initial phase of the epidemic, jiang et al. successfully provided deep insight into the genomic characterization of sars-cov-2. the sars-cov-2 genome initiates at the 5' utr region, followed by the coding region for a set of 15 non-structural proteins, stretching two-thirds in length, and the remaining one-third region being translated into four structural and eight accessory proteins. the rna genome of novel coronavirus is translated through fourteen open reading frames (orf) and produces polyproteins. the two polyproteins, pp1ab and pp1a are generated via translation of single orf at 5' terminus, and collectively generated 15 non-structural proteins. the downstream region contains genes for s, 3a, 3b, e, m, p6, 7a, 7b, 8b, n, 9b, and orf14 proteins, which are synthesized under different orfs26. sars-cov-2 lack the hemagglutininesterase (he) gene, that has been reported in other members of betacoronavirus16. moreover, mutations and genetic variations at different sites within the genome are responsible for the emergence of various strains of sarscov-2. phan identified 93 mutations during nucleotide sequence analysis of 86 strains of sars-cov-2, from china, australia, america, south korea, singapore, belgium, and england27. wang et al. also reported distinct variations at 13 sites in orf1a, orf1b, 3a, m, 8 and n genes of different strains of sars-cov-2. in their study, the mutation rate of orf8 was the highest (30.5%), followed by the mutation rate of orf1a (29.5%)28. the four structural proteins of sars-cov-2 include spike surface glycoprotein (s), small envelope protein (e), membrane protein (m), and nucleocapsid protein (n) (fig. 2)29. the s glycoprotein contains 1,273 amino acid residues and is made up of two subunits, s1 and s2. the subunit s1 exhibited a structural resemblance to the s1 unit of sars-cov. it consists of one signal peptide (sp), one n-terminal domain (ntd), and three c-terminus domains (ctd1, ctd2, and ctd3). the subunit s2 consists of fusion peptide (fp), heptapeptide repeat sequence-1 (hr1), heptapeptide repeat sequence-2 (hr2), transmembrane domain (tm) and c-terminal domain30. the s-protein is of prime importance since s1 plays a crucial role in interacting with human cell receptor, and s2 plays an important role in membrane fusion during infection2. the receptor binding domain (rbd) is located in ctd1 region of sars-cov-2 and exhibits 75% amino acid sequence homology to sars-cov. this striking resemblance have emphasized the involvement of same host cell receptor during virus-host interaction i.e., angiotensin converting enzyme-2 (ace-ii) and consequently similar clinical manifestations as of sarscov18. the binding sites in the rbd of both sars-cov and sars-cov-2 with 83.3% sequence similarity conserved 13 hydrophobic amino acid residues involved in protein-protein interaction31. moreover, cryo-em of s-protein indicated that rbd of sars-cov-2 has 10 and 20 folds higher affinity to ace-ii receptor of human cells. sars-cov-2 binds ace-ii with higher affinity as the binding free energy of the sars-cov-2 rbd-ace2 interaction is -50.43kcal/mol, which is significantly lower than the binding free energy of the sars-cov rbd-ace2 interaction (-36.75kcal/mol)32. moreover, zhou et al. also confirmed the binding affinity of s-protein with ace-ii and non-binding affinity towards dipeptidyl peptidase-iv (receptor of mers-cov) and aminopeptidase-n (receptor of other coronaviruses)33. the accessory proteins of sars-cov-2 also possessed structural variation as compared with sars-cov. these include elongated 8b (121 amino acids) and shrunken 3b (22 amino acids) in comparison to sars-cov 8b (84 amino acids) and 3b (154 amino acids), whereas the accessory protein 8a was not global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 87 found in sars-cov-226. this study provides ground for further investigation of the significance of these variations in viral pathogenesis. p a t h o l o g y o f s a r s c o v 2 the pathogenesis of coronaviruses involves the interaction of viral surface proteins with the host cell receptor, fusion of viral membrane and host cell membrane, release of viral genome into the intracellular environment of host cell, translation of the viral genome into polypeptides, increase in the copy number of viral genome, assemblage of viral particles in compartmentalized organelles, and release into the extracellular matrix by exocytosis34. the presence of sprotein not only imparts unique morphological feature, but also plays a significant role in virus-cell fusion35. sarscov-2 interacts with ace-ii present on the surface of cells lining the nasal cavity, epithelial cells of the airways, and alveolar type ii cells36,37. after binding of rbd to ace-ii, the s-protein is cleaved at polybasic furin cleavage sites (prrar) by host cell’s type ii transmembrane serine protease (tmprss2) into s1 and s2 subunits38,39. figure 2. types of proteins translated from sars-cov-2 positive sense rna genome. global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 88 the multiple cleavage sites give distinctive characteristics to sars-cov-2 because these are associated with enhanced infectivity and the highly contagious nature of the virus40. the exposure of fp in s2 causes conformational changes in the s2 unit41. this is followed by folding back of hr1 and hr2 to form a six-helix structure responsible for bringing the viral membrane and host cell membrane close enough to fuse34. like sars, mers, and other coronaviruses, the compartmentalization of sarscov-2 occurs via endocytosis 42. in the double-membrane vesicles, the viral proteins are dissolved and the viral rna is released in the cytoplasm43. the viral genomic rna (grna) is directly translated into pp1a and pp1ab polypeptide. the coronavirus main protease (mpro) cleave the pp1a and pp1ab polypeptide, which results in the release of multisubunit rnadependent-rna-polymerase (nsp12) and other nonstructural proteins (nsp1-nsp16). nsp1/nsp3, nsp2, nsp15, and nsp16 are actively involved in evading the immune response. the coordination of nsp12, nsp13, and nsp14 forms a replicase-transcriptase-complex (rtc), which proceeds rna synthesis, rna proofreading, and rna modification. the rtc is responsible for the transcription of grna in three ways. the rtc transcribed grna into a nested set of sub genomic rna (sgrna), positive-sense single-stranded rna (+ssrna), and negative-sense single stranded rna (-ssrna)16,34. nested sgrnas are translated into the structural and accessory proteins, negative-sense rna strands act as templates to synthesize more complementary positive-sense rna strands, and positive-sense rna strands interact with nprotein and form nucleocapsid in host cytoplasm44. the accessory protein nsp8 works as a primase and generate a 7-8 nucleotide short sequence complementary to 3’ of grna so that new rna strands can be synthesized. the nsp7nsp8 complex increases the binding of rdrp (nsp12) to rna and enhances the enzymatic activity of rdrp45. the structural proteins s, m, and e are involved in the formation of the viral coat16. these viral proteins are insulated in the endoplasmic reticulum and are released as endoplasmic reticulum-golgi-intermediate compartment (ergic)44. the nucleocapsid is infused with ergic, and an assemblage of viral particles take place. during assembly, the nucleocapsid is further stabilized by interactions with the m protein. the m protein also interacts with the s protein and is responsible for its incorporation into the new virion16. in coronaviruses, e protein is the outermost protein layer and is involved in several aspects of the virus life cycle and pathogenesis46. once virions are assembled, they are released into the extracellular matrix through exocytosis47. in covs, the s-protein has also been reported to mediate the fusion of membranes of adjacent normal cells to the infected cell, form multinucleated giant cells, and mechanistically facilitate the rapid spread43. d i a g n o s t i c t e s t s f o r c o v i d 1 9 specimens for sars-cov-2 detection the detection of sars-cov-2 in humans is correlated with the type of sample, maintenance temperature, isolation techniques, and handling during testing. the specimen for viral detection can be blood, sputum, nasal swabs, pharyngeal swabs, anal swabs, bronchoalveolar lavage fluid (blf), and secretions of the lower respiratory tract48. nasal swabs and throat swabs have been extensively used for diagnostics owing to simplicity of sampling technique, conventional storage and transfer feasibility of specimens49. collection of blf had been highly recommended in severe and critical cases as blf has a rich source of viral particles in comparison to nasal and pharyngeal compartments. moreover, detection rate of sars-cov-2 in blf has the highest success rate (100%)50. the choice of the specimen also correlates to the number of days post-onset of the disease. the maximum viral load in throat swabs can be detected within 4-7 days; in sputum specimens within 7-10 days, and in stool samples within 0-11 days. precautionary measures and safety are highly emphasized and strictly observed while collecting and processing the samples51. detection of sars-cov-2 in laboratory and clinical settings during the initial days of the covid-19 outbreak, individuals were suspected as per who guidelines based on their exposure to covid-19 infected areas, and symptoms of fever and cough52. for detection of sarscov-2 in the laboratory, nucleic acid amplification test (naat) is conducted either through reverse transcriptase polymerase chain reaction (rt-pcr) or high throughput sequencing (hts). in case of rt-pcr primers of highly global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 89 conserved regions, rdrp gene, s gene, e gene, and n gene were designed, and protocols were optimized. the highest analytical sensitivity for real-time amplification has been observed in the rdrp assay53. hts is extensively used to test the presence of the viral genome of sarscov-2 in test samples. however, large-scale htsbased testing has not been adapted globally as it is not costeffective49. in clinical settings, primary confirmation is obtained by a computed topographyscan (ct-scan) of the chest, reporting morphological abnormalities of the lungs54. infected patients are reported to suffer from damaged lungs, indicating ground-glass opacity, bilateral patchy shadows, and segmentations of lungs55,56. clinical evidences suggested that it takes almost 10 days for severe lung abnormalities to get noticed after the initial onset of disease50. molecular techniques for detection of sars-cov-2 once the extraction procedure is completed, the presence of the sars-cov-2 genome is detected in the test samples. for this purpose, the following molecular techniques based on nucleic acid detection have been customized. reverse transcriptase polymerase chain reaction (rt-pcr) sars-cov-2 is a single-stranded rna virus. during rtpcr, ssrna is firstly converted into a complementary dna (cdna) with the help of reverse transcriptase. reverse transcriptase uses rna template to synthesize a hybrid dna molecule by extending the 3´ end of the annealed primer. once cdna molecules are synthesized, denaturation takes place and single stranded dna (ssdna) molecules are generated, primers are annealed to the template, and elongation takes place by the addition of complementary a, t, c, and g nucleotides to a specific region. amplification is monitored with the help of a fluorescent dye or sequence-specific dna probe. sequence-specific dna probe is labeled with a fluorescent molecule and a quencher molecule57. primers are designed complementary to highly conserved region of the genome and are usually 17-22 nucleotides long. amplification of targeted region is carried out in one step or two steps. during one-step rt-pcr, the amplification process is either carried out in the same vial in which cdna is synthesized. in two-step rt-pcr, the cdna template is transferred to a new vial, and the pcr master mix is added to increase the copy number of cdna. single-step rt-pcr is time-efficient, whereas, two-step rt-pcr is comparatively sensitive58. in the case of rt-pcr for sars-cov-2, the genomic regions of orf1b, orf8, n, s, rdrp, and e genes were preferred. based on the urgency of test results, single-step rt-pcr has been extensively used for covid-19 testing. rt-pcr based commercialized diagnostic kits included covid-19 rtpcr (labcorp), 2019-novel coronavirus real-time rtpcr diagnostic panel, taqpath covid-19 combo kit (thermofisher applied biosystems), allplex 2019-ncov assay (seegene), and cobas sars-cov-2 (roche). simplexatm covid-19 direct assay does not require extraction of viral rna from dna/rna sample solution and selectively amplifies only the targeted regions of sarscov-258. rt-pcr diagnostic method is limited as it is sensitive to handle, and time-consuming. the protocol requires rt-pcr thermocycler for temperature variations and is dependent on the sample processing capacity of the thermocycler57. isothermal nucleic acid amplification isothermal nucleic acid amplification is a recently developed technique that does not require a variation in temperature as required for rt-pcr. it is the working principle of reverse transcription loop-mediated isothermal amplification (rt-lamp) and transcriptionmediated amplification (tma). sars-cov-2 detection kit developed by id now covid-19 (abbott diagnostics) is based on rt-lamp. the technique takes only 13 minutes per sample to detect viral genome, however, the apparatus is limited to a single tube run57. hologic’s platform has successfully developed high throughput transcriptionmediated amplification, which is capable of processing 1,000 samples per day. at the molecular level, hologic panther fusion platform captures the target sequences, amplify specific regions, and detects rna amplicons transcribed from single-stranded cdna. the viral rna fragments are hybridized to oligonucleotides probes and t7 promoter primer7. the oligonucleotides contain a complementary sequence of magnetic micro particles, which are separated from an ocean of fragments by applying magnetic field. the captured rna molecule is reversed transcribed for cdna and the rna strand is degraded. the ssdna molecule is replicated and global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 90 denatured to produce templates for rna amplicons. rna amplicons are detected with the help of single-stranded torch conjugated to fluorophore and quencher and emit photons upon hybridizing to the rna amplicon and the signal is read by detectors36. the hologic panther sarscov-2 has shown greater sensitivity to minute amounts of viral genome present in solution. gorzalski et al., have retested 116 samples of sars-cov-2 through tma and found 98.2% detection accuracy as compared to 96.2% for rt-pcr. tma has given zero false-positive results with 100% specificity59. the platform has been highly recommended by food and drug regulatory authority for testing of large sample size under emergency situation. crispr assays clustered regularly interspaced short palindromic repeats (crispr) are the nucleic acid sequences present in prokaryotic organisms and are the recognition sites of crispr-associated set of enzymes (cas9, cas12, and cas13). gootenberg et al. have previously used cas13a and combined allele-specific sensing ability of crispr and recombinase polymerase amplification to detect rna fragments of zika virus and other pathogens60. cas12 has also been modified to recognize and cut viral rna of african swine fever virus61. crispr-based assays have also been established to qualitatively detect sars-cov-2. for this purpose, sherlock biosciences had strategized crispr detection technique capable of detecting 10-100 copies per microliter of the sample. synthetic rna fragments of covid-19 were used to perform the test-run. s gene and orf1ab gene were selected as targeted regions. isothermal rna amplification was carried out and amplified fragments were detected through lwacas13a crispr guided rnas. the qualitative analysis was confirmed by dipstick method and no additional instrument for detection was needed. the breakthrough of crisprbased assays had significantly shortened the detection time to 40-60 minutes as compared to 4-6 hours of rtpcr for detection of sars-cov-2 genome62. the detectr (sars-cov-2 dna endonuclease-targeted crispr trans-reporter) have designed rt-lamp for amplification of extracted rna from nasopharyngeal and oropharyngeal swabs. the procedure is followed by detection of predefined targeted regions of e gene and n gene by cas12. the cas12 fluorescent signal is detectable in <1 min and a visual signal is achieved within 5 mins. the protocol is highly efficient as no cross-reactivity of the n gene of sars-cov-2 with other infectious coronaviruses (oc43, hku1, 229e, and nl63) is noticed. the cas12a based crispr assay needs no special instrument for read-out and takes only 45 minutes. thus, crispr-based assays provide a breakthrough for larger capacity of covid-19 testing in a limited time duration. c l i n i c a l f e a t u r e s o f s a r s c o v 2 i n f e c t e d i n d i v i d u a l s incubation period and critical days the studies conducted by xu et al., in china have reported incubation period of 3-5 days63. guan et al., studied 1,099 patients in china and have reported median incubation period of 4 days. an exceptional incubation period of 24 days had also been observed, which highlighted the importance of quarantine and social distancing to avoid human to human transmission56. pooled analysis of confirmed covid-19 patients from 4 january, 2020 to 24 february, 2020 has determined incubation period of 5 days64. systematic review and meta-analysis of 18 studies have generated mean incubation period of 6 days with 2 to 14 days critical for onset of illness. studies concluded by wang et al., have declared 7 to 13 days critical times after the onset of illness and have analyzed clinical parameters including average age, gender ratio, and underlying diseases in covid-19 patients65. sex-specific and age-specific dominance during 2020, sars-cov-2 infection remained dominant in men as compared with women. the mortality rate of covid-19 was higher in male patients than in female patients66. kopel et al., provided a biological explanation for this higher rate of incidence, as kidneys in the male body have more aceii receptors in comparison to the female body. the immunological mechanism of the female body produces more titer of antibodies67. progesterone and 17 β-estradiol are exclusively produced in females and exhibit immunomodulatory roles. they enhance immune tolerance, deregulate innate immune inflammatory response, and prevent “cytokine storm”. these attributes contribute to mild and non-severe progression of covid19 in women68. the average age of patients was determined 49-70 years69. global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 91 clinical manifestations of covid-19 the clinical manifestations of novel coronavirus have been reported to vary from person to person. the confirmed infected individuals may show no symptoms at all i.e. asymptomatic condition. common symptoms include fever, dry cough, muscular pain, anorexia, dyspnea, and fatigue 70. less common symptoms include headache, sore throat, sputum production, vomiting, and diarrhea. patients in critical condition suffer from pneumonia, a lower respiratory rate, lymphopenia, and hypoalbuminemia65. sustainment of pathological conditions in patients admitted to intensive care unit (icu) leads to severe pneumonia, cardiac malfunctioning, shock, acute respiratory distress syndrome (ards), acute kidney injury (aki), and multiple organ dysfunction syndrome (mods)71. ards is type-i respiratory failure (t1fr) and is marked by the presence of hypoxia (pao2 < 8000 pa), tachypnea, tachycardia, cyanosis, and absence of hypercapnia72. moreover, secondary infection by bacterial sp. (staphylococcus caprae, and acinetobacter buamanni) has also been reported in covid-19 patients, which insists the compromised immunological setup and multiple culprits for additional severity of pneumonia65. autopsy report of the covid-19 deceased in china indicated the presence of gray-white patchy lesions in the lungs and alveolar damage with cellular fibromyxoid exudates, bilateral lung tissue damage and ards63. barton et al., conducted autopsy in the u.s.a. that confirmed the alveolar damage, airway inflammation, and acute bronchopneumonia in nonsurvivors73. sars-cov-2 infection in pregnant patients changes in the immune system, increase the susceptibility to respiratory infections during pregnancy74. the clinical manifestations of covid-19 are not different in pregnant women when compared to the general population75. fever (36.5°c-38.8°c), cough, shortness of breath and myalgia are the main symptoms reported in sars-cov-2 positive pregnant patients. lymphopenia, elevated levels of c-reactive proteins, aspartate aminotransferase and alanine aminotransferase have also been noticed76. pregnant women have reported pulmonary complications during ct scan, including focal, scattered or bilaterally distributed lesions in the lungs, lymphadenopathy and ground-glass opacity75. covid-19 can also develop complications in the form of fetal distress77. pregnant females developing severe clinical conditions in the old age group, had higher bmi, diabetes and exhibited gastrointestinal disturbances including diarrhea, vomiting, and nausea78. in covid-19 pregnant women, standardized care requires additional protection during the antenatal period, vaginal or cesarean delivery and breastfeeding74. a study was conducted on 33 neonates born to covid-19 mothers under strict preventive infection measures, which indicated positive results for only 3 neonates (9%), confirming vertical transmission of sars-cov-279. viral particles remained unidentified in the breast milk sample of eight patients, however, the possibility of superficial transfer through droplets has not been ruled out, demanding extreme precautions during breastfeeding80. the vaginal secretions of pregnant women also reported virus-free suggesting vaginal delivery as safe as cesarean recommended to avoid transfer of infection through any means81. covid-19 is also responsible for the increase in the mortality rate among pregnant women. in non-pregnant females, sars-cov-2 infection can cause disturbance in the female reproduction cycle, and the menstrual cycle. it may develop infertility as aceii are also present on the ovaries, uterus, vagina, and placenta82. children are considered more vulnerable to sars-cov-2 infection, however, clinical manifestations of covid-19 cases in children are generally less severe than adults83. role of underlying diseases and comorbidities hypertension, cardiovascular diseases (cvd), and diabetes added additional burden to covid-19-associated severity and death rate. hypertensive patients are at greater risk to develop complications during sars-cov-2 infection as ace-ii is common to both renin-angiotensinaldosterone system (raas) induced hypertension and covid-1984. hypertension is the most dominant comorbidity (16.9%), followed by diabetes (8.2%) in chinese covid-19 cases85. in a study on 212 chinese patients, pre-existing injuries to kidneys (27.3%), liver (8.7%), and heart (6.1%) have been related to poor prognosis of covid-19, and patients may encounter lifethreatening situation85. in new york hypertension is reported in 56.6% of 5700 patients admitted to 12 hospitals around the city. other underlying conditions, including global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 92 diabetes (33.8%), coronary artery disease (11.1%), congestive heart disease (6.9%), cancer (6%), chronic obstructive pulmonary disease (5.4%), chronic kidney disease (5%), and liver disease (0.6%) are also observed86. diabetes is a metabolic condition and is marked by elevated levels of glucose in the blood. diabetic patients are at a higher risk for sars-cov-2 infection as observed in the sars-2003 and mers-2012 pandemics87. diabetes also plays an indirect role in viral pathology by facilitating viral entry into cells and impairing the immune system88. guo et al., reported that covid‐19 diabetic patients are at a higher risk of developing severe pneumonia, release of tissue injury‐related enzymes, hypercoagulable state associated with dysregulation of glucose metabolism and uncontrolled inflammation responses89. moreover, in diabetic patients, therapeutic concern is limited to cq but not for ace inhibitors, angiotensin receptor blockers, or thiazolidinediones. thus, during treatment of sars-cov-2 diabetic patients, regular blood glucose monitoring and patient-tailored therapeutic strategies are highly recommended90. patients with chronic obstructive pulmonary disease (copd) are also at a higher risk of poor prognosis and covid-19-induced mortality. alqahtani et al., have analyzed the role of “smoking” and have reported it as a potential risk factor associated with covid-19 progression, as both active smokers (22%) and exsmokers (46%) progressed to severe cases91. the underlying comorbidities do not confer the same degree of risk; hypertension, cvd, and diabetes significantly assist in the development of complications in covid-19 patients, whereas, cerebrovascular diseases, chronic liver, and renal disorders have shown non-significant relation to progression of severity in covid-19 patients92. they can also cause therapeutic intervention in covid-19 treatment, thus limiting the range of therapeutic agents to be used for treatment of sars-cov-2 infection93. immunological assessment of covid-19 the human body initiates innate immune system upon recognizing foreign antigens displayed on the surface of the infected cells. the innate immune response is followed by an adaptive immune response. dendritic cells and macrophages initiate a cascade of inflammatory cytokines and chemokines, which attract granulocytes and macrophages94. tumor necrosis factor-α (tnf-α), interleukin-1 (il-1), interleukin-6 (il-6), interleukin-8 (il-8), interleukin-12 (il-12), interferon-α (ifnα), interferon-β (ifnβ), interferon-γ (ifnγ), ifn-γ-induced protein-10 (ip10), and monocyte chemoattractant protein-1 (mcp-1), macrophage colony-stimulating factor (gm-csf) and granulocyte colony-stimulating factor (g-csf) act as inflammatory cytokines and promote infiltration of inflammatory cells95. cd4+ t cells and cd8+ t cells play a major role in clearance of viral infection94. during sarscov-2 infection, activation of the immune system and recruitment of monocytes, dendritic cells, natural killer, t cells and b cells cause fever and mild symptoms of covid1996. the over-production of inflammatory cytokines during combating the virus, termed as “cytokine storm”, damages pulmonary tissue at the site of infection. this deterioration of the lungs develops into ards and also causes multiple organ failure and death97. asymptomatic individuals have lower levels of pro-inflammatory and anti-inflammatory cytokines, particularly, tumor necrosis factor-related apoptosis-inducing ligand (trail), growth-related oncogeneα (gro-α). g-csf and il-6, indicating a weaker immune response to sars-cov-298. during the early phase of pandemic, several studies for immunological characterization of covid-19 patients were conducted. in wuhan, the immunological characterization of 107 patients has indicated significant reduction in lymphocytes with severity of clinical conditions1. comparative analysis of clinical and immunological features of moderate cases and severe cases has also reported significant decline in levels of absolute t-lymphocyte, cd4+ t cells, and cd8+ t cell in severe cases as compared to moderate cases70. in new york, 90% of hospitalized patients were initially diagnosed with lymphopenia99. chen et al., have extensively evaluated cytokine levels in plasma as an immunological indicator and have noticed highly significant values of il-2r (p < 0.001), il-10 (p = 0.001) and significant value for tnf-α (p = 0.02). however, no significant differences among severe and moderate patients have been reported for il-1b (p= 0.40), il-6 (p= 0.04), and il-8 (p=0.22). immune profiling of covid-19 has revealed heterogeneous immune response with severity of sars-cov-2 induced illness. mathew et al., performed high dimensional flow global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 93 cytometry of immune components in covid-19 patients and identified three groups of patients. patients with severe clinical manifestations of covid-19 have shown elevated levels of activated cd4+ t cells and lower levels of exhausted cd8+ t cells, and plasmoblasts. patients with mild clinical conditions either have reduced cd4+ t cells and elevated level of memory b cells or have highly insignificant levels of activated t cells and memory b cells100. in recovered patients, lymphopenia and levels of cd4+ t cells and cd8+ t cells were within normal range70. production of sars-cov-2 specific antibodies the time duration of infection to immune response via antibodies production varies from disease to disease and shows variation among patients with the same viral infection. the infected person produces igm, igg, and iga antibodies against n-protein and glycoproteins of s-protein of the enveloped viruses, as observed in the neutralization response to human immunodeficiency virus (hiv)101. the serologic studies on sars and mers have shown that in patients, virus-specific antibodies are detectable on day 14 after onset of disease102. in the case of covid-19, igm and igg antibodies can be detected in one to two weeks of sars-cov-2 infection103. hu et al., observed the level of igm and igg in covid-19 patients, which was 73.5% collectively on day 13-15 onset of disease and 97.8% on day 16-18 onset of the disease. mild and severe patients produced the same amount of igm, but there was a significant difference in production levels of igg, as patients with more severity of sars-cov-2 infection produced more titer of igg104. the seroconversion of igm can occur before, simultaneously, or after the seroconversion of igg105. sars-cov-2-specific viral antibodies have also been successfully identified in asymptomatic individuals; however, the level of igg is reported to be lower than in acute symptomatic patients98. the titer of antibodies against sars-cov-2 infection increases with the severity of disease irrespective of age, gender, and underlying comorbidity106. in severe cases of covid-19, antibodies have failed to generate an effective response against viral particles of sars-cov-2. the inefficiency of these antibodies to display significant viral neutralization has been suggested to be associated with cytokine storm, antibody-dependent enhancement (ade) of viral uptake by macrophages, and enhanced inflammation resulting in immunopathology107. in follow-up studies of recovered discharged patients, igg concentration were reduced by 21% and igm concentrations declined by 23%104. the decline in titer concentrations of sars-cov-2 specific antibodies is suggested to provide protection for limited a time period in recovered patients and increases chances of reinfection. t r e a t m e n t s t r a t e g i e s f o r c o v i d 1 9 p a t i e n t s in general, covid-19 patients are treated with antiviral therapy, corticosteroid therapy, intravenous immunoglobulin therapy, and oxygen therapy. during covid-19 pandemic, randomized and non-randomized clinical trials faced challenges regarding sample size, variation of clinical severity among patients and time constrain for effective treatment yet significant progression to continue or discontinue a particular therapy was reported108. antiviral treatment of covid-19 patients during the early days of the pandemic, the exact pathogenic pathway of sars-cov-2 was not fully understood, thus existing antiviral agents were used for treating covid-19 patients. in china, oseltamivir, ganciclovir, arbidol, lopinavir, and ritonavir have undergone clinical trials for the treatment of the novel coronavirus infection3. lopinavir and ritonavir are licensed protease inhibitors of hiv, which have shown promising results against sars-cov in 2003-2004. these drugs are also recommended for safe treatment of covid-19 in pregnant women109. however, it has been noticed that these drugs failed to reproduce an effective response in other patients as no significant difference in treatment of sars-cov-2 infection was obtained in standard care and experimental group110. remdesivir is a nucleotide analog rna polymerase inhibitor with broad-spectrum antiviral activity, and has previously been used for treatment of ebola virus infection (2014-2016)111,112. the preliminary report on a clinical trial of remdesivir indicated contraction in recovery period of covid-19 patients to 11 days as compared to 15 days in the control group113. moreover, the final report revealed that remdesivir had a median recovery time of 10 days as compared to 15 days in the control group. moreover, patients treated with remdesivir required a lower global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 94 proportion of supplementary oxygen through mechanical ventilation or extracorporeal membrane oxygenation (ecmo). during the trail, adverse side-effects common to remdesivir group and placebo group were pyrexia, hyperglycemia, anemia, reduced hemoglobin levels and decline in glomerular filtration rate113. favipiravir is an rna-dependent rna polymerase inhibitor, which is used for treatment of influenza viruses a, b and c. favipiravir has been found effective as etiotropic treatment for sars-cov-2 infection. it has been revealed that 62.5% patients treated with avifavir (favipiravir) showed viral clearance on day 5 and 92.5% showed viral clearance on day 10 of drug intake. moreover, it is highly effective in normalizing body temperature in symptomatic covid-19 patients as early as day 2 of drug intake. the adverse side effects of avifavir included chest pain, nausea, and gastrointestinal disorders including diarrhea and, vomiting114. cai et al., have reported that time duration for viral clearance by favipiravir is less than that of lopinavir/ritonavir in covid-19 patients115. in june 2020, in china randomized controlled trial for testing the therapeutic effectiveness of favipiravir in patients who, after testing negative once tested positive again for sarscov-2 rna through reinfection, reactivation of original virus or new infection under nct04333589116. chloroquine (cq) and its derivatives are a class of fdaregistered antimalarial drugs which also exhibit antiviral activity. during the sars epidemic, studies have revealed the antiviral role of cq as it interfered with glycosylation of aceii in vitro. suggesting the prophylactic and therapeutic use for sars-cov infection117. the use of cq in a pilot study of chronic patients possessing hepatitis-c virus (hcv) infection also reported significant reductions in viral load of hcv rna118. however, the antiviral property of cq was also found to be ineffective during in vivo. analysis for therapeutic role in hiv infection, highlighting its controversial antiviral role and low safety index119. hydroxychloroquine (hcq), a derivative of cq has shown more potency physiologically based pharmacokinetic models (pbpk) in vitro. than cq120. gautret et al., have reported a significant reduction of sars-cov-2 viral load in 20 patients treated with hcq as a primary drug and azithromycin as a reinforcing drug121. in u.s.a., an extensive protocol for randomized controlled trial was designed to study the post-exposure prophylaxis role of hcq in close contact, with results expected in later half of 2020122. another randomized controlled trial was designed in spain to access prophylactic role of hcq in pregnant women under nct04410562123. zinc exhibits antiviral activity through multiple mechanisms. it plays an important role in the immune system through viral recognition, restoration of depleted immune cell function and prevention of cytokine storm124. it inhibits viral attachment to host cell receptors and interferes with viral replication by altering the proteolytic processing of replicase polyprotein and rdrp in sarscov, hcv, rhinoviruses and influenza viruses. zinc showed synergistic effect when co-administered with antiviral therapy in hiv, hcv and sars-cov125. zinc is crucial for maintenance of respiratory epithelium and ionbalance system. covid-19 patients also share risk factors common to zinc deficiency including old age, gender, and diabetes126. case study on four covid-19 patients reported significant improvement in covid-19 patients provided with highdose zinc therapy. thus, it is highly recommended to further evaluate the association of zinc deficiency with severity of covid-19 infection127. corticosteroid treatment of covid-19 patients therapeutic corticosteroids are administered as an adjuvant therapy for viral infections, particularly influenza and pneumonia. they consist of synthetic glucocorticoids (gc) and mineralocorticoids (mc). gc exhibit antiinflammatory and immunosuppressive properties128. gc receptors are located on the surface of epithelial cells and renal cells; upon ligand binding, they enter the nucleus and downregulate genes of pro-inflammatory cytokines and chemokines including ap-1 and nf-kb, and also activate anti-inflammatory genes129. however, long-term gc therapy can induce severe side effects, metabolic diseases, cardiovascular anomalies and may attribute to inflammation128. corticosteroid treatment has been associated with sustained viral load in blood, delayed viral clearing, and elongated hospitalization in mers130. boudreault et al., indicated that the adaptation of corticosteroid therapy provides controversial information, as immunomodulation via corticosteroids has varying effects depending upon the specific respiratory virus and rises concern for safety and efficacy of treatment131. based on past clinical evidence, russell et al., also deemed the global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 95 treatment as non-beneficial and unqualified to be specifically recommended for sars-cov-2132. due to contradictory evidence of corticosteroid therapy in viral infections, the who recommended to discontinue by 28 january 20207. corticosteroid therapy reduced the mortality rates in hematopoietic cell transplant patients infected by seasonal influenza virus131. chen et al., reported a reduction in hospitalization period for corticosteroid treatment of sarscov infection and lower mortality rate. these results were contrary to those previously reported by auyeung et al., who reported 20.7-fold increase in icu administration (severity of clinical conditions) and increased death rate133,134. methylprednisolone has been associated with survival in ards patients135. during the initial phase of covid-19 pandemic, corticosteroid therapy was used in 20-44.9% of chinese severe patients136. low-dose and short-term administration of methylprednisolone was associated with improvement of oxygen saturation (spo2), and better clinical outcomes in covid-19 patients134. furthermore, corticosteroid treatment also alleviated pulmonary fibrosis and prevented further pathological deterioration in lung injury in covid-19 patients with ards137. in june, u.k. reported the clinical trials of dexamethasone (steroid) under ‘recovery’ to be effective as one-third reduction in the mortality rate of critical covid-19 patients was observed138. antibiotic treatment in covid-19 patients in humans, antibiotic treatment is carried out for illnesses caused by pathogenic bacteria. in covid-19 patients, a compromised immune system could lead to secondary infection by bacteria. risk factors for secondary infections include old age, underlying diseases, and a suppressive immune system139. the antibiotic treatment was used to cope with such infections65. antimicrobial (antibacterial and antifungal) assessment in sputum is an important indication of superinfection by bacterial and fungal pathogens140. acinetobacter baumannii, klebsiella pneumoniae, pseudomona aeruginosa, enterobactor cloacae, serratia marcescens, aspergillus fumigatus, aspergillus flavus, candida albicans and candida glabrata were reported as causative agents of secondary infection in various studies67,92. in china, intravenous antibiotic treatment was provided to 58% of patients (637/1099), where 78% had mild conditions and 22% exhibited severe clinical manifestations56. huang et al., also adapted antibiotic therapy in 81(100%) patients in the study cohort, even though only 10% had secondary bacterial infection136. in pregnant patients, administration of intravenous ceftriaxone was recommended to combat bacterial pneumonia and sepsis141. in france, co-administration of azithromycin with hcq generated better results than hcq, the significance of azithromycin in the prevention of secondary bacterial infection. azithromycin also assisted hcq in the rapid clearance of viral load in 100% of patients as compared to 12.5% of patients in control group142. essential monitoring of immunological parameters in covid-19 patients is highly recommended to strategize antibiotics use in the treatment of secondary microbial infections140. oxygen therapy of covid-19 patients for better outcome, therapeutic approach is combined with mechanical medical assistance. non-invasive ventilation is provided by nasal cannula and an oxygen mask to covid19 patients with mild symptoms66,136. mechanical invasive ventilation and extracorporeal membrane oxygenation (ecmo) assist breathing in covid-19 patients who have developed severe lower respiratory deterioration143. the niv has proved effective in increasing arterial blood gas tensions in patients with impaired central respiratory drive. the niv is conducted either through continuous positive airway pressure (cpap) or bi-level positive airway pressure (bipap). bi-pap ventilators and volumecontrolled ventilators have been recommended for severe pulmonary injury and ards144. moreover, selection between cpap and high-flow nasal oxygen (hfno) is based on clinician’s choice and depends on airway pressure, o2 consumption, and patient tolerance. in practice, cpap is usually preferred to hfno owing to adjustable airway pressure, provision of 50% fracture of inspired oxygen (fio2) at 5-6l/min, patient-ventilator synchrony, and precautions during setting. invasive mechanical ventilation is connected to a mechanical ventilator and includes tracheal tube, laryngeal mask, and tracheostomy to bypass the upper respiratory tract. it is used to stabilize patients suffering from global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 96 hypoxemia, hypoventilation, hypercapnic respiratory failure, acute lung injury, and ards. mechanical ventilation is guided by clinical condition, comorbidities of patients, optimization of delivered tidal volume (tv), and respiratory rate (rr). the mechanism of invasive mechanical ventilation is based on trigger, target, and cycle. the trigger is initiated either through a patient’s breath or is set as time-triggered in the absence of patient’s breath. target refers to breath delivery strategy and cycle determines how breath is terminated145. in patients with severe pneumonia or ards, ac-vc ventilators are the best choice as airway pressure is not maintained externally in these ventilators, but is a function of compliance, and resistance of the respiratory system of a patient. ac-pc type ventilators are also proposed for ards patients admitted to icu, however, there is risk of barotrauma formation in some patients. pressure regulated volume control (prvc) system does not satisfy ards patient’s respiratory needs, is only recommended in recovering patients. walter et al., suggested ideal mechanical conditions for ards as rr of 20 breaths per min, inspiratory rate flow of 80 lmp, fio2 at 7-8ml/kg ibw, peep 5cm h2o and pplt< 30cm h2o and pao2 5580mmhg145. a detailed precise study focusing on the ventilation system best aligned to patients’ respiratory need and survival for covid-19 is highly recommended to be conducted as oxygen therapy is of prime importance alongside therapeutic treatment. synthesis and production of sars-cov-2 vaccines the vaccine is a product capable of inducing an immune response by generating antibodies once administered into the body of an organism. vaccination immunizes that individual or boost pre-existing immunity against specific bacterial or viral infections. large-scale production of vaccines is an elongated process. it is initiated with the selection of appropriate viral targets, in vitro studies, preclinical trials in animal models, clinical trials, establishment of pipeline and production of vaccine in large-scale146. once sufficient pre-clinical data is obtained, three phases of clinical trials are conducted. phase i clinical study is conducted in small sample size, phase ii is done to optimize dosage and phase iii is conducted on larger sample size particularly, a demography at risk. large-scale production of vaccine requires the approval of fda, setup of good manufacturing practices, and production platforms147. during the early phase of the pandemic, insufficient funding, non-availability of pre-clinical data, non-optimized pipelines, and lack of capacity for mass production of sars-cov-2 vaccines resulted in global challenge146. in order to develop sars-cov-2 vaccines for global population, study, trial was designed, regional, national and international demographics were targeted, rigid and flexible clinical endpoints were determined, traditional and non-traditional technological platforms were adapted, and vaccines were produced in large quantities. under the umbrella of the coalition for epidemic preparedness and innovations, and who, vaccine r&d confirmed 115 vaccine candidates initially, and the number was increased to 321 in the latter half of 2020. pre-clinical studies and clinical trials were initiated by china, usa, germany, australia, south africa, india and south korea. human first-line immune response is initiated fully against spike protein antigen, a primary protein for the interaction of virus-host cell receptor, thus, offering “target antigen” for vaccine development147. based on chemical composition, vaccines for viruses are categorized into protein subunit vaccines, live-attenuated virus vaccines, whole inactivated viral vaccines, viral vector vaccines, virus-like particle (vlp) vaccine, dna vaccine, and rna vaccine. sars-cov-2 has shown a genomic resemblance to coronaviruses; however, no fda approved commercial vaccine is available against coronaviruses until covid-19148. protein subunit vaccines comprise of viral antigenic fragments (responsible for generating immune response) and adjuvants (enhance immunogenicity)149. s-protein of sars-cov and merscov offer better targets for inducing immune response as compared to n and m structured proteins. protein subunit vaccines for sars-cov and mers-cov are based on sprotein targets. during clinical trials, both rbd-based vaccines for sars-cov and mers-cov induced high-titer neutralizing antibodies without causing pathogenic effect. rbd-based vaccines for sars-cov also induced longlasting neutralizing antibodies for 12 months and that of mers-cov for 6 months150,151. however, full length sprotein and trimeric s protein (trispike) of sars coronavirus also contained non-neutralizing epitopes, and triggered fcγ receptor-ii (fcγrii)-dependent sars-cov global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 97 infection in vitro. protein subunit vaccine of sars-cov-2 targets full length s protein or rbd of s protein. during 2020, novavax (sars-cov-2 rs/matrix-m1 adjuvant) entered phase ii trial (nct04533399) in south africa. anhui zhifei longcoms’s recombinant new coronavirus vaccine contains rbd of sars-cov-2 (nct04466085) has also entered phase ii clinical trials. these trials were designed to evaluate the efficacy, immunogenicity and safety of sars-cov-2 recombinant vaccines149. virus-like particle (vlp) vaccines present similar conformation of epitopes as in the original virus, but they lack a viral genome. vlp vaccines for human papillomavirus (cervarix and gardasi) and hepatitis-b virus (engerix and recombinvax hb) have been successfully commercialized152. in case of sars-cov, chimeric vlp consisting of s protein and influenza virus m1 protein are capable of inducing an immune response against sars-cov in the experimental group of mice. the in vivo study of mice model also revealed an effective immune response induced by chimeric rbd (mers-cov) and vp2 structural protein of canine parvovirus152. however, vlp vaccines also induce pulmonary immunopathology in preclinical mice models153. the evaluation of immunogenicity of sars-cov-2 vlp vaccine (nct04450004) has been registered for clinical trial phase i. in contrast to vlp, dna vaccines contain genes encoding viral antigen components that are expressed by plasmid vectors. dna vaccines are administered through electroporation of individual’s cells149. s-protein based dna vaccines exhibited the greatest immunogenicity in sars-cov and mers-cov. gls-5300 (ino-4800), dna vaccine of mers-cov produced an immune response in 85% participants recruited in the clinical trial registered under nct04447781 and nct04336410154. full length dna vaccine for sars-cov-2 designed by inovio’s (ino4800) has induced t cell immune response in 94% participants with no major adverse effect. rna vaccine production platforms are mainstreamed exclusively for covid-19. rna vaccines contain mrna of the antigens that translates inside the human cell and consequently initiates the immune response149. moderna mrna-1273 vaccine encodes a modified perfusion sprotein, which is more stable and prevents activation of interferons155. the phase iii clinical trial (nct04470427) exhibited 94.5% efficacy with no significant safety concerns. biontech and pfizer’s mrna was initially focused on four candidates bnt162b1 (modified rna translated into trimer-rbd), bnt162b2 (modified rna encoding full-length s-protein), bnt162a1 (uridine mrnabased vaccine), and bnt162c2 (self-amplifying rna based vaccine). bnt162b2 exhibited maximum efficiency of 95% against sars-cov-2 and least systematic reactogenicity in the old age group149. viral vector vaccines are recombinant vaccines capable of encoding viral antigens in modified viruses. viral vector platforms include adenovirus, measles virus, parainfluenza virus, rabies virus, modified vaccinia virus ankara (mva), and vesicular stomatitis virus (vsc). the manufacturing process of these vaccines is complex since it requires custom optimization of cellular systems, exclusion of contaminants, and safety concerns regarding integration of genome into vaccinated human149. in the case of sars-cov, the adenovirus vector expressing s1 fragment, and mva expressing s-protein-induced immune response in animal models156,157. mers-cov s-protein vaccine based chimpanzee adenovirus vector (chadox1) induced high titer of antibodies and was determined safe and well tolerated during clinical trial for mers-cov vaccine149. covid-19 viral vector vaccines include azd1222 (chadox1 ncov-19), gam-cov-vac (rad26s+rad5-s), and ad5. azd1222 was developed by the collective efforts of astrazeneca and oxford university. gam-cov-vac, also known as sputnik-v is developed by gamaleya research institute, and ad5 is developed by cansino biological inc. and beijing insitute of biotechnology. azd1222 (chadox1 ncov-19), expresses a sars-cov-2 s protein. the interim report published in july 2020 stated that it can initiate immune response with no severe adverse side effect158. moreover, the results of phase ii/iii clinical trial in uk (2020-0012288-32) reported average efficiency of 70% in participants. sputnik-v is designed on the recombinant viral vector system in which adenovirus type 26 vector encoding sars-cov-2 spike glycoprotein, and adenovirus type 5 vector encoding sascov-2 s-glycoprotein have been integrated. the phase i/ii trial results for sputnik-v have revealed the vaccine efficacy after dose-i is 91.4% and after dose-ii is 95%. moreover, no adverse effect was noticed in the participants. ad5 vector covid-19 vaccine also induces significant immune response after single dose. it has global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 98 entered phase iii (nct04526990 and nct04540419) at multiple centers around the globe149. live attenuated vaccines are formed after weakening the pathogenicity of viruses by mutations and deletions. these vaccines are commercially available against measles, mumps, polio, and yellow fever. no live attenuated sarscov and mers-cov vaccines have undergone clinical trials. moreover, none of the vaccine candidates for covid-19 are based on live attenuated vaccine platform as the pathogenesis of disease is still not clear and global incidence of reinfection has been noticed149. whole inactivated viral vaccines are generated by chemical or radiological treatment of virions. studies indicated that sars-cov-inactivated particles by formaldehyde or uv are capable of inducing an immune response159. in case of mers-cov, γ-irradiated mers-cov vaccine adjuvant with mf59 causes eosinophil-induced pulmonary destruction along with generating neutralizing antibodies160. sinovac inc. have developed coronavac by inactivating sarscov-2 after treating it with beta-propiolactone. phase i and phase ii trials of coronavac are registered under nct04383574 and nct04352608. sinopharm inc. and wuhan insitute of biological products have collectively introduced inactivated vaccine candidate, which has demonstrated initiation of antibody response with low occurrence of adverse side effects161. p r e v a l e n c e o f c o v i d 1 9 i n 2 0 2 0 epidemiological parameters of covid-19 the transmissibility and severity of a pandemic are characterized by epidemiological parameters including basic reproduction number (ro), case-fatality rate (cfr) and mortality rate. basic reproduction numbers (ro) are an important parameter for determining the expansion potential of an epidemic162. it is defined as the number of secondary infections expected to generate from a single patient in a community where the disease has not occurred previously and no herd immunity has been established. the estimated ro ≥ 1 in particular demography indicates rapid spread of human-to-human transmissions and infection162. ro values can be estimated by stochastic markov chain monte carlo method, dynamic compartmental model, statistical exponential growth model, statistical maximum likelihood estimation, mathematical transmission model, stochastic simulations of early outbreak trajectories, and mathematical seirtype epidemiological model163. the establishment of an epidemic is dependent on modes and routes of transmission. transmission of viral particles is initiated through asymptomatic individuals, pre-symptomatic individuals, symptomatic individuals, and the environment. on the basis of origin of covid-19 transmission, cases were classified into local transmission, community transmission, sporadic cases, imported cases and clusters of cases. the estimated ro levels of covid-19 (1.4-5.7) are significantly higher than those for mers-2012 (0.453.9) but have a similar index to sars-2003 (1.7-3.6). the cfr is another important parameter to understand the seriousness of a pandemic and is defined as the conditional risk of death for patients with the disease164. china observed a cfr of 2.3%, whereas italy reported cfr of 7.2% was during the first half of 2020165. republic of korea and germany successfully reduced cfr by 0.7%1.2% in an infected population by adapting frequent testing equipped with smart lockdown policies, and provision of healthcare facilities7,166. gradual decline in cfr was reported after the first wave of covid-19 pandemic but it was restored during second wave of pandemic after ease in social restrictions was adapted. global incidence of covid-19 in 2020 at the beginning of the 21st century, the first coronavirus epidemic broke out in guangdong, china occurred (2003) by sars. sars showed the dominant presence for 8 months in 29 countries with 8,096 cases, 774 deaths, and 9.6% mortality rate. after a decade, the mers outbreak in the epicenter of saudi arabia infected 2,494 humans in 27 countries and reported a mortality rate of 34%, corresponding to 858 deaths17. sars-cov-2-induced covid-19 is a large-scale global pandemic that has infected the highest number of people in the history of infectious diseases. over the span of the year till 29th december 2020, it has infected 72 million people and has induced 17 hundred thousand deaths with the death rate of 2.2%, a number much lower than sars and mers, yet a great concern in the global population of 7795 million. the outbreak of the covid-19 and its global distribution has been strictly monitored by the government global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 99 organization “center of disease control and prevention”, responsible for analyzing incidence rates of diseases and devising preventive measures for national health. “covid19” is the first pandemic which has been successfully digitalized in real-time167. the chinese center for disease control and prevention successfully identified novel coronavirus on 7th january 2020, causative agent of pneumonia infection in wuhan10. in collaboration with internationally recognized states and regions, who played a crucial role and successfully generated “situation reports” indicating new infections, the total number of cases, and cumulative deaths in covid-19 patients on a daily and weekly basis. who declared very high-risk levels for china and risk alerts for remaining countries, on the 23rd january 2020 as export of novel coronavirus through cross-border travelers became evident7. during the first half of 2020, it has been reported that the total number of globally infected individuals was 9,826 in january 2020, whereas, 5,934,936 cases were later confirmed in june, 2020168. during february 2020, novel coronavirus spread to 54 countries, causing who to declare “high-risk levels” for the covid-19 pandemic as a 769% increase in global cases was observed. number of cases increased drastically in march by 779% and who declared “global pandemic”. the mitigation of viral spread was carried out by adopting safety measures, and partial or complete lockdown by countries around the globe. this resulted in a relative decline in the percentage of new cases by 467% in april, 220% in may, and 20% in june 2020169. china, republic of korea, and japan in western pacific; u.s.a in american region; iran in eastern mediterranean and u.k., spain, italy, france and russian federation in europe; india in southeast asia were highly ranked countries with covid-19 cases in june 2020. china successfully contained the virus with a few additions of cases per month, whereas the number of cases escalated in other counties, particularly france, spain, italy, india, russia and usa. in the latter half of 2020, from july 1st to december 29th, 2020, additional cases were reported around the globe. by 30th july, who reported an increase of 7 million cases (17, 106,007) and 165 thousand (668,910) covid-19-induced deaths, proposing a stern need to strictly monitor physical distance and preventive measures. in august, the who reported 24,854,840 confirmed cases and 838,924 nonsurvivors. in september, the global number of covid-19 confirmed cases infected 0.41 percent of the global population. in october, global cumulative cases were reported as 43,341,451. in november, as escalation of 43% marked in second wave of pandemic. at the end of 2020, 79 million people were infected by sars-cov-2 infection, a number of great concern for 7.8 billion people around the world (fig. 3-4; table 2-3). throughout the third and fourth trimester, south africa reported the highest number of cases in the african region. france, spain, and italy reported peak infections in the european region for covid-19, a resurgence owing to enhanced social interactions during vacations and ease of social restrictive measures. usa and india reported the highest number of covid-19 cases in 2020, highlighting challenges faced in the world’s most populated countries. figure 3. distribution of covid-19 cases from january 2020 to december 20207. western pacific south-east asia europe americas eastern mediterranean global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 100 table 2. detailed epidemiological presence in who distributed regions in the months of january 2020 to december 2020. jan 2020 feb 2020 mar 2020 apr 2020 may 2020 jun 2020 western pacific 9782 82,941 104,686 147,743 181,665 215,566 south-east asia 17 47 4,215 54,021 260,579 784,931 europe 14 1119 423,946 1,434,649 2,142,547 2,692,086 america 9 79 163,014 1,246,190 2,743,793 5,136,705 eastern mediterranean 4 510 50,349 182,417 505,001 1,058,055 africa 0 2 3,786 24,713 100,610 297,290 other 0 705 712 712 741 741 worldwide 9826 85,403 750,890 3,090,445 5,934,936 10,185,374 jul 2020 aug 2020 sep 2020 oct 2020 nov 2020 dec 2020 western pacific 306,052 487,571 600,891 715,300 874,705 1,059,751 south-east asia 2,009,963 4,073,148 6,720771 8,969,707 10,738,733 71,842,422 europe 3,333,300 4,205,708 5,662,875 9,664,042 18,495,511 25,271.220 america 9,152173 13,138912 16,233,110 19,737,794 26,216,515 34,403.371 eastern mediterranean 1,533,357 1,903,547 2,340,215 2,955,552 4,045,906 4,823,157 africa 770,421 1,044,513 1,172,342 1,298,315 1,49,524 1,831,277 other 741 741 741 741 worldwide 17,106,007 24,854,140 32,730,945 43,341,451 61,866,635 79,231,893 table 3. differential percentage increase in the number of cumulative cases in consecutive months from january 2020 to december 2020. months-2020 total number of cases cases in previous month new cases each month percentage increase january 9,826 february 85,403 9,826 75,577 769% march 750,890 85,403 665487 779% april 3,090,445 750, 890 2,339,555 312% may 5,934,936 3,090,445 2,844,491 92% june 10,185,374 5,934,936 4,250,411 72% july 17,106,007 10,185,374 6,920,633 68% august 24,854,140 17,106,007 7,748,133 45% september 32,730,945 24,854,140 7,876,805 32% october 43,341,451 32,730,945 10,610,506 32% november 61,866,635 43,341,451 18,525,184 43% december 79,231,893 61,866,635 17,365,258 -6% global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 101 figure 4. total number of sars-cov-2 positive cases in each month and relative percentage increase from january 2020 to december 20207. table 4. percentage increase in the number of deaths indicating a rise in mortality rate with the spread of covid19 from january 2020 to december 20207. months-2020 total number of deaths no. of deaths in previous month percentage increase w.r.t previous month january 213 february 2,924 213 1264% march 36,405 2,924 1011% april 217,769 36,405 498% may 367,166 217,769 67% june 503,862 367,166 37% july 668,910 503,862 33% august 838,924 668,910 25% september 991,224 838,924 18% october 1,157,509 991,224 17% november 1,448,990 1,157,509 25% december 1,754,574 1,448,990 21% covid-19 induced deaths in 2020 in the early phase of the pandemic, the disease was concentrated in mainland china and a total number of covid-19-induced deaths on 31st january were reported 213. however, with the spread of the disease to other countries, the number of non-survivals also increased drastically, making covid-19 a highly deadly pandemic. in february, 3,924 deaths were reported, with a maximum number of non-survivals (72%) belonging to china. during march, the european region marked the highest death rate of 73% for covid-19 patients; the highest number of deaths being reported in italy (11,591) and spain (7,340)7. in the situation report for 30th april, 2020, it was recorded that 62% covid-19 induced deaths occurred in europe global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 102 where spain, italy, united kingdom, and france were among the top four. in may, the highest number of deaths were noticed in 21% in the u.k. (21%), italy (18%), and france (16%) and spain (16%). in june, the number of deaths in the american region contributed to 49% of global covid-19-induced deaths and was followed by 39% european. in the third trimester, the highest number of death rate was observed in the usa (1,414), brazil (1,595), india (779), and russia (161). in august, covid-19 produced 170,014 non-survivors and in october the death toll crossed 1.1 million (table 4). covid-19 induced 187,251 in october in who classified seven regions169. due to variants of sarscov-2 and the second wave of pandemic across the world, a 25% increase in death rate was observed and 291,481 deaths were reported. an additional 21% rise in the number of deaths were reported in december 2020 and total number covid-19 induced deaths reached 1.7 million. during the third quarter of 2020, the highest number of deaths were reported in america, european and southeast asian regions, particularly, u.s.a., u.k., and india7. prevalence of covid-19 in china during 2020 ferretti et al. has estimated ro=2 in the epicenter of covid-19 disease. most transmissions are conducted through pre-symptomatic individuals (46%), in comparison to symptomatic individuals (38%), asymptomatic individuals (10%) and environmental contamination (6%). the pre-symptomatic condition was case-specific and distinguished from the asymptomatic condition in which no signs and symptoms of viral infection were noticed at all89. read et al., provided a comprehensive estimation of infected individuals in wuhan before 23rd january, 2020 and estimated average 6,510-25,095 new cases and maximum increase of 33,490 cases in chinese population168. mizumoto et al., applied statistical analysis, and estimated ro= 3.49 with dataset available till 11 february 2020. the estimated number of infections were 18,967 and covid-19 induced deaths were 821. the estimation was close to officially record, positive cases of 19,559 covid-19 infection and 821 covid-19 induced deaths170. the pooled ro value of covid-19 epidemic in china was estimated to be 3.32, indicating an infected person can transmit disease to at least four other persons. sanche et al., adopted case count approach and estimated ro between 4.7-6.6171. mathematical modelling of ro estimation provided a range of 1.4-6.49 indicating the exponential role of human-to-human transmissions172,173, highlighting underestimation of the covid-19 spread and the importance of strict social preventive measures. in china, the outbreak of respiratory novel infection of unknown etiology was officially confirmed on 31st december 2019. by 21st january, 278 cases were reported, and 93% were concentrated in wuhan, hubei province. on january 31st, the number of sars-cov-2 positive cases reached 9,720. the cases were initially studied with respect to the exposure to the hunan seafood market, but later drastic increases confirmed spread through human contacts. china reported 50,580 laboratory confirmed cases on 15th february and 157% increase till 29th february, 20207. in march, 3,151 new confirmed cases were reported as a result of implementation of restriction policies in hubei province and chinese region. moreover, in the first half of 2020, an additional 2,682 cases were reported in china, highlighting the importance of nonpharmaceutical measures for containment of the virus in the world’s highest populated country162. in the third trimester of 2020, china reported a 3% increase in covid19 infections. however, the number of cases reached 96,324 on december 29th, 2020, and a 6% increase was noticed during last trimester of 2020. prevalence of covid-19 in american region who defines american region as north america (united states of america) and south america. in the region, local transmissions through community interactions and cluster cases have extensively been reported, and sars-cov-2 viral containment has proved challenging. the epidemiological incidence of covid-19 was highest in usa, followed by brazil, mexico, and canada. in u.s.a, six imported cases from china were confirmed in january and the number of confirmed positive cases increased to 62 in february7. however, the incidence rate escalated in march when a 236,416% increase was noticed, resulting in 146,640 infections. implementation of preventive measures and an increase in lockdown duration could not significantly contain the virus in a population of 331 million. with rapid and enhanced testing facilities, u.s.a. reported 1,003,974 cases in april, 1,716,078 cases in may, and 2,537,636 cases in june 2020. the escalation listed usa global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 103 as the most infected country in the world, with the highest number of covid-19 individuals in the first half of 20207. the trend was sustained during the latter half of 2020 and usa noticed 18 million covid-19 infections in december. during june to july, covid-19 cases increased by 73%. the following months of august and september reported a reduction of 20% and 25% in covid-19 cases. during the second wave of pandemic, initiated in october, covid-19 incidence rates increased by 44%. it reached peak value of 177% during november and an increment of 30% during december. the cumulative cases of covid-19 in december 2020 were 18,648,989, contributing 54% of infections in american region. in u.s.a, community transmissions were the major reason for the escalation of covid-19 positive cases. canada reported 3 infections in january, whereas, brazil reported only 1 infection, and mexico reported only 2 infections in february. canada confirmed the first covid19 imported case in january 2020 and only 14 on 29th february, 2020. international and national transmissions established an epidemic in canada, reporting 6,303 new infections in march. the epidemic sustained throughout 2020, with new infections rising each month, april (44,046), may (39,108), june (13,509), july (11,920), august (11,888), september (23,098), october (65,648), november (142,960) and december (180,234). gradual reductions in the relative number of confirmed cases were observed in may, june, july, and august as strict lockdown was implemented. the escalation in october, november and december correlated to the second wave of pandemic and the evolution of virulent strains of sars-cov-2. the average number of new cases per day also increased in brazil, march (137), april (2,254), may (12,686), june (29,299), july (38,972), august (40,404), september (29,493), october (23,484), november (28,141) and december (39,039). mexico observed relative percentage increases in april (697%), may (405%), june (156%), july (88%), august (43%), september (23%), october (24%), november (22%) and december (26%). on december 29th, mexico reported 1,372,243 cumulative cases of covid-19. overall, no country in the region was included in covid-19 free zone7. prevalence of covid-19 in europe united kingdom, france, finland, germany and italy imported covid-19 infection in january, 20205. seir mathematical models indicated ro= 6.94 with 95% ci, 475,000 expected cases, and 50,000 cumulative deaths in the absence of preventive measures and lockdown policy for the public in european countries174. linka et al., also estimated ro= 4.22 ± 1.69 for european region and the highest ro was estimated in the cases of germany (6.33) and netherlands (5.88)175. sars-cov-2 positive confirmed cases escalated by local, community and clustered patterns of transmissions. italy reported 888 infections, germany and france reported 57 infections, spain reported 32 infections and u.k. reported 20 infections in february. italy, germany, france, spain and uk reported a rapid increase in the number of sars-cov-2 positive cases in march and april (italy=2,035,059, germany=195,062, france=1,277,009, spain=212,885, uk=165,205). the relative decrease in subsequent number of new cases were observed in germany (35,140), spain (36,053), france (29,864) and italy (7,772) as time transitioned from 1st may to 30th june. contrary to this, u.k. reported 146,744 new cases of covid-19 in june. in the second half of 2020, italy and spain reported peak infections in september, u.k. reported peak infections in october, germany and russian federation reported peak infections in november, france and turkey reported peak infections in december. the variation in peak infections highlights the fact that the start of the second wave of covid-19 varied in different countries. these rises in the number of new cases were the manifestation of ease in social restrictions, and virulent strains of sars-cov-2. russian federation and turkey are assigned to europe under who regional distribution. russian federation reported only 2 imported cases in february; however, the number of covid-19 cases increased drastically in a population of 143 million. in march 1,837 cases were confirmed and the number increased to 647,849 in june 2020. the highest number of new cases were reported in april (5697%). russian federation noticed a rise in the number of new infections in july (30%), august (18%), september (16%), october (35%), november (47%), and december (35%). the rapid surge was associated with delayed adaptation of restriction policies by the public. turkey was among the first european countries to report an escalation of covid-19 cases after the first infection. during march 10,827 covid-19 cases were reported and a drastic increase of 986% was observed in the following global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 104 month. the spread of disease in may (39%), june (22%), july (16%), august (16%), september (17%), october (16%), november (35%), and december (170%), indicated that the second wave of the pandemic was initiated in november in the country. prevalence of covid-19 in western pacific region who classified china, republic of korea, japan, vietnam, singapore, new zealand, australia, malaysia, cambodia, philippines, mongolia, republic of fiji, brunei darussalam and papua new guinea in western pacific region? republic of korea and japan were on the list of countries which confirmed presence of imported cases on 20th january, 20207. philippines reported the highest number of cumulative cases, reporting 469,005 infections in the region. philippines reported only 3 cases in january; 2020 however, rapid increase in the number of cases was noticed each month, march (1,543), april (6,666), may (9,012), june (19,214), july (52,936), august (123,757), september (88,125), october (70,274), november (56,167) and december (75,659). the ease of preventive measures by authorities and neglect on the part of the population were related to emergence of new cases during the first and second wave of pandemic. japan reported only 230 cases of covid-19 in february. during the first wave of pandemic, peak infections were observed during april (12,135). during the second wave of pandemic, japan reported peak infections in november (47,155) and december (72,659). south korea reported 11 cases on 31st january 2020. local transmissions and clustered infections were the cause of more cases in february (3,150), march (6,636), april (979), may (1,132), and june (1,332), july (1,505), august (5,394), september (3,912), october (2,432), november (7,740), and december (23,109). throughout the year, korea reported a higher number of cases per day in august, november and december. australia also observed a continuous rise in the number of covid-19 cases; however, it was not as high as in philippines, japan, and korea. in australia, the highest number of cvoid-19 cases were confirmed in august (9,244). in contrast to these three countries, it reported only 511 new sars-cov-2 infections in october, 358 in november and 411 in december. these results are the manifestations of a complete shutdown of cross-border traveling of people. new zealand reported 1 case of covid-19 in february. the adaptation of lockdown policies during earlier stages of the pandemic was responsible for only 529 cases in april 25 in may, 24 in june and 32 in july. unlike other countries, peak infections of covid-19 were reported in august (168) and no exponential rise was noticed in the following months september (99), october (108). november (111) and december (92). in the region, viet nam noticed more cases in march (187), august (531) and november (172) and reported 1440 cumulative cases on 29th december, 20207. in these countries, strict lockdown policies, nonpharmaceutical interventions, contact tracing, and shutdown of borders limited the spread of covid-19. prevalence of covid-19 in eastern mediterranean the eastern mediterranean region comprises 23 countries. iran, pakistan, saudi arabia, united arab emirates, egypt and qatar collectively reported 764,330 cumulative cases in june and 2,370,306 cumulative cases in december 2020. iran confirmed 338 infections in february and sustained the highest number of cases in the region throughout the year. the percentage increase in the number of cases per month was as high as 12177% in march and as low as 19% in september 2020. pakistan confirmed 2 imported cases of covid-19 in february. pakistan observed a continuous surge of infections through pilgrims from iran, and local transmissions. the estimated ro for covid-19 cases in pakistan from 1st march to 28th may, 2020 was approximated to 1.87176. pakistan reported a percentage increase of covid-19 cases in april (754%), may (341%), june (201%), july (33%), august (6%), september (5%), october (6%), november (63%) and december (20%). the second wave of covid-19 was initiated in november 2020 as new strains of sars-cov-2 were reported. saudi arabia reported a percentage increase of 1373% in april, followed by 290% in may, 124% in june and 33% in july. the rise was mitigated by closing borders for international visitors to the state. consequently, a percentage increase as low as 4% was reported in the months of october and november. u.a.e. is an international hub of business and tourism. u.a.e. confirmed 611 infections with covid-19 in march and 11,929 in april. u.a.e reported a 184% increase in may global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 105 and 42% in june. covid-19 infections drastically increased 39% in october, marking the start of the second wave of covid-19 pandemic. iraq, yemen, syrian arab republic, and afghanistan reported 14% cumulative cases of covid-19 in the region, a number too high for the poverty and hunger-driven population of these countries. prevalence of covid-19 in southeast asia the southeast asian region comprises india, bangladesh, thailand, sri lanka, nepal, indonesia, myanmar, maldives, bhutan and timor-leste. early prediction of covid-19 outbreak in india estimated ro=1.41, a number of concern in world, the second largest populated country 177. india confirmed 1,071 covid-19 cases in march 2020. however, the rapid spread of sars-cov-2 reported the second highest number of infections in the world after u.s.a. the covid-19 cases increased every month, april (31,979), may (140,093), june (384,697), july (1,072,030), august (1,903,863), september (2,449,799), october (1,953,897), november (1,446,490), and december (794,931). india contributed to 86% of the cumulative cases in the southeast asian region. bangladesh reported its first case of covid-19 on march 8, 2020, and later confirmed 49 cases on 31st march 2020. the estimated ro was equivalent to 1.82 for a duration of 65 days178. it contributed to the second highest number of covid-19 cases in the region, preceded by india. the number of new cases per month increased in april (7,054), may (37,505), june (97,193), july (93,088), august (74,036), september (48,948), october (42,378), november (60,368), and december (47,480). in the region, thailand was the first state to report 14 imported cases on 31st january 2020. the number of new cases per month increased in february (42), march (1,482), april (1,430), may (127), june (90), july (139), august (101), september (112), october (0), november (520), and december (2054). during july to november, thailand only reported 3,966 infections. the number of covid-19 cases dramatically increased to 6,020 in december, coinciding with the second wave of the pandemic. prevalence of covid-19 in african region african region was marked covid-19 free zone until 29th february, when nigeria and algeria confirmed the presence of the first imported case. covid-19 spread rapidly through local, clustered, and community transmissions. the estimation of ro provided a value of 2.37 (ci 95%, 2.2-2.5). south africa ranked first in africa for reporting the highest number of sars-cov-2 infections during the first and second half of 2020. in june, south africa confirmed 144,264 cumulative cases, nigeria 25,133, algeria 25,133, ghana 17,351. democratic republic of congo, the hub of an ebola virus outbreak in 2009, reported 98 cases in march and 6,938 by the end of june 20207. during the second half of 2020, algeria reported a 228% increase, south africa reported 106% cases, nigeria reported a 96% rise in the number of covid-19 cases. democratic republic of congo reported an increase of 80% and ghana reported an increase of 55% in covid-19 cases. the relative percentage increase was the highest in december, 2020 and correlated to a second wave of covid-19 pandemic7. p r e c a u t i o n a r y m e a s u r e s a g a i n s t c o v i d 1 9 a n d s o c i a l i m p a c t covid-19 has a significant impact on the socio-economic life of mankind. the pandemic has challenged human interactions, which form the basic network of a dynamic society, and has threatened the existence of a global community. cultural and societal norms are the backbone of any nation, whereas education and industrial setup strengthen the socio-economic dimensions of nations. covid-19 is a communicable disease which spreads through viral droplets in the air. the virus enters the body of a healthy person through inhalation of contaminated air or land onto the body’s surface19. it is also transmitted through fomites, and contaminated water 7. direct contact with covid-19 positive individuals enhanced rates of transmission and sars-cov-2 infection179. precautionary measures and non-pharmaceutical interventions after the announcement of “ global high-risk level” of covid-19 by who in march, countries adapted precautionary measures, and non-pharmaceutical interventions to combat the larger-scale community spread, and cope with demanding medical equipment and the healthcare facilities180,181. these include frequent cleaning of hands with alcohol-based sanitizers, washing of hands with soap underwater for at least 20 seconds, global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 106 avoiding hand-shakes during greetings, following social distancing, avoiding unnecessary traveling, and community activities, and taking precautionary measures for religious gatherings182. the use of mask was made mandatory. cloth masks were used by the general public to commute, and they provided efficient protection against sars-cov-2 and other viral droplets. protection from viral droplets is dependent on the type of fabric used and the number of layers of clothing. the implementation of regional lockdown by government authorities significantly declined real-time infections as compared to the estimated rise in curves for covid-19 cases in china, europe, uk, and india175,183. in china, public gatherings and activities have been banned since january. the global community observed 13% reduction in expected global cases of covid-19 by imposing strict lockdown policies and limiting community transmissions184. moreover, china also closed schools and workplaces, and expected delay of the succeeding waves of pandemic185. high values of estimated ro for covid-19 in european region correlated to the social infrastructure and characterization of european nations175. germany, u.k., and india officially implemented lockdown policies on march 24, 2020174. restrictive movement of the masses, social distancing and contact tracing in india were adapted to mitigate the transmission rates of covid-19183. some countries adopted strict lockdown policies, including pakistan, iran, and italy, whereas, south korea implemented soft lockdown and adapted a widespread testing strategy180. air travel restrictions from china were implemented to combat high-risk levels for usa, taiwan, hong kong, singapore, south korea, japan, and vietnam169. impact of covid-19 on vulnerable population the shutdown of outdoor activities, quarantine, social isolation of the masses and unemployment have increased depression, stress-related gender violence, and abuse in vulnerable children and women176,186. the underprivileged class of society living in poor-sanitary and unstable residences faced insufficient primary health care before the pandemic, and the situation became worse during the pandemic. vulnerable people in society, including retarded individuals, homeless people, internally displaced persons, and low-class migrants, have been devastated by covid19 closures and restrictions 187. covid-19 has a direct impact on children of low and middle-income countries, as it interrupted administration of vaccines of other diseases, and lacks access to food during school hours188. closure of educational institutions and day-care centers in italy still protected majority of children from sas=rs-cov-2 infection as only 1.5% pediatric covid-19 cases were reported189. the ro estimations for covid-19 have declared the people of africa vulnerable as most countries have underdeveloped health care systems190. the lack of proper infrastructure, meager access to national health facilities, and congested residential units make “refugees” a highrisk population for sars-cov-2 infection. in bangladesh, one million rohingyan refugees, staying at kutupalongbalukhali expansion site and vicinities, were highly prone to covid-19 throughout the year191. in lebanon, syrian refugee settlements reported poor access to clean water and sanitation. contaminated water and untreated sewage materials can induce self-inoculated infection in masses192. in a framed time, radical decisions for the development of special health care facilities are needed to reduce infectious contacts in these societies. psychological impact of covid-19 natural disasters, terror attacks, and pandemics of the past have shown associations to anxiety, depression, drug addiction, and post-traumatic stress syndrome (ptsd)78. people also suffered anxiety, depression, and fear of stigmatization after the sars and ebola pandemics. cooke et al., reported that one in four adults suffered from covid-19-induced stress. pooled data indicated 23.8% world population-developed ptsd and 24.8% adults suffered from covid-19-associated stress193. the highstress levels were concentrated in epidemiologically infection-dominant countries194,195. patients suffering from covid-19-developed depression, whereas healthy population developed fear of getting sick and suffocated from long-term homestays. hage et al. reported increased risks of mental stress in health care professionals regarding their performance under limited medical resources and transfer of sars-cov-2 infection from hospitals196. the coverage of covid-19 pandemic by media outlets provided by the real-time situation and management of international health crisis. however, the pandemic was global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 107 either understated or exaggerated on social media platforms, which escalated waves of fear among the general public197. on social platforms, the pandemic was followed by infodemic, a situation of prime misinformation and fake news on social media platforms. the situation jeopardized the efforts of national health institutions and induced mental distress in communities198. efforts were made to create awareness for public health and safety regarding social consciousness, and psychological pressure induced by covid-19 in every age group of all countries199. educational impact of covid-19 covid-19 has also put pressure on educational institutions. in order to prevent the transmission of disease in children, public and private schools around the world started offering online classes200. during the initial phase of the pandemic, schools and institutions readily adapted distant teaching and learning mechanisms by offering online classes through google classrooms, zoom, and learning management system (lms)201. during marchaugust, the tech industry played a crucial role in aiding to the online educational activities, yet regional, national, and international communities faced several challenges during the pandemic. the several unresolved hindrances include non-availability of electricity, distorted internet connections, and deprivation of computers and electronic gadgets in low-income families and people living in remote areas. the deprivation of on-campus study has rendered girls’ education inaccessible and created difficulties in achieving sustainable developmental goals of gender equality and women empowerment in developing countries202. a plethora of international students also developed financial and psychological stress in the pandemic. the problems were primarily caused by the cancelation of oncampus classes, restricted movements in the hostel’s accommodation, quarantine, and uncertainty of return to and from their homeland203. in july, international students who resumed research-based studies had shown higher productivity and prioritization of research activities as they had restricted access to available funding186. countries including northern ireland and pakistan postponed examinations204,205. in the third trimester, pakistan reopened schools by adapting strict preventive measures and sops. however, due to a second wave of the pandemic, the shutdown was implemented again to prevent a hike in the number of covid-19 cases in children. provision of basic healthcare facilities and management of outpatients owing to its distinctive origin, exponential transmissions, and rapid spread the focus of medical professionals and researchers shifted to the study of covid-19. during the pandemic, most hospitals restricted visiting hours or completely closed facilities for outpatients. moreover, mental health care facilities were also partially or completely closed. in usa, the cancelation of non-urgent outpatient activities, follow-ups, and operations of cancer patients gave rise to problems regarding management of prior health services166. the decision of closure was also finalized by the managing authorities of the public and private health sectors in infection-prominent regions of france in europe and pakistan in asia206. covid-19 situation has been associated with worse outcomes for diabetes due to the disruptions caused by the pandemic in the diet, care routine, and lifestyle during social restrictions207. in pakistan, italy, and france, physical access to outpatient departments (opd) of hospitals was not approved by authorities. in order to cope with the situation, telemedicine was emphasized, and distant communication between doctors and patients through video-conferencing was held208. covid-19 and healthcare professionals while handling the pandemic, health care providers and medical staff were obligated to wear personal protective equipment (ppe), masks, and gloves to protect themselves from viral transmission through the coughing or sneezing of patients209. in health care professionals, n95 respirators were extensively used to avoid any contamination while providing health care services210. surgical masks have previously successfully hindered the transmission of respiratory viruses, including influenza virus and rhinovirus. they also proved effective as they reduced aerosol and droplet viral contamination of area211. initially, the selection of facial masks (medical or/and surgical) and respirators (n95, p2, and ffp2) aroused concerns regarding maximum protection of healthcare workers, medical and non-medical hospital staffs. the type of mask to be used primarily depended upon the availability global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 108 of stock and preferred choice of workers212. the constant use of n95 respirators by health care professionals, assigned to covid-19 patients provided benefit against covid-19213. however, sars-cov-2-induced healthcareassociated infection in nurses and doctors214. throughout the sustained crisis, inadequate supply of ppe put doctors and nurses at higher risks of contamination and viral threat. i m p a c t o f c o v i d 1 9 o n i n t e g r a t e d g l o b a l e c o n o m y covid-19 induced economic downfall the 20th century marked the era of globalization, which transvers into the 21st century. in the neo-liberal contemporary period, the world economy was highly intercalated and interdependent215,216. the economic repercussions of covid-19 have encompassed the whole world217. the infectious spread of covid-19 has disturbed the businesses, industries, and financial structure of the entire world. economists have declared that the virus is as contagious economically as it is medically. in the first half of 2020, strict national and international policies, including the shutdown of industrial and economic sectors, closed international borders, and delayed trade gave birth to a global financial crisis close to a recession. the global shutdown rendered people jobless increasing the burden on domestic and international economic growth indicators including gross domestic product (gdp), consumer spending and income, industrial production, labor market, inflation, and balance of trade. the disturbance in the export-import cycle has caused inflation in prices of basic commodities like food and medicines217. the pandemic also incurred a burden on the global healthcare sector. in u.s.a., the cost to fight the pandemic for two months was estimated 2.14 trillion dollars218. during sars (2003), global economic loss was estimated 30-100 billion usd219. the initial loss associated with covid-19 has been estimated to $1.1 trillion, compared to $25.2 billion loss in 2014 during ebola virus epidemic. estrada mathematical modeling of economic indicators has concluded that the world economy will eventually be restored by 2025. during the first trimester, in january and february, china reported a peak number of cases of covid-19, resulting in decline of the chinese economy which led to the distortion of global supply chain. in march, the economic recession was shifted to europe and u.s.a., which reported the highest covid-19 cases, and adapted lockdown policies, and shutdown businesses. the second trimester of 2020 sustained the crippled economic events giving rise to inflation, the rise in unemployment rate, and shut down of national revenues across the globe. the chinese authorities eased the lockdown for businesses and industries to recuperate the economic losses. during the third and fourth trimester, countries adapted standard operating procedures (sops) specifically designed for the pandemic and reopened revenuegenerating national sectors including restaurants, recreational places, road and rail transportation, and aviation. irrespective of social distancing measures and sops, a fluctuation in the number of new cases per day was observed in the second half of 2020. overall, covid-19 has put pressure on advanced, developed, developing, and under-developing nations of the world. reduction in national and global gdp the national economy is an important component of the financial growth of a state. stock markets, manufacturing capacity, unemployment rate, consumer price index (cpi), currency strength, gross domestic product (gdp), and trade are important indicators extensively studied to determine the economic standing of a nation and its contribution towards global gdp. thus, global gdp is an economic indicator of world economic progress which is created by the national gdp of the countries across the world. international monetary fund (imf) has estimated a reduction of 3.5% to 4.8% in global gdp in 2020 and an expected rise of 4.9% in 2021 (fig. 5). the top hit countries of the covid-19 pandemic have contributed to 55% global gdp in 2019. these include u.s.a. (24%), china (16%), japan (6%), germany (5%), france, india, u.k. (3%), italy, brazil, and canada (2%). clark has estimated that the growth rate in the gdp of eu may decrease by 7.4% and a forecasted economic recovery in 2021 will be unable to balance the present economic recession created by covid-19 situation220. world economic outlook (weo) have reported a reduction of 3.5% global gdp as compared to pre-pandemic gdp of 2.8%. moreover, the advanced economies u.s.a (-4.9%), canada (-5.5%), spain (-11.1%), u.k. (-10%), italy (-9.2%), france (-9%), germany (-5.4%), japan (-5.1%) contributed significantly global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 109 to the decline in global gdp. the emerging markets and economies india (-8.0%), brazil (-4.5%), and asean (-3.7%) also faced challenges during the pandemic. the economic downfall was prominent in the second quarter of 2020, as states followed lockdown policies across the globe. in u.s.a, during the first quarter of 2020, stalled manufacturing operations resulted in -1.3% gdp. during the second quarter, gdp hit the lowest with -9.0% downfall. in the third quarter, after ease of restrictions and booming of the industrial sector, a positive rise of 7.5% was observed, and in the fourth quarter, additional gdp growth of 1.1% was noticed221. following u.s.a., india reported the highest number of cases in the world. the gdp growth of india observed a rise of 3.1% in the first quarter; however, the country noticed a decline of -23.9% in the second quarter, and -7.5% in third quarter of 2020. the abrupt decline was the manifestation of a nationwide lockdown to control the surge of covid-19 pandemic. india observed positive growth of 0.4% during the fourth quarter of 2020. the economy of japan primarily relies on the manufacturing units, private consumption sector and exports. japan was among the first few countries that reported imported cases of covid-19 in january 2020. the growth of japanese economy contracted in the first quarter (gdp= -0.9%) and the second quarter (gdp= -27.8%) in second quarter of 2020. however, with the growth of private consumption and increased exports, japan regained its economic strength reporting a 21.4% rise in gdp during the third quarter and 9% gdp during the fourth quarter of 2020. crash of stock markets the stock market is one of the leading indicators of the future economic direction of a state. the strength of the stock market provides information about the growth potential and thriving capability of companies and economies222. in the long run, a strong market is associated with high earnings and a prosperous economic situation, whereas a weak market is an indication of future economic decline. the exponential spread of sars-cov2, lack of knowledge, and treatment facilities, and uncertainties associated with the covid-19 sustained during the first quarter of 2020. in order to reduce the transmission of the virus, governments enforced lockdown of commercial markets, macro-businesses and microbusinesses. in the first and second quarter, the fluctuating interests of investors led to covid-19 induced stock market shocks. owing to the demanding needs of technology during the lockdown, global hike in investment in tech companies was observed. however, the stock markets of u.s.a, u.k, japan, and italy disproportionately suffered, and manufacturing companies met aggressive recession. american, european, and asian stock markets plunged with the implementation of strict preventive measures. gormsen and koijen reported a rise in risk levels from 0.0071 to 0.0196 from february to march, establishing a severe negative impact on the global stock market 223. during the pandemic industrial setups, crude oil, and cooperate sector markets generated more loss for investors, whereas gold and food commodities proved safe-haven assets. in the initial days of february, the index points of chinese stock market hit low. during march, the stock market of u.s.a. hit circuit breaker mechanism and chicago stock market hit its lowest peak during 9th to 16th march, 2020222. u.s.a. stock market reported an intense reduction in equity values of petroleum, real-estate, entertainment, and hospitality divisions, while it noticed high returns for natural gas, food, healthcare, and software stocks221. following u.s.a., u.k. also reported a 10% decline in index points on march 12, 2020. the japanese stock market recorded a 20% loss in march. during the lockdown period, the asian stock markets of singapore and south korea did not report such fluctuations as compared to american and european stock markets221. covid-19 had a short-term negative impact on stock markets of countries marked by advanced and progressive industrial sectors such as china, italy, south korea, spain, germany, japan, and u.s.a224. in the third quarter of 2020, japanese stock markets reported 71% stock returns, strengthening its position as a major economic center225. these stock markets gradually revived after the uplift of the lockdown and ease in social parameters for the containment of the virus. global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 110 figure 5. reduction in gdp of 227 countries reported by imf. impact on international supply chain contagion and market disruption the manufacturing sector of any state is an indicator of a flourishing economy. this establishment plays a significant role in providing services and products to the customers. it is also associated with the rate of employment; the bigger the manufacturing capacity is, the more workers will be required to operate it successfully. the financial impact of the reduced or stalled manufacturing sector is amplified globally through international supply chain contagion. supply shocks in one industry of a country are transmitted to another industry of another country when the output of the first is an input of the latter. in addition to the stalled manufacturing process, the non-interest of investors and compromised purchasing power of domestic and international customers affect the production sector severely when compared to the service sector226. the impact of covid-19 on the production sector differentiates itself from other natural disasters (earthquakes, tsunamis, and flooding) where loss incurred can be estimated by analyzing physical damage227. u.s.a., china, germany, japan, britain, india, france, and italy have been responsible for 65% of the world’s manufacturing in 2019 and are important members of the international supply chain228. in the first quarter of 2020, the shutdown of chinese industrial sector created a void in the global supply chain, which was filled by india, vietnam, south korea, and japan. during january to february 2020, chinese industrial production reported a decline of 13.5%. the most negatively impacted production sectors included transport equipment (-28%), general equipment (-28%), textiles (-27%), and machinery (25%)229. during januaryfebruary, the purchasing managers index (pmi) of the chinese manufacturing department declined significantly, whereas, during march-may the pmi of other dominant manufacturing countries reported significant reduction as the production sector was shuttered down226. the hyper-specialization of the european industrial sector faced the challenges of domestic production of medical equipment, as the shortage from chinese suppliers intensified during the pandemic229. the automobile industry of italy specializes in the production of automobile parts, suffered financial losses during the pandemic from foreign purchases that were interrupted due to trans-national borders shut down. the loss of active income impacted domestic household incomes and the healthcare system of italy, reducing gdp to -9%. japan, china, south korea and taiwan are the major exporters in the information and technology communication industries and collectively contribute to 50% of imports of u.s.a. hubei province is the hub of electronics and optical fiber industry, caused a global reduction of 10% in smartphones production and shippment226. during the pandemic, researchers and policy makers supported the strengthening of resilience, and global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 111 sustainability of supply chains in domestic and international arena to cope with covid-19-induced disruptions in the markets228. impact of covid-19 on global oil and petroleum demand owing to the covid-19 mitigation policies, shutdown of production units, manufacturing industries reduced global demand for oil. this incurred economic loss of oil and petroleum exporting countries, particularly saudi arabia, kuwait. initially, the brent oil prices dropped to $20 per barrel to the previous rate of $69 in 2019226. the reduced oil demand directly impacts the industrial and aviation sector, it also affects the value of the stock market. the crude oil supply and demand shocks to affect the stock market sector and energy-intensive sectors. the restricted international travels and closure of the aviation industry further reduced the extraction of oil and refining activity. before covid-19 the economic boom in china made it the largest importer of crude oil. the pandemic had risen the risk of spillover between global and chinese crude oil futures230. prior to the widespread sars-cov-2 infection, the us was already facing an oil price slump. the covid19 situation further added to the oil price shocks and sent a blow to the usa economy231. arezki and nguyen highlighted the fact that the export and recovery of global oil prices in the international market depending on the control of pandemic in china, usa, and germany. impact of covid-19 on other sectors of revenue covid-19 induced a negative impact on the tourism and transport industry232. albulescu proved that the sustainment of the covid-19 situation in the first half of 2020 had a negative impact on us financial volatility and disturbed global financial cycle, as us is the biggest economy in the world232. during the first wave of covid19, the sales of agricultural commodities reduced by 20% because of implementation of public safety laws. panic buying and mismanagement of food sources caused nonavailability of vegetables, fruits, and meat233. the tourism sector reported increased revenue after the rise of globalization and became a significant sector of economic growth. according to the 2016 statistical data, 9.5% of the european workforce has been in earning through the tourism sector and generated revenue of 2.4 million dollars. they faced challenges of sustenance of businesses during covid-19 pandemic owing to national and international travel restrictions. tourism contributed to 4.2% of the gdp of usa in 2019 and 1.9% of the gdp of egypt in 2019. covid-19 incurred irreversible revenue losses to egyptian peninsula. the impact of restrictions on air travels and international air transport association has been estimated to reduce global revenue by 44%. the global travel business association reported that the business travel sector would lose $820 billion in revenue due to the coronavirus pandemic. the airline business hit the lowest during march, when sudden flights cancellation was devised by authorities and some businesses reported to exist on the verge of bankruptcy including u.k airline flybe. the global film industry incurred a $5 billion loss during the coronavirus outbreak. the us higher education sector reported a loss of $600billion, with the closure of oncampus activities. the fear of lack of investments in nonbanking firms leading to insolvencies and bankruptcies was significantly mitigated by the country’s government policies for economic emergencies and relief packages. foreign direct investment (fdi) is an important indicator of economic development. before the global recession of 2008, an fdi influx us $ 1971 billion in 2007 was reported, the highest amount of last decade, was hit hard as most funds were utilized to assist health care programs and vaccine development against sars-cov-2. goodell further elaborated the eminent economic consequences of covid-19 on domestic banks, health care management finances, and financial markets227. overall, the diversion of funds for the management of health care facilities and resources during highly prevalent infections have burdened the economic capacity of middle, low-middle, and low-income countries, as was observed in the case of hiv227 234. i m p a c t o f c o v i d 1 9 o n e n v i r o n m e n t positive impact of covid-19 on environment most countries tried to control the expansion of sars cov2 by implementing lockdown, social distancing measures, strict traffic restrictions, and self-quarantine measures. these measures reduced air pollution in brazil and lowered the concentrations of nitrogen dioxide, carbon dioxide, carbon monoxide, and particulate matter that have a global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 112 diameter of less than 2.5μm or less than 10μm235. the lockdown in china also caused 30% to 50% reduction in the levels of air pollutants nox, pm2.5, pm10, and so2236. the study of tropospheric no2 in east china using ozone monitoring instrument (omi) and tropospheric monitoring instrument (tropomi) revealed significant reduction in concentration of no2237. lockdown during covid-19 caused a great drop in the global consumption of oil and coal, and subsequent decrease in air pollution. the air quality of cities improved due to limited use of vehicles. new york reported 50% and china observed a decrease of 25% in the emission of greenhouse gases238. european countries, including spain, italy and u.k. reported decreased emissions of nitrogen dioxide239. the lockdown also resulted noticeable reduction in water bodies, canals, and rivers in urban areas. harmful impact of covid-19 on environment the adverse effect of covid-19 on the environment is also a major concern. the healthy and safe air and water environment are of great importance for life on earth239, 240. sars-cov-2 resulted in an increased load of medical, inorganic, and plastic waste241. extensive use of face masks generated 129 billion waste face masks that are the most noticeable item responsible for polluting ocean water and threatening the life of marine animals241. the disposal of medical waste gave rise to a challenging problem as transport and disposal infrastructure needed to incorporate large quantities of waste in china, italy, france, and netherlands. on february 2020, wuhan produced 200 tons of medical waste which were four times the waste it produced in pre-pandemic situation238. the use of biocidal agents, soaps and simple water used in washing of contamination material possessed threat to the sanitation system. several treatment technologies have been introduced for pharmaceutical, domestic and industrial wastewater242. the european union classified healthcare waste generated during the pandemic as highly infectious and philippines built a disposal facility on the island of luzon to handle covid-19 induced medical waste238. ppe waste in bangladesh reported disastrous and its management was very challenging243. moreover, in order to manage medical waste in china, u.s.a and south korea, on-site incinerators, mobile incinerators, and autoclave systems had been provided to manage health care waste and ensure safety of health care professionals238. c o n c l u s i o n s sars-cov-2, with a zoonotic lineage and transmission ability via human contact, and environmental contamination, causes respiratory ailments in humans. infected individuals may stay asymptomatic or show mild and severe symptoms with disease progression. the first wave of covid-19 was successfully mitigated by adaptation of social distancing, quarantine, strict lockdown, and non-pharmaceutical hygienic measures, contact tracing and regular temperature checkups. the economic fatality of the sars-cov-2 induced pandemic could not be denied and its impact on the financial burden of advanced, middle-income, lower-middle-income, and low-income countries was handled through the respective government’s preparedness and management policies. during the first half of the 2020, ongoing therapeutic clinical trials and production of vaccine pipelines were the main events. during the second half of the 2020, the ease of lockdown policies alleviated the social and economic stress, and establishment of herd immunity in masses, and functioning of industrial sectors under sops were the main events. the second wave of covid-19 pandemic and new variants of sars-cov-2 which were initiated during october and november resurfaced the global concern against the covid-19. during the year, international organizations, who, world trade organization, international monetary fund, g20, and asia development bank provided monetary packages. the assistance provided by a health care professional, researchers, testing facilities, and governments’ policies helped combating the second wave of pandemic observed with ease of restrictions globally. overall, the challenges posed by covid-19 have been so far and could only be handled through public awareness, public safety measures, and sufficient allocation of funding for vaccine development. the progression of a pandemic will persist in the following year, and the productivity of the global community will highly depend on vaccines-driven social and economic makeup. global overview of sars-cov-2 induced covid-19 in 2020 vol. 13 (1), june 2022 issn (print): 2305 – 8722 issn (online): 2521 – 8573 r a d s j . b i o l . r e s . a p p l . s c i . 113 c o n f l i c t o f i n t e r e s t the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article. f u n d i n g s o u r c e none. a c k n o w l e d g m e n t s the authors wish to acknowledge faculty fellowship program brain pool fellowship program through the national research foundation of korea (nrf) funded by the ministry of science and ict (grant no.: 2019h1d3a1a02071191) under which the corresponding author is serving. l i s t o f a b b r e v i a t i o n s ards acute respiratory distress syndrome blf bronchoalveolar lavage fluid crispr clustered regularly interspaced short palindromic repeats ctd c-terminus domain cpap continuous positive airway pressure covid-19 coronavirus disease-2019 cq chloroquine gc glucocorticoids ddp gross domestic product hcv hepatitis-c virus he hemagglutinin-esterase imf international monetary fund mers middle eastern respiratory syndrome nsps non-structural proteins ntd n-terminal domain opd outpatient department orf open reading frames pcr polymerase chain reaction ptsd post-traumatic stress syndrome sard-cov-2 severe acute respiratory syndromerelated coronavirus-2 sp signal peptide who world health organization r e f e r e n c e s 1. chen n, 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