chauhan & agarwal nootropics: boon or bane vol 1 | issue 2 | jul – sep 2022 indian j pharm drug studies | 71 letter to editor piracetam induced psychotic symptoms in a preadolescent girl with intellectual disability nidhi chauhan1, sonali agarwal2 from, 1department of psychiatry, postgraduate institute of medical education and research, chandigarh, 2junior resident, department of psychiatry, government medical college & hospital, chandigarh, india correspondence to: nidhi chauhan, assistant professor, department of psychiatry, postgraduate institute of medical education and research, chandigarh, india. email: dr.nidhichauhan@gmail.com iracetam, a nootropic agent, is believed to improve higher brain functions, like learning & memory justifying use in individuals with intellectual disability. it has a gaba-mimetic action, activating ampa type glutamate receptors and its uses extend beyond the nootropic effects. it’s commonly used as an off-label drug, usually prescribed for learning difficulties, poor memory and speech disorders [1]. due to its purported effects as intelligence enhancers/memory boosters, nootropics are also referred to as ‘smart drugs’ and are construed as wonder drugs with ‘only beneficial effects’ and no side effects [2]. literature reports that this is a serious public health concern similar to that associated with anabolic steroid use [2]. since, piracetam crosses blood brain barrier, its central nervous system stimulatory effectspsychomotor agitation, aggression, irritability, dysphoria, sleep disturbances, headache, decreased appetite are reported in literature [3,4]. in this background, we discuss the case of a girl with intellectual disability who developed auditory and visual hallucinations in addition to cns adverse effects on prescription of piracetam. till date, to the best of our knowledge no such case with piracetam induced psychotic symptoms has been reported. an 11 years old girl was admitted to the psychiatry emergency room for recent onset change in behaviour i.e., irritability, agitation and hallucinatory behaviour. history exploration and assessments revealed that she had a preterm birth, delayed cry, delayed development in all domains. she had learning difficulties since early childhood but no help was sought. in may 2021, for persisting academic difficulties and poor academic performance, parents consulted a private pediatrician who prescribed her syrup piracetam (syrup noofit 500 mg/ 5 ml) 500 mg bd. within 5-6 days of its initiation, she was observed to remain fearful, complained of hearing and seeing things which others could neither see nor hear, was agitated and confused, with sleep and appetite disturbances. attributing these behaviors due to the new drug started, parents discontinued it. within next 45 days, improvement was observed in agitation, confusion, hallucinatory behaviour and sleep. parents restarted syrup piracetam but this was associated withreemergence of earlier symptoms. they again discontinued piracetam following continued worsening for 5-6 days and brought her to psychiatry emergency services. no history of seizures, other neurodevelopmental disorders, substance use, skin rash (porphyria), drug use (steroids) was obtained. mental status examination revealed, confused state, increased psychomotor activity, perseveration, echolalia, palilalia and impaired attention-concentration. naranjo algorithm score was 5 indicative of probable drug indued adverse effect. a provisional diagnosis of piracetam induced psychotic symptoms with moderate intellectual disability was kept. she was admitted and kept drug naïve. investigations (serum lactate, urine for ketones/ porphyrins, eeg, mri) did not reveal any abnormalities. she improved within 4-5 days of ward stay and within a week was her premorbid self. the family members were educated about intellectual disability, and that medicines/supplements would not improve her memory, rather constant practice in doing things will bring about some change. she was discharged in a satisfactory condition with attainment of premorbid level of functioning. the index case highlights indiscriminate use of piracetam for intelligence enhancer/ memory booster effect in a girl with intellectual disability despite literature reporting thatpiracetam therapy does not improve cognitive function rather is associated with adverse effects.4 it is approved for use in europe for treatment of seizure disorder; however, it is not us fda approved for any indication and its sale as a dietary supplement is also prohibited [5]. despite having insufficient evidence to establish its safety, piracetam is available in markets and being used as off label/over the counter drug [6]. its use in dose of 500 mg twice/day (higher range of usual dose 40 mg/kg/d; body weight 27 kg) in a girl with already compromised brain functions exposed her to the unwarranted cns adverse effects, to the extent that she experienced visual and auditory hallucinations in addition to irritability, p mailto:dr.nidhichauhan@gmail.com chauhan & agarwal nootropics: boon or bane vol 1 | issue 2 | jul – sep 2022 indian j pharm drug studies | 72 psychomotor agitation, confusion, sleep and appetite disturbances which are reported in literature. it was further supported by a score of 5 in naranjo algorithm scale for adverse drug reactions. cns adverse effects are explained on the basis of its neuroexcitatory effect on ampa receptor induced calcium influx, thus increasing maximal density of ampa glutamate and acetylcholine receptors and potassium induced glutamate release in the brain. it has also been postulated to have a role in increasing the availability of oxygen, permeability and fluidity of the mitochondrial cell membrane in the intermediate stages of the krebs cycle, increasing activity of adenylate kinase, thus increasing atp production in the brain. [7]. adverse effects like anxiety, insomnia, agitation, irritability is identical to symptoms of excess acetylcholine/glutamate neuroactivity as seen in one of the previous case reports8 and are in sync with the index case. clinical research on clinical use of nootropics is yet inconclusive. however, the use of nootropics to treat cognitive problems among healthy as well as in children with learning difficulties/ memory disturbances/ poor academic performance cannot be prevented. casual, unsupervised, nonmedical use of nootropic drugs to improve academic performance could have unanticipated negative mental health effects. health professionals need to be cognizant of the fact that nootropics may be to the human mind what steroids are to the body (misuse/abuse for muscle building, improving performance/physical shape despite dverse physical & psychological effects) and thus promote judicious use. references 1. donma mm. clinical efficacy of piracetam in treatment of breathholding spells. pediatrneurol 1998;18:41-5 . 2. canterbury rj, lloyd e. smart drugs: implications of student use. j primprev 1994;14:197-207. 3. winblad b. piracetam: a review of pharmacological properties and clinical uses. cns drug rev 2005;11:169-82. 4. lobaugh nj, karaskov v, rombough v, rovet j, bryson s, greenbaum r et al. piracetam therapy does not enhance cognitive functioning in children with down syndrome. arch pediatradolesc med 2001;155:442-8. 5. wong c. what you should know about piracetam. said to enhance memory and cognition, this drug is not approved by fda. holistic health 2021 https://www.verywellhealth.com/getsmart-withpiracetam-89499. 6. u.s. food and drug administration: office of nutritional products, labeling and dietary supplements. 75-day premarket notification of new dietary ingredients: piracetam. updated march 3 2004 https://www.fda.gov/food/new-dietary-ingredientsndinotification-process/submitted-75-day-premarketnotificationsnew-dietary-ingredients. 7. colucci l, bosco m, rosario ziello a, rea r, amenta f, fasanaro am. effectiveness of nootropic drugs with cholinergic activity in treatment of cognitive deficit: a review. j exppharmacol 2012;4:163-72. 8. talih f, ajaltouni j. probable nootropicinduced psychiatric adverse effects: a series of four cases. innovclinneurosci 2015;12:21-5. how to cite this article: chauhan n, agarwal s, piracetam induced psychotic symptoms in a preadolescent girl with intellectual disability. indian j pharm drug studies. 2022;1(2) 71-72. funding: none conflict of interest: none stated https://www.verywellhealth.com/get-smart-withpiracetam-89499 https://www.verywellhealth.com/get-smart-withpiracetam-89499 https://www.fda.gov/food/new-dietary-ingredients-ndinotification-process/submitted-75-day-premarketnotifications-new-dietary-ingredients https://www.fda.gov/food/new-dietary-ingredients-ndinotification-process/submitted-75-day-premarketnotifications-new-dietary-ingredients https://www.fda.gov/food/new-dietary-ingredients-ndinotification-process/submitted-75-day-premarketnotifications-new-dietary-ingredients bhoge et al. pharmacological importance of benzothiazole lead vol 1 | issue 1 | apr jun 2022 indian j pharm drug stud | 15 review article a comprehensive review on synthetic strategy of benzothiazole lead and pharmacological importance bhoge nd1, mohite pb2, deshmukh vk2, magare bk3 from, 1mula education society’s shri dnyaneshwar mahavidyalaya, newasa, ahmednagar, maharashtra, 2mula education society’s college of pharmacy, sonai talnewasa, distahmednagar maharashtra, 3shivaji arts, commerce and science college, kannad, aurangabad, maharashtra, india correspondence to: n.d. bhoge, shri dnyaneshwar mahavidyalaya newasa talnewasa, dist. ahmednagar, maharashtra, india. email: nitinbhoge4550@gmail.com abstract background: in recent years heterocyclic compounds analogues and derivatives have attracted wide attention due to their useful biological and pharmacological properties. benzothiazole is among the usually occurring heterocyclic nuclei in many marines as well as natural plant products. benzothiazole is a privileged bicyclic ring system with multiple applications. objective: to review and explore the synthetic strategy of lead and pharmacological importance of benzthiazole derivatives. materials and methods: a literature search was conducted on various database sources (like pubmed, science direct) with the help of a combination of different keywords: benzothiazole, thiazole, antitumor, anti-inflammatory activity and anti-convulsant, antioxidant, antimutagenic, anti-diabetic, anti-hyperplasia, and antimicrobial. the search was customized by applying the appropriate filter to get the most relevant articles to meet the objective of this review. conclusion: it is known to exhibit a wide range of biological properties including anticancer, antimicrobial, and anti-diabetic, anticonvulsant, anti-inflammatory, and anti-viral, anti-tubercular activities. a large number of therapeutic agents are synthesized with the help of benzothiazole nucleus. during recent years there have been some interesting developments in the biological activities of benzothiazole derivatives. these compounds have special significance in the field of medicinal chemistry due to their remarkable pharmacological potentialities. this review is mainly an attempt to present the research work reported in the recent scientific literature on different biological activities of benzothiazole compounds. key words: benzthiazole, anticancer, anticonvulsant, anti-inflammatory, antimicrobial etc. antzsch and waber first described thiazolein 1887 and its structure confirmed by popp in 1889. in thiazole, moiety numbering starts from the sulfur atom. the basic structure of benzothiazole is the combination of a benzene ring fused with 4, 5 positions of thiazole. benzothiazole is one of the most important aromatic heterocyclic compounds having a chemical formula c7h5ns.it plays a very important role in medicinal chemistry as well as in organic chemistry. today`s most of the drug molecule contains a benzothiazole as an important scaffold and because of this scaffold, it has therapeutic activity. benzothiazole and its derivatives have tremendous applications in synthetic chemistry as well as in pharmaceutical chemistry because of its unique structure, potent and significant pharmacological activities. benzothiazole is a combination of two rings six-member mono atomic and five-member hetero atomic and both rings are responsible for the therapeutic activity.[1] (fig.1) benzothiazole analogues have also been screened as potential therapeutical applications which include antiviral, anti-microbial, anti-diabetic, anti-cancer, antiinflammatory, anti-oxidant, anti-fungal, anti-psychotic, anti-leishmanial etc [2]. because of amyloid-binding property derivatives of 2aryl benzothiazoles are screened for radioactive imaging moieties in neurodegenerative disorders [3, 4]. fig.1 structure of benzothiazole material and methods a literature search was conducted on various database sources (like pubmed, science direct) with the help of a combination h bhoge et al. pharmacological importance of benzothiazole lead vol 1 | issue 1 | apr jun 2022 indian j pharm drug stud | 16 of different keywords: benzothiazole, thiazole, antitumor, anti-inflammatory activity, and anticonvulsant, anti-oxidant, anti-mutagenic, anti-diabetic, anti-hyperplasia, and antimicrobial. the search was customized by applying the appropriate filter to get the most relevant articles to meet the objective of this review synthetic and biological active strategy of benzothiazole derivatives many researchers are using various methodologies. benzothiazole derivative can be synthesized by using conventional, microwave method or by other green chemistry method. in this review article we are focusing on recent methods which are available for the synthesis of benzothiazole derivatives and their biological activities. hebishy synthesized a bisand poly (benzimidazoles) and benzothiazoles derivatives by using corresponding bisand poly (aldehyde) with ophenylenediamine or 2aminothiophenol, respectively, in ethanol at boiling temperature in presence of sodium hydrogen sulphate, further these synthesized derivatives were screened for anticancer activities against human breast adeno carcinoma cell line (mcf-7), liver cancer cell line (hepg-2), and epithelial colorectal adeno carcinoma cells (caco-2). but among all derivatives hexakis (benzothiazole) showed a highest activity against hepg-2 cell lines with ic50 values of 21.16 and mcf7 cell lines with ic50 values of 13.25 μm [5]. racanéa et al., reported about a synthetic method for the synthesis of benzothiazole based derivative and most of the derivatives showed cytotoxic activity which performed using the mtt assay method. compounds 2a, 2b, 2c, and 2d (fig. 2) were then selected for examining their in vitro enzyme inhibitory activities against egfr, her2 and ts enzymes using lapatinib and 5fu as standards. furthermore, cell cycle analysis and apoptosis induction detection were also evaluated [6]. fig.2 compounds chosen for mtt assay asatkar et al., reported that the aqueous extracts of rice husk derived chemically activated carbon acts as a catalyzed for the synthesis of benzothiazole, benzoxazole and n-benzimidazole in room temperature by converting 2-aminothiophenol, 2amino phenol and ortho-phenylene di-amine with aromatic aldehydes with 98%yeild [7] (fig. 3) fig. 3 synthesis of benzothiazole, benzoxazole and nbenzimidazole from rice husk a series of novel pyrido [2, 1-b] benzothiazole and pyrido [2, 1-b] benzoimidazole derivatives were synthesized by reacting with n-aryl-2-cyano-3,3-bis (methylthio) acrylamide with benzothiazole acetonitrile and benzoimidazole acetonitrile, respectively, while nsubstituted 2-pyridyl-benzothiazole derivatives were synthesized by reacting 2benzo [d] thiazol-2yl) -3-(dimethylamino) acrylo nitrile with either cyanoacetamide, aryl cyanoacetamides or 2-cyano-n'(4substitutedbenzylidene) acetohydrazide (azzam et al). furthermore, synthesized compound tested for fluorescence measurements, the compound n-substituted 2 pyridylbenzothiazole derivatives shows fluorescence properties with high quantum yields upto 0.29. the antimicrobial study of synthesized compound revealed that triazolo-pyridone had the highest potency among all tested compounds against escherichia coli, klebsiella pneumonia and staphylococcus aureus while pyrido [2, 1-b] benzoimidazole derivatives 4a and 4b had the highest potency over other compounds against candida albicans [8] (fig.4). fig.4 (3) triazolo-pyridone, (4) benzoimidazole derivatives 4a and 4b with more efficacy against candida albicans ermis et al described the synthesis of a series of thiophenebenzothiazole derivative amine compounds in good yields by bhoge et al. pharmacological importance of benzothiazole lead vol 1 | issue 1 | apr jun 2022 indian j pharm drug stud | 17 reduction of corresponding imine derivative which is obtain from aromatic aldehyde and 2aminobenzothiazole by microwave reaction. the characterizations of all compounds were performed by ftir, 1h and 13c nmr and single crystal x-ray diffraction method [9] (fig.5). azzam et al. prepared a series of n-sulfonamide-2-pyridone derivatives which contains benzothiazole moiety via the reaction of benzothiazole sulfonyl hydrazide with sodium salts of both (hydroxymethylene) cycloalkanones and unsaturated ketones, as well as) hethoxymethylene derivatives. the prepared compounds were screened for in vitro for their antiviral activities against the hsv-1, havhm175, hcvcc genotype 4, cbv4, and hadv7 viruses. among prepared compound, five compounds were found to possess viral reduction of 50% or more against cbv4 with significant ic50, cc50, and si values. in the case of hsv-1 and hav hm175 viruses, three compounds have shown more than 50% reduction, while in the case of hcvcc genotype 4 and hadv7 viruses, only two compounds demonstrated more than 50% reduction. the two most potent compounds against hsv-1 virus, 6a and 6b, were evaluated for their inhibitory activity against usp7 [10] (fig. 6). a series of 2-pyrimidylbenzothiazoles derivatives containing amino or sulfonamide moieties at the c2 position of the pyrimidine ring were synthesized by azzam et al. by subsequent reaction of guanidine or naryl sulfonated guanidine with different derivatives of ylidene benzothiazole. the newly synthesized compounds were evaluated for their antiviral activity against hsv-1, cob4, hav hm 175, ed43/sg-feo (vyg) replicon of hcv genotype 4a, and hadv7. nine derivatives were shows high cytotoxicity concentration and more than 50% viral reduction. another five compounds against hsv-1 have been also evaluated against hsp90α with their activities compared to that of the reference drug acyclovir. the five potent compounds 7a, 7b, 14b, 14g, and 14h against hsv-1 have also presented inhibitory activity against the hsp90α protein with ic50 in the range of 4.87−10.47 μg/ml[11] (fig.7). fig. 7 synthesis of 2-mercapto -benzazole derivatives a green protocol given by vessally et al. for the synthesis of 2-mercapto-benzazole derivatives 9a by using low-cost and nontoxic potassium thiocyanate in water. the reactions proceeded smoothly under catalystand ligandfree conditions to give the corresponding products in good to excellent yields [12] (fig.7). abdelgawad et al. reported a synthesis of new series of benzothiazole/benzoxazole and/or benzoimidazole substituted pyrazole derivatives 10, 11 and 12 and it were evaluated for anti-proliferative agents. this work revealed that substituting pyrazole ring with an acetyl or phenyl moiety at n-2 of the pyrazo linone nucleus as shown in the target candidates 11 and 12 increased the anti-proliferative activity of these compounds comparing with unsubstituted derivatives 10. moreover, 2acetyl-4[(3-(1h-benzimidazol-yl) phenyl] hydrazono-5methyl2,4-dihydropyrazol-3-one (11) was the most active compound against both mcf-7 and a549 cell lines with half maximal inhibitory concentrations (ic50) = 6.42 and 8.46 lm, respectively [13] almehmadi et al. synthesized a novel macromolecule encompassing benzothiazole1, 2, 3-triazole containing hydrazone. the synthesized compounds were evaluated for anticancer activities with a549 and h1299 lung cancer cell bhoge et al. pharmacological importance of benzothiazole lead vol 1 | issue 1 | apr jun 2022 indian j pharm drug stud | 18 lines. the anticancer activities ranged from 55 to 90%. dna binding study was also carried out to see the mechanism of action and the dna binding constants were of good value ranging from of 2.0 ×10 5 and 14.7 ×10 5 m −1; indicating good interactions of the reported molecules with dna. it was observed that compound 13, 14, 15 and 16, 17 &18 were quite good active as anticancer drugs [14] (fig.9) asgarshamsi et al. were performed a reaction between para hydroxyl benzaldehyde and amino benzothiazole derivatives. the synthesized derivative was further evaluated for scavenging activity. various electronic and energetic descriptors such as high occupied molecular orbital and low unoccupied molecular orbital energy gaps, bonding dissociation enthalpy of oh bond, ionization potential, electron affinity, hardness, softness, and spin density of the radical and neutral species were calculated. ft calculations with b3lyp hybrid functional and 6-311++ g** basis set in the polarizable continuum model were utilized to obtain these descriptors. ascorbic acid showed the best dpph scavenging activity. but 19th band showed a promising anti-oxidant activity. the values of ehomo for 19a and 19b were closer to zero, thus, they showed the best scavenging activities. dadmal et al. reported the synthesis of a series of new triazole and isoxazole linked benzothiazole derivatives were synthesized. anticancer evaluation against two different cell lines revealed that all these derivatives showed significant anticancer activity against hela and a549 cell lines. fig. 8 novel macromolecules encompassing benzothiazole fig. 9 para hydroxyl benzaldehyde and amino benzothiazole derivative showing antoxidant activity reviewing these recent advances in benzothiazole derivative we will go for synthesis of some novel benzothiazole derivatives and synthesized compound will be evaluated for possible biological activities such as anti-fungal, anti-bacterial, anti-viral, anti-microbial, anti-diabetic, anticancer, anti-inflammatory, antioxidant etc. fig. 10 new triazole and isoxazole linked benzothiazole derivatives highly effective compounds (20a-b and 21a-c) have shown less than 4.5 μm concentration in their ic50 values on human cervical (hela) and lung (a549) cancer cell line and these compounds inducing apoptosis to cancerous cells through caspase dependent apoptotic process via the mitochondrial pathway. from these study compounds 21a-chas identified as most promising compounds which show higher cytotoxicity in human cervical and lung cancer cell line than the other compounds and can be taken up for further in vivo cancer studies that may be of interest in cancer chemoprevention [16] (fig.10). conclusion the present review highlights the use of benzothiazole moiety as a template for development of newer therapeutic agents. biological properties of the nucleus include anti-cancer, antidiabetic, analgesic, anti-inflammatory and antimicrobial. with proper designing and structure activity relationship studies of known benzothiazole, prospective compounds can be designed and synthesized for a variety of biological activities. benzothiazole scaffold is a versatile and multifunctional molecule which possess therapeutic effect in various disease like cancer, diabetes. references 1. srivastava a, mishra p, chandra s, et al. benzothiazole derivative: a review on its pharmacological importance towards synthesis of lead,” int. j. pharm. sci. res.2019; 10(4):1553–66. 2. ali r and siddiqui n “biological aspects of emerging benzothiazoles: a short review,” j. chem. 2013. 3. henriksen g, hauser ai, westwell ad, et al. metabolically stabilized benzothiazoles for imaging of amyloid plaques. j med chem. 2007; 50:1087-9. 4. chhabra m, sinha s, banerjee s, et al. “an efficient green synthesis of arylbenzothiazole analogues as potent antibacterial and anticancer agents,” bio. med. chem. lett.2016; 26(1):213– 217. bhoge et al. pharmacological importance of benzothiazole lead vol 1 | issue 1 | apr jun 2022 indian j pharm drug stud | 19 5. hebishy ams, abdelfattah ms, elmorse, et al. “synthesis of novel bisand poly (benzimidazoles) as well as bisand poly (benzothiazoles) as anticancer agents. j heterocycl. chem.2020; 57(5): 2256–2270. 6. racané l, ptiček l, fajdetić g, et al. green synthesis and biological evaluation of 6-substituted -2-(2-hydroxy/methoxy phenyl) benzothiazole derivatives as potential antioxidant, antibacterial and antitumor agents. bio. chem. 2020; 95:103537. 7. asatkar a, lambat tl, mahmood s, et al “facile protocol for the synthesis of benzothiazole, benzoxazole and n-benzimidazole derivatives using rice husk derived chemically activated carbon,” mater. today proc. 2019; 29:738–742. 8. azzam ra, elgemeie gh, oman rr. “synthesis of novel pyrido [2,1-b] benzothiazole and n-substituted 2-pyridyl benzothiazole derivatives showing remarkable fluorescence and biological activities,” j. mol. struct.2020;1201:127194. 9. ermiş e, durmuş k. novel thiophene-benzothiazole derivative azomethine and amine compounds: microwave assisted synthesis, spectroscopic characterization, solvent effects on uv–vis absorption and dft studies. j mol struct. 2020; 1217: 128354. 10. azzam ra, elboshi ha, elgemeie gh, “novel synthesis and antiviral evaluation of new benzothiazole-bearing sulfonamide 2-pyridone derivatives as usp7 enzyme inhibitors,” acs omega. 2020; 5(46):30023–30036. 11. azzam ra, osman rr, elgemeie gh. “efficient synthesis and docking studies of novel benzothiazole -based pyrimidine sulfonamide scaffolds as new antiviral agents and hsp90αinhibitors,” acs omega. 2020; 5(3):1640–1655. 12. vessally e, monfared a, eskandari z, et al. “a new strategy for the synthesis of 2-mercaptobenzazole derivatives by green chemistry metrics,” phosphorus, sulfur silicon relate. elem.2021; 196 (1):1–5. 13. abdelgawad ma, bakr rb, omar ha. “design, synthesis and biological evaluation of some novel benzothiazole/benzoxazole and benzimidazole derivatives incorporating a pyrazole scaffold as antiproliferative agents,” bioorg. chem. 2017; 74: 82– 90. 14. almehmadi ma, aljuhani a, alraqa sy, et al. design, synthesis, dna binding, modeling, anticancer studies and dft calculations of schiff bases tethering benzothiazole-1,2,3 trizoleconjugates. j mol strct. 2021; 1225: 129148. 15. asgarshamsi m, fassihi a, hassanzadeh f, et al. “synthesis, antioxidant activity, and density functional theory study of some novel 4[(benzo[d]thiazol-2-ylimino) methyl] phenol derivatives: a comparative approach for the explanation of their radical scavenging activities. res pharm sci.2021;16 (1):35–47. 16. dadmal tl, appalanaidu k, kumbhare rm, et al. “synthesis and biological evaluation of triazole and iso-oxazole tagged benzothiazole/ benzoxazole derivatives as pontent cytotoxic agents. new j. chem. 2018; 42(19):15546–15551. how to cite this article: bhoge nd, mohite pb, deshmukh vk, et al. a comprehensive review on synthetic strategy of benzothiazole lead and pharmacological importance. indian j pharm drug stud. 2022; 1(1): 15-19. funding: none; conflict of interest: none stated shaikhmulani et al. rp-hplc method development and validation of ben.hcl & ctd vol 1 | issue 3 | oct apr 2022 indian j pharm drug studies | 102 original article rp-hplc method development and validation for simultaneous estimation of benidipine hydrochloride and chlorthalidone in pharmaceutical dosage form hasan i shaikhmulani, ashpak m tamboli, naziya a tamboli, rohan t kshirsagar, harshvardhan t suryawanshi, omkar khandare from, department of pharmaceutical chemistry, sahyadri college of pharmacy, sangola, solapur, maharashtra, india correspondence to: hasan i shaikhmulani, department of pharmaceutical chemistry, sahyadri college of pharmacy, methwade, sangola, solapur, maharashtra, india 413307 email: shaikhmulani9881@gmail.com abstract a simple, precise, rapid and accurate reverse phase high performance liquid chromatography (hplc) method was developed for the estimation of benidipine hydrochloride and chlorthalidone in bulk and tablet dosage form. c18 c-18 agilent zorbax bonus – rp (250 x 4.6 mm, particle size -5 micron) was used in this method. the mobile phase comprises of methanol and 0.1% orthophosphoric acid (45:55v/v) with flow rate 1 ml/min (photodiode array detector). the retention time for benidipine hydrochloride was 1.09 min and for chlorthalidone was 3.52 min. the detection concentration was linear over 32-48 µg/ml for benidipine hydrochloride and 100-150 µg/ml for chlorthalidone. the regression equation of benidipine hydrochloride and chlorthalidone were found to be y = 50184x30070 and y = 37102x-74642 respectively with regression coefficient of benidipine hydrochloride and chlorthalidone were 0.999 and 0.999. so, the present work is aimed for development of simple, reproducible chromatographic rp-hplc method for simultaneous estimation of benidipine hydrochloride and chlorthalidone. the developed method was successfully validated in accordance to ich guideline. hence the method can be conveniently adopted for routine analysis in quality control laboratories. key words: benidipine hydrochloride, chlorthalidone, rp-hplc, method development, validation enidipine hydrochloride is a 5-o-[(3r)1benzylpiperidin-3-yl] 3-o-methyl (4r)-2, 6dimethyl-4-(3-nitrophenyl)-1, 4-dihydropyridine3, 5dicarboxylate; hydrochloride [1-2]. benidipine hydrochloride is a long acting anti-hypertensive drug class of ca2+channel blocker (dihydropyridine derivative) its longer duration of action to slow dissociation from the dhp receptor on smooth muscle cell [3].it is a triple l, t and n channel blocker for treatment of hypertension and angina pectoris mostly used in japan and india [1-3]. chlorthalidone is a 2chloro-5-(1-hydroxy-3-oxo-2hisoindol-1-yl) benzenesulfonamide [4]. it is a long-acting thiazide, diuretic drug used in treatment of hypertension and certain kidney disorder [5]. fig. 1structure of benidipine hydrochloride molecular formula: c28h32cln3o6 molecular weight: 542 g/mol chlorthalidone has longest duration of action but similar diuretic effect at maximum therapeutic doses. it reduces reabsorption of sodium and chloride primarily through inhibition of the na+ / clsymporter in the apical membrane of distal convoluted tubule cells in kidney [6-7]. chlorthalidone was first introduced in switzerland in1959 it is also available as generic medicine [4]. fig. 2 – structure of chlorthalidone molecular formula: c14h11cln2o4s molecular weight: 338.8g/mol b shaikhmulani et al. rp-hplc method development and validation of ben.hcl & ctd vol 1 | issue 3 | oct apr 2022 indian j pharm drug studies | 103 materials and methods chemicals used: a pure drug in powder form benidipine hydrochloride and chlorthalidone received from alkumentis pharmaceuticals, mumbai and ipaca pharmaceutical, pvt. ltd mumbai, methanol (ar grade) and orthophosphoric acid utilized as diluent and received from fine chem laboratories, mumbai and loba chemie pvt. ltd. and a commercial product manufactured by alkumenties pharmaceuticals “benitowa-ch” which contain both the benidipine hydrochloride and chlorthalidone received from market. instrument used: agilent 1260 infinity quaternary hplc device equipped with photodiode array detector and the output signal was checked and processed using openlab ezchrome software. chromatographic conditions were as follows: oven temperature: 300 c, flow rate was:-1ml/min, run time was: 10 min, injection volume: 10µl, wavelength: -238 nm, column: c-18agilent zorbax bonus –rp (250 x 4.6 mm, particle size -5 micron) and diluent: methanol: 0.1% orthophosphoric acid (50:50). preparation of mobile phase: the mobile phase was prepared by mixing methanol and 0.1% orthophosphoric acid in the ratio of 55:45 (v/v). the resultant solution filtered through micron filter 0.45µm pore size by means of vacuum pump and then degassed by using ultasonicator to remove gases in solvent. preparation of standard solution: standard solution of both the drug were prepared by weighing 5mg and 12.5mg of benidipine hydrochloride & chlorthalidone and transfer in to 10ml of volumetric flask and make volume up to 10ml using mobile phase and prepared solutions concentration were 500µg/ml and 1250 µg/ml respectively. preparation of working standard solution: the working standard solution of both the drug were prepared by weighing 5mg and 12.5mg of benidipine hydrochloride & chlorthalidone and transfer in to 10ml of volumetric flask and make volume up to 10ml using mobile phase then pipette out 0.8ml and 1.0ml respectively and add into another 10ml volumetric flask and make up volume up to 10ml using mobile phase. the resultant solution was 40µg/ml and 125µg/ml, respectively. selection of wavelength: the sample was scanned from 200400 nm photodiode array detectors. the wavelength selected for analysis chosen was 238 nm on basis of appropriate intensity of both the drug. analysis of marketed formulation take 5 tablets each tablet contains 8 mg of benidipine hydrochloride and 12.5 mg of chlorthalidone and weighed and powdered from that transfer 5mg of benidipine hydrochloride and 12.5 mg of chlorthalidone in 10 ml of volumetric flask and make up volume up to 10 ml with help of diluent and further dilution would make final concentration of solution were 40µg/ml and 125 µg/ml respectively. then solution was filtered by 0.45 µm nylon membrane filter by using vacuum filter. tablet formulation analysis was carried out as mentioned under section tablet formulation analysis. procedure was repeated for 5 times. sample solution was injected and area was recorded for each drug concentration and percentage purity was determined as shown in table 1. analysis is the most important aspect of any drug development; a suitable method must be developed so as to ensure that any drug in dosage from. with help of method development ensure that the amount of particular drug can easily determine. the validation parameter confirms that the developed method is precise, accurate and reproducible and can be used for used for routine evaluation of benidipine hydrochloride and chlorthalidone in combined dosage form [812]. in the present study suitable for rp-hplc method was developed with the aim of making detection of benidipine hydrochloride and chlorthalidone more accurate and precise with addition of validation parameters like specificity, linearity, accuracy, and lod & loq [8]. table 1 assay result with benidipine hydrochloride & chlorthalidone s.no benidipine hydrochloride chlorthalidone peak area amt. recovered in µg/ml % recovery peak area amt. recovered in µg/ml % recovery 1 1612593 32 98.54 3800032 100 100.41 2 1612895 31.97 98.56 3765482 99.81 99.67 3 1623583 31.82 99.22 3768221 99.98 99.57 4 1624896 32.03 99.31 3798523 100 97.67 5 1628369 32 99.52 3799213 100 100.39 mean 1620467 31.96 99.03 3766294 99.95 99.54 % rsd 0.44 0.23 0.45 1.09 0.08 1.12 shaikhmulani et al rp-hplc method development and validation of ben.hcl & ctd vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 104 table 2 concentration and area of benidipine hydrochloride % level conc (µg/ml) area 80 32 1584314 90 36 1762564 100 40 1979466 110 44 2181734 120 48 2378413 table 3 concentration and area of chlorthalidone chlorthalidone % level conc (µg/ml) area 80 100 3800057 90 112.5 4214430 100 125 4721514 110 137.5 5199512 120 150 5626385 table 4linearity values of benidipine hydrochloride and chlorthalidone parameter benidipine hydrochloride chlorthalidone range 32-48 µg/ml 100-150µg/ml slope 50184 37102 intercept 30070 74642 correlation coefficient 0.999 0.999 method validation 1. linearity: to get desired analyte concentrations the standard solution was created by adding 5 mg of benidipine hydrochloride and 12.5mg of chlorthalidone in 10 ml of volumetric flask and make up volume up to 10 ml using diluent then further dilution was made to get 32-48µg/ml of benidipine hydrochloride and 100-150µg/ml respectively. the correlation coefficient for calibration curve benidipine hydrochloride and chlorthalidone was found to be 0.999 and 0.999 respectively (table 4). fig. 3 chromatogram of standard mixture of benidipine hydrochloride & chlorthalidone 2. precision: precision of an analytical procedure express the closeness of agreement between a series of measurement obtained from multiple sampling of same homologous sample under précised condition. intraday precision for benidipine hydrochloride and chlorthalidone are shown in table no. (5&6). the %rsd for benidipine hydrochloride and chlorthalidone was found to be 0.161and 0.390, respectively. interday precision for benidipine hydrochloride and chlorthalidone are shown in table no. (7&8). the %rsd or benidipine hydrochloride and chlorthalidone was found to be 0.27and 0.41, respectively. fig. 4 linearity graph of benidipine hydrochloride 3. lod & loq: limit of detection (lod) means the detection of an individual analytical procedure express the closeness of agreement between a series of measurement obtained from multiple sampling of same homologous sample under précised condition and limit of quantitation (loq) means the quantitation of an analytical procedure is the lowest amount of an analyte in a sample which can be quantitatively determine with suitable precision and accuracy. it can be calculated using formula 3.3 σ/s and 10 σ/s, respectively. where σ is sd of yintercepts of regression line and s is slope of calibration curve (table 13). 4. robustness: this was done by minute changes in the chromatographic condition and found to be unchanged by minute changes like ±2% changes in volume of organic solution of mobile phase. fig. 5 linearity graph of chlorthalidone 5. accuracy: accuracy is the closeness of agreement between the values found. the value accepted as the conventional true value or the accepted reference value. the accuracy of the y = 37102x + 74642 r² = 0.999 3400000 3900000 4400000 4900000 5400000 5900000 80 130 180 concentration (µ g/ml) linearity of chlorthalidone y = 50184x 30070 r² = 0.9993 1200000 1400000 1600000 1800000 2000000 2200000 2400000 2600000 28 38 48 58 concentration (ug/ml) linearity of benedipine shaikhmulani et al rp-hplc method development and validation of ben.hcl & ctd vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 105 method was confirmed by recovery study from the marketed formulation at three level of standard addition (table 9 & 10). 6. system suitability parameters: 7. specificity: specificity is ability to assess the analyte for the presence of various components that may be present. specificity of the rp_hplc method was determined by entire separation of benidipine hydrochloride and chlorthalidone with parameter like retention time (rt), resolution (rs) and tailing factor (t). the peak obtained for benidipine hydrochloride and chlorthalidone were sharp and have a clear baseline separation. the results are shown in table 12. table 5 intraday precision study of benidipine hydrochloride concentration in µg/ml area % recovery mean % recovery ± sd mean % recovery ±rsd 1612694 98.55 32 µg/ml 1613005 98.57 98.59±0.064 1614658 98.67 1825799 99.39 99.21±0.161 36 µg/ml 1836154 99.97 99.79±0.34 1837001 100.01 2023149 99.28 40 µg/ml 2024153 99.33 99.25±0.08 2020746 99.16 table 6 intraday precision study of chlorthalidone concentration in µg/ml area % recovery mean % recovery ± sd mean % recovery ±rsd 3794817 100.26 99.53±0.66 100 µg/ml 3745628 98.94 3767898 99.41 4256897 100.19 99.75±0.49 122.5 µg/ml 4215783 99.21 99.75±0.39 4243162 99.86 4712539 100 99.98±0.02 125 µg/ml 4710986 99.96 4711656 99.98 table 7 interday precision study of benidipine hydrochloride concentration in µg/ml area % recovery mean % recovery ± sd mean % recovery ±rsd 32 µg/ml 1611936 98.50 98.45±0.04 97.93±0.270 1611127 98.45 1610456 98.41 36 µg/ml 1796321 97.76 97.48±0.44 1795854 97.73 1789500 96.97 40 µg/ml 1999120 98.04 97.88±0.33 1887664 97.49 1998664 98.06 table 8 interday precision study of chlorthalidone concentration in µg/ml area % recovery mean % recovery ± sd mean % recovery ±rsd 3689541 97.48 97.79±0.354 100 µg/ml 3698627 97.73 3715496 98.18 4158946 97.89 98.62±0.637 122.5 µg/ml 42011993 98.93 98.67±0.421 4207234 99.05 4699137 99.75 99.62±0.273 125 µg/ml 4678789 99.31 4701856 99.81 shaikhmulani et al rp-hplc method development and validation of ben.hcl & ctd vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 106 table 9 recovery study of benidipine hydrochloride std area 1968846 %level reps spiked conc. (µg/ml) area amount recovered µg/ml) %recovery avg stdev rsd 80% rep 1 32 1584314 32.19 100.59 99.86 0.702637 0.70 rep 2 32 1572215 31.94 99.82 rep 3 32 1562213 31.74 99.18 100% rep 1 40 1979466 40.22 100.54 99.72 0.726323 0.73 rep 2 40 1957783 39.78 99.44 rep 3 40 1952473 39.67 99.17 120% rep 1 48 2378413 48.32 100.67 99.62 1.149973 1.15 rep 2 48 2324579 47.23 98.39 rep 3 48 2357896 47.90 99.80 table 10 recovery study of chlorthalidone table 11 system suitability parameters results and discussion in this method development 100 ml of mobile phase was prepared by mixing 550 ml of 1% orthophosphoric acid and 450 ml of methanol. the mobile phase filtered through 0.45µm filters to avoid the column clogging due to smaller particle size. the flow rate was found to be 1 ml/min resulting in short retention time, good baseline stability with low noise level. in this present developed rp-hplc method, the standard and sample preparation required less me. by the use of proposed method the retention time of benidipine hydrochloride and chlorthalidone was found to be 1.09 and 3.52 minutes respectively. table 12 specificity parameter parameter benidipine hydrochloride chlorthalidone tailing factor 1.09 1.05 resolution 0.00 9.62 retention time 1.09 3.52 the assay of benidipine hydrochloride and chlorthalidone in bulk drug and combined dosage form was found to be 100.99% and 100.15% respectively. a good linear relationship, r2 = 0.999 for benidipine hydrochloride and r2 = 0.999 for chlorthalidone was observed between the concentration range of 32-48µg/ml and 100-150µg/ml, respectively. the lod and loq values for benidipine hydrochloride was found to be 2.05µg/ml and 6.22 µg/ml and for chlorthalidone 7.71 µg/ml and 23.38 µg/ml, respectively. the low values of standard deviation of retention time and peak area indicates high precision of method from recovery study data it has found to be mean % recovery was within a limit, indicates high accuracy of proposed method. there are no additional peaks are observed in the chromatogram which indicates no interference of common ingredients used in formulation. no marked changes were observed in % assay of the optimized condition with that of alternate condition in the robustness study indicating that the method is robust. std area 4735952 % level reps spiked conc. µg/ml) area amount recovered (µg/ml) % recovery avg stdev rsd 80% rep 1 100 3800057 100.30 100.30 100.58 0.282482 0.28 rep 2 100 3821454 100.86 100.86 rep 3 100 3810244 100.57 100.57 100% rep 1 125 4721514 124.62 99.70 100.06 0.444878 0.44 rep 2 125 4732156 124.90 99.92 rep 3 125 4762145 125.69 100.55 120% rep 1 150 5626385 148.50 99.00 98.96 0.047238 0.05 rep 2 150 5621020 148.36 98.91 rep 3 150 5623887 148.44 98.96 sr.no parameters benidipine hydrochloride chlorthalidone 1 retention time (rt) 1.09 3.52 2 area 1979466 4731515 3 theoretical plates 5344 8346 4 resolution 0.0 9.62 shaikhmulani et al rp-hplc method development and validation of ben.hcl & ctd vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 107 table 13 lod & loq parameter name of drug lod (µg/ml) loq (µg/ml) benidipine hydrochloride 2.05 6.22 chlorthalidone 7.71 23.38 conclusion a simple competent method was developed for the simultaneous equation of benidipine hydrochloride and chlorthalidone within a short analysis time with no interference of common additives present in tablet formulation. the proposed method can be applied for routine quality control and applied for bulk and in tablet dosage form. references 1. wikipedia contributors. benidipine [internet]. wikipedia, the free encyclopedia. available from: https://en.m.wikipedia.org/wiki/benidipine 2. drugbank.com. [cited 2022 jul 28]. available from: https://engo.drugbank.com/drug/db09231 3. tripathi k.d, essential of medical pharmacology 7th edition jaypee brother’s medical publishers, new delhi p.n-549-551. 4. wikipedia contributors. chlortalidone [internet]. wikipedia, the free encyclopedia. available from: https://en.m.wikipedia.org/wiki/chlorthalidone 5. tripathi k.d, essential of medical pharmacology 7th edition jaypee brothers medical publishers, new delhi p.n.559-560 6. chaudhary br. development and validation of stability indicating gradient rp-hplc method for simultaneous estimation of telmisartan and chlorthalidone in bulk api and fixed dose combination. world j pharm res [internet]. 2017; 1015 29. available from: http://dx.doi.org/10.20959/wjpr201710-9417 7. bhoomi d patel, ankit chaudhary, sudhir gam. rphplc method development and validation for simultaneous estimation of benidipine hydrochloride, telmisartan and chlorthalidone in tablet dosage form, journal of emerging technologies and innovative research, 6(3) 2019, 110 124 8. vhanmane a, tamboli a, more s. rp-hplc method development and validation for the simultaneous estimation of gabapentin and amitriptyline hydrochloride in pharmaceutical dosage forms. jddt [internet]. 15 june 2019 [cited 28 july 2022]; 9(3-s): 62 8. available from: http://jddtonline.info/index.php/jddt/article/view/2756 9. ich q2a text on validation of analytical procedure, international conference on harmonisation. tripartite guideline. 1994; 1-5. 10. bekett ah, stenlake jb, practical pharmaceutical chemistry, cbs publisher and distributers, new delhi, india part 2 2002; 237 275. 11. ich q2b text on validation of analytical procedure, international conference on harmonisation. tripartite guideline. 1994; 1 10. how to cite this article: shaikhmulani hi, tamboli am, tambili na, kshirsagar rt, suryawanshi ht, khandare o. rp-hplc method development and validation for simultaneous estimation of benidipine hydrochloride and chlorthalidone in pharmaceutical dosage form. indian j pharm drug studies. 2022: 1(3) 102-107. funding: none conflict of interest: none stated fig. 6 chromatogram of 80% accuracy fig. 7 chromatogram of 100% accuracy. fig . 8 chromatogram of 120% accuracy. https://engo.drugbank.com/drug/db09231 https://en.m.wikipedia.org/wiki/chlorthalidone http://dx.doi.org/10.20959/wjpr201710-9417 http://dx.doi.org/10.20959/wjpr201710-9417 varshini n et al. a comprehensive study and overview of novel norovirus vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 37 review article exploring the novel norovirus: a comprehensive study and overview varshini n1, himasvi m m1, ramdas bhat2, ar shabaraya3 from 1 ug student, department of pharmacy practice, 2 assistant professor, department of pharmacology, 3 principal and head of department of pharmaceutics, srinivas college of pharmacy, valachil, post farangipete, mangalore, karnataka, india-574143 correspondence to: ramdas bhat, assistant professor, department of pharmacology, srinivas college of pharmacy, valachil, mangalore 574143 india. tel: +91 7795772463 email: ramdas21@gmail.com abstract norovirus is a highly contagious virus that is the leading cause of acute gastroenteritis worldwide. it is a small, non-enveloped rna virus that is classified as a member of the caliciviridae family. norovirus is spread through contaminated food or water, close contact with infected individuals, or by touching contaminated surfaces and then ingesting the virus. symptoms of norovirus infection include nausea, vomiting, diarrhea, and abdominal cramps. the virus primarily infects the epithelial cells lining the small intestine, leading to inflammation and damage of the intestinal lining, and decreased absorption of nutrients and fluid. the inflammation and damage can lead to dehydration, especially in young children, elderly individuals, and those with weakened immune systems. norovirus also triggers an immune response in the body, which can further contribute to inflammation and damage of the small intestine. in severe cases, the inflammation and damage can lead to a temporary decrease in the body's ability to absorb nutrients, leading to malnutrition. prevention measures include regular handwashing, proper food preparation and storage, and disinfection of contaminated surfaces. those who are sick with norovirus should stay home and avoid close contact with others until they have been symptom-free for at least 48 hours. additionally, it is important to educate the public about the importance of proper hygiene and the steps they can take to prevent norovirus transmission. in conclusion, norovirus is a highly contagious virus that is a leading cause of acute gastroenteritis worldwide. the virus primarily infects the small intestine, leading to inflammation and damage, and decreased absorption of nutrients and fluid. prevention measures include proper hygiene, food preparation and storage, and avoiding close contact with infected individuals. key words: norovirus, acute gastroenteritis, reverse transcription polymerase chain reaction, diarrhoea oroviruses are positive-sense, single-stranded rna viruses that are non-enveloped and belong to the caliciviridae viral family. the norovirus is a highly contagious virus that causes gastroenteritis (inflammation of the stomach and intestines). it is one of the leading causes of foodborne illness and gastroenteritis a norovirus infection. contaminated food and drink, intimate contact with infected people, or touching contaminated surfaces and then putting your hand in your mouth are all ways for the virus to spread [1]. norovirus is a worldwide epidemic, particularly in closed environments where it can spread quickly, such as schools, nursing homes and cruise ships. economic losses from disease and quarantine measures can accompany outbreaks, causing great distress and suffering to individuals and communities. routine hand washing, careful food preparation and storage, and appropriate disinfection and cleaning of contaminated surfaces are all effective preventative measures [2]. even though the majority of people recover within a few days from norovirus infection, it can be especially dangerous for vulnerable groups such as young children, the elderly, and people with weakened immune systems. to prevent the spread of the norovirus, if you experiencesymptoms, take care and seek medical attention if ecessary, especially if you belong to a high-risk group [3]. definition and history norovirus was named after being discovered in 1972 during a gastroenteritis outbreak in norwalk, ohio. over 100 people were affected by the outbreak at a school, and researchers were able to identify the virus using electron microscopy. it was discovered that the virus was a small, round, structured virus that was resistant to common disinfectants. norovirus has since been identified as a leading cause of gastroenteritis outbreaks worldwide. it is estimated that it kills over 200,000 people each year, primarily in developing countries with limited access to clean water and sanitation. norovirus outbreaks are common in developed countries in places where large numbers of people are in close contact, such as hospitals, nursing homes, cruise ships, and restaurants [4]. despite the significant health and economic impact of norovirus, there is still much to learn about the virus and how it spreads. researchers continue to study the virus to develop better n mailto:ramdas21@gmail.com varshini n et al. a comprehensive study and overview of novel norovirus vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 38 methods for prevention, treatment, and control of norovirus outbreaks [5]. epidemiology the norovirus, which is extremely infectious, is a major source of disease all over the world. every year, the number of norovirus cases in the united states is expected to reach 21 million. the true number of occurrences is likely to be higher because many people with mild symptoms may choose not to seek medical attention. acute gastroenteritis-induced stomach and intestine inflammation accounts for approximately 18% of all cases worldwide. when comparing high and low mortality rates in developing countries, developed countries (20%) have a higher prevalence of it. in comparison to hospital inpatients (17%), who are frequently impacted by other factors, norovirus has a much greater impact in the community and among hospital outpatients (24% and 20%, respectively). to prevent the spread of the norovirus, good hygiene practises such as routine hand washing and avoiding direct contact with sick people are required. in addition to sanitising infected surfaces, proper food handling and preparation techniques can help to prevent the virus from spreading [6]. transmission the norovirus virus is extremely contagious and spreads quickly from person to person. the virus can be transmitted through a number of routes, including contaminated food or drink, close contact with sick people, and touching contaminated surfaces. foodborne norovirus transmission is a common mode of transmission. the virus can spread when sick food workers prepare food without properly washing their hands, or when food is contaminated with norovirus-infested faeces. this can happen as a result of contaminated product washing water or during food preparation. close contact is another major mode of norovirus transmission. the virus is highly contagious and spreads quickly through close physical contact, such as shaking hands, hugging, or sharing utensils or food. this mode of transmission is most common in enclosed communities, such as schools, nursing homes, and cruise ships. surface transmission is another important mode of norovirus propagation [7]. the virus can survive on surfaces for several days, and touching your mouth after coming into contact with a contaminated surface can result in infection. this type of transmission is especially dangerous because multiple people may come into contact with contaminated surfaces in public places such as restrooms. norovirus can also be transmitted through the air in tiny droplets released during vomiting, though this occurs less frequently. it is still possible in confined spaces such as aircraft, classrooms, or hospitals. to prevent the spread of the norovirus, it is critical to practise good hygiene, which includes routine handwashing, safe food preparation and storage, and disinfecting contaminated surfaces. people who are sick with the norovirus should stay at home and avoid close contact with others until they have been symptom-free for at least 48 hours. the general public must also be educated on the importance of good hygiene and the precautions they can take to prevent the spread of the norovirus [8]. pathophysiology noroviruses are among a class of viruses that are challenging to grow in a lab environment. it has been challenging to predict with accuracy how norovirus spreads among humans. numerous studies have found that the complicated norovirus infection involves a wide variety of cell types in the human intestine. the single layer of intestinal epithelium made up of enterocytes, the main cell type lining the human gut, is present there. it comprises a range of immune cells and is hidden deep within the enterocytes. numerous studies have shown that immune cells such b cells, dendritic cells, and macrophages are infected and where norovirus replicates. theoretically, noroviruses may infect people without passing through enterocytes. among the hypothesised entry sites are lymphoid follicles and m cells in the stomach (specialised cells that coat peyer patches). because m cells do not exude mucus and lack microvilli on their surfaces, norovirus may readily enter the host and infiltrate immune cells. other research studies suggest that norovirus may actively infect enterocytes lining the gut lumen, while there isn't enough evidence to draw a definitive conclusion. there has also been research about how the host's gut flora affects norovirus spread. according to one theory, norovirus works with gut microorganisms to increase infection and multiplication. norovirus symptoms typically last 1-3 days, with clinical symptoms presenting 1-2 days after viral injection. even after their symptoms have subsided, humans may continue to excrete the virus for up to 60 days. patients with weakened immune systems may be able to excrete the virus for months or even years [4]. diagnosis norovirus (nov) is a prevalent cause of gastroenteritis, and recognising outbreaks and managing public health requires accurate diagnosis. the electron microscopy (em) approach is the traditional nov diagnosis method, but it is not widely used due to its high cost and technical limitations. the immuneenzymatic approach (elisa) detects the virus in faeces through an interaction between norovirus capsid proteins and an enzyme, although its diagnostic sensitivity is limited. the rt-pcr (real-time reverse transcriptase polymerase chain reaction) molecular technology, which is commonly used for detecting nov and researching epidemics, is a more sensitive and specific method. the rt-pcr technology quantifies varshini n et al. a comprehensive study and overview of novel norovirus vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 39 particular dna or rna sequences in clinical samples in real time and offers various benefits over traditional pcr, including enhanced sensitivity, specificity, and repeatability, as well as lower contamination. the pol gene region's phylogenetic analysis can be utilised to detect nov genotypes and aid in epidemiological research [4]. management and treatment the norovirus, commonly known as the winter vomiting bug, is a highly infectious virus that causes acute gastroenteritis, resulting in symptoms like vomiting, diarrhoea, and stomach cramps. managing norovirus sickness necessitates a twopronged strategy that focuses on both treating the patient's symptoms and limiting the likelihood of an epidemic. in order to prevent the spread of disease, infection control is a key concern, and the foundations of infection management are hand washing, surface cleaning, and avoiding contact with human fluids. the most important issue in treating the patient's symptoms is managing the patient's hydration level. because dehydration is a common complication of norovirus infection, oral rehydration therapy is the mainstay of treatment. to assist replace the body's fluids, both the american academy of paediatrics and the world health organization (who) recommend oral rehydration solutions including electrolytes and glucose. oral rehydration treatments are suggested for paediatric patients over sports drinks and juices, which have a high osmotic and carbohydrate load and might aggravate diarrhoea [9]. in dire situations, such as when the patient is unable to stop vomiting or is very dehydrated, intravenous hydration and hospitalisation may be required. antibiotics are normally not advised for norovirus, unless a bacterial infection is detected. adults may benefit from anti-motility drugs, and antiemetics may help ease vomiting symptoms. ondansetron, in particular, has been demonstrated to be useful in managing vomiting in youngsters. because of the predicted economic and public health benefits, the development of a norovirus vaccine is a major priority. however, developing a vaccine has been difficult due to the virus's intricate structure, human immune responses, the difficulty of replicating the virus, and a lack of acceptable animal models for vaccine development [10]. several vaccinations are now being tested in pre-clinical studies, and one has completed adult phase ii clinical trials. because of the virus's capacity to mutate, development efforts have concentrated on multivalent immunisations, comparable to influenza vaccines [11]. prevention norovirus is a highly contagious virus that can cause acute gastroenteritis outbreaks with symptoms such as vomiting, diarrhoea, and stomach cramps. it is easily transmitted from person to person and through contaminated food, water, and surfaces. to prevent the spread of norovirus, it is critical to follow a set of guidelines that include frequently washing hands with soap and warm water, avoiding contaminated food and water, disinfecting contaminated surfaces, practising good hygiene, wearing gloves when caring for someone who has norovirus, staying home if you have norovirus, and avoiding self-medication with antibiotics [12]. hand washing is a critical step in preventing the spread of norovirus, as it can be transmitted through fecal matter that can be present on contaminated surfaces. avoiding contaminated food and water is also important, and it is recommended to cook shellfish thoroughly, wash fruits and vegetables, and only drink water that is safe and clean. disinfecting contaminated surfaces, such as doorknobs, faucets, and countertops, with a chlorine bleach solution or a disinfectant that is effective against norovirus is also recommended. additionally, it is important to practice good hygiene, such as avoiding close contact with people who are sick and covering your mouth and nose when coughing or sneezing. if caring for someone with norovirus, wearing gloves can help avoid contact with their vomit or feces. finally, it is crucial to stay home and avoid contact with others if you have norovirus, as it can easily spread to others. it is important to avoid self-medication with antibiotics, as they are not effective against norovirus and can contribute to the spread of antibiotic-resistant infections [13]. prognosis despite the fact that the majority of patients have no significant side effects from norovirus infection. the biggest risk of negative outcomes, including death, is among elderly and immunocompromised people [14]. in particular, research demonstrate that elderly individuals with norovirus infection have an elevated death rate. neonates with norovirus infection are at risk for necrotizing enterocolitis. additionally, immunocompromised patients run the danger of having diarrhoea that lasts for months to years in some cases, as well as a sickness with greater severity and a longer clinical course [15]. conclusion norovirus is a highly contagious virus that causes acute gastroenteritis, characterised by symptoms such as vomiting, diarrhoea, and stomach cramps. there is no cure, and treatment consists of oral rehydration therapy. norovirus spreads quickly in crowded areas, so good hygiene practises such as handwashing, avoiding close contact with infected people, and disinfecting contaminated surfaces can help prevent infection. food that is properly cooked and stored can also reduce the risk of infection. high-risk groups should take varshini n et al. a comprehensive study and overview of novel norovirus vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 40 extra precautions, and in severe cases, hospitalisation for intravenous fluid replacement may be required. overall, people must be vigilant and proactive in protecting themselves and their communities from norovirus. references 1. vinjé j, estes mk, esteves p, et al. ictv virus taxonomy profile: caliciviridae. the journal of general virology. 2019 nov; 100(11):1469. 2. sell j, dolan b. common gastrointestinal infections. primary care: clinics in office practice. 2018 sep 1; 45(3):519-32. 3. sadkowska-todys m, zieliński a, czarkowski mp. infectious diseases in poland in 2016. przegl epidemiol. 2018 jan 1; 72(2):129-41. 4. robilotti e, deresinski s, pinsky ba. norovirus. clinical microbiology reviews. 2015 jan; 28(1):134–64. 5. kambhampati a, koopmans m, lopman ba. burden of norovirus in healthcare facilities and strategies for outbreak control. journal of hospital infection. 2015 apr; 89(4):296–301. 6. belliot g, lopman ba, ambert-balay k, pothier p. the burden of norovirus gastroenteritis: an important foodborne and healthcare-related infection. clinical microbiology and infection. 2014 aug; 20(8):724–30. 7. xiao s, tang j, li y. airborne or fomite transmission for norovirus? a case study revisited. inter.national journal of environmental research and public health. 2017 dec 14; 14(12):1571. 8. barclay l, park gw, vega e, et al. infection control for norovirus. clinical microbiology and infection. 2014 aug; 20(8):731–40. 9. chen y, hall aj, kirk md. norovirus disease in older adults living in long-term care facilities: strategies for management. current geriatrics reports. 2017 feb 24; 6(1):26–33. 10. cohen r, raymond j, gendrel d. antimicrobial treatment of diarrhea/acute gastroenteritis in children. archives de pédiatrie. 2017 dec 1; 24(12):s26-9. 11. krammer f, palese p. advances in the development of influenza virus vaccines. nature reviews drug discovery. 2015 feb 27; 14(3):167–82. 12. greening ge, cannon jl. human and animal viruses in food (including taxonomy of enteric viruses). viruses in foods. 2016:5-7. 13. guix s, pintó r, bosch a. final consumer options to control and prevent foodborne norovirus infections. viruses. 2019 apr 9; 11(4):333. 14. cardemil cv, parashar ud, hall aj. norovirus infection in older adults. infectious disease clinics of north america. 2017 dec; 31(4):839–70. 15. yates mv. norovirus. microbiology of waterborne diseases. 2014; 515–22. how to cite this article: varshini n, himasvi m m, ramdas bhat, ar shabaraya. exploring the novel norovirus: a comprehensive study and overview. indian j pharm drug studies. 2023: 2(2) 37-40. funding: none conflict of interest: none stated mandumpala & sebastian barriers to hospital pharmacy information system vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 84 review article barriers to hospital pharmacy information system – an emerging concern janice jacson mandumpala, stephy sebastian from, pharm d intern, department of pharmacy practice, nirmala college of pharmacy, muvattupuzha, kerala, india. correspondence to: janice jacson mandumpala, pharm d intern, department of pharmacy practice, nirmala college of pharmacy, muvattupuzha, kerala, india – 686661 email: janice.jacson@gmail.com abstract pharmacy plays an important role in keeping up with the clinical care of patients. in a hospital setting the pharmacies revol ve around medication demands and catering to their everincreasing prices. such factors cause inefficiencies in the availability of information systems. pubmed and google scholar databases were studied and extracted for getting information on the technology used in pharmacies. ongoing vendor involvement, acknowledgement of technology limitations, and attempts to address them were crucial in overcoming technology barriers. this review aims to provide the importance of information systems and implement them across various hospitals. key words: hospital pharmacy, pharmacy, information system, healthcare. he concept of pharmacy travels back to the ancient times of the greeks and the egyptians. the pharmacy was once a part of medicine but later it separated as a different functional unit with a unique service. one of the major drawbacks in the antique era is the absence or underdeveloped stage of computerized sophistication and systematic handling of data. with the fast pacing health technology, it is high time that the health sector also progresses at the same speed [1]. there is an unsatisfactory performance in the health sector technology in both cost and quality. healthcare informatics is constantly working on improving itself along with a specific focus on technologies. in this review, we are focusing on healthcare informatics which is defined by himss as “the scientific field that focuses on medication-related data and knowledge within the continuum of healthcare systems— including its acquisition, storage, analysis, use, and dissemination—in the delivery of optimal medication-related patient care and health outcomes.” [2]. pharmacy plays a major role in hospitals. the review particularly focuses on acute hospital pharmacy in central care. in acute hospitals, every admitted person receives at least one dose of medication while a majority of the patients are prescribed from five to nine dosage forms some patients receive up to thirty dosage forms. frank et al performed a crosssectional study on pharmacy, this study proved how polypharmacy has increased from the year 1997 to 2021 [3]. as the number of prescriptions increased in the year the concern of improper diagnosis and improper treatment has also increased. by the year 2007, the number of excessive prescriptions decreased in comparison to the year 1997 after controlling the prescriptions through informatics. a recent irish study performed by o’sullivan et al revealed that around 84.5 % of the patients had polypharmacy while the rest 43% had a prescription of more than 10 medications that are superpolypharmacy [4]. this showed the number of complex medications administered by patients. the budget of a hospital is more than 300 million per annum. in a 2014 study, 1.8 billion irish healthcare was spent on medications. these high costs of healthcare prove the need for information systems in healthcare. powsey et al conducted a study that demonstrated that information systems can play a major role in simplifying patient data [5]. patient safety has become an emerging concern in media during recent years. patient injuries are most commonly due to adverse drug events. many of these are caused by medication errors which are preventable. they can occur at any stage of the medication use process including ordering, transcribing, dispensing, administering, and monitoring. most of the solutions to medication errors, such as computerized physician order entry systems, have focused on reducing errors at the medication ordering stage [6]. this reduces the stress on clinical pharmacists for processing paper and gives a satisfactory performance by the information system. therefore we are taking a closer look at information systems that aid in improving the effectiveness across all the sectors along with the healthcare sector. before we implement information systems in healthcare let us look at t mandumpala & sebastian barriers to hospital pharmacy information system vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 85 the barriers present in healthcare. this review article focuses on various barriers in hospital pharmacy information systems. case studies we present three case studies given by noel carroll et al [7] in their research paper. two case studies were presented in public hospitals one case study was presented in a private hospital fig 1overview of the case studies 1, 2 and 3 case study 1 this case study was managed by the irish government health service executive (hse). this hospital included various healthcare branches like urology, oncology, cardiology, etc. the pharmacist provided direct care to all the branches. the doctors went on various rounds for all the patients to provide care and improvement in the medications. the hospital system did not have an epharmacy. despite lacking epharmacy services in the hospital the hospital recognized how e-pharmacy could improve patient health care. as there were various patients included in this case student the use of e-pharmacy could give accurate information and medications for each patient. case study 2 this study was also managed by the irish government hse. this case study is presented in an acute care hospital along with a 24-hour emergency department. similar to case study 1, the pharmacy provided medications for all patients and doctors went for rounds to check on patients and improve their medications. this hospital recognized the importance of epharmacy and implemented a business plan to run the epharmacy. later this plan remained unexecuted. case study 3 this case study was presented in a private hospital. the private hospital had medical and surgical care for various specialities. the doctors provided advanced surgical and radiotherapy safekeeping for patients. the doctors were also supported with modernized technology. the pharmacy department purchased, stored, formulated, and compounded all the required medications. the clinical pharmacists regularly monitored and reviewed the prescriptions of patients. they also provided medical counselling for community pharmacists to ensure the safety and efficacy of the pharmacy. the private hospital discussed how they were interested in the information system for pharmacy. figure 1 represents the overview of these case studies. nelson et al outlined various techniques such as using the writing locations, key themes, and verification of stakeholders in the pharmacy and larger hospital staff. a total of four visits were observed which were followed by interviews of the staff members. a total walkthrough of the hospital pharmacy was done which led to an interaction with the key staff which included pharmacists, nurses, and clinical pharmacists delivering services. the pharmacy is comprised of six to eight members. a snapshot of the pharmacist was taken regularly. questions about various pharmacy processes and sub-process were also asked. the observation of staff interaction was done and how patients were administered medications. data analysis was performed with coding techniques. initial first open coding determined themes of transcripts. open coding was followed by axial coding which determined the categories or subcategories. the relationship between the two codings was determined. the approval of ethics was performed for these studies through hse. alissa et al conducted one study to determine challenges in the pharmacy electronic information system. the interview was done from october 2007 to march 2008. semi-structured interviews were conducted in this process to illustrate how the positive and negative impacts of information technology affect the work of pharmacy on healthcare workers. the employee interviews were taken at the united states veterans affairs medical center (vamc). both men and women were recruited for the study. table 1it gives an overview of software packages, which are used in coordination with the electronic information system abbreviation software function reference cp hemo clinical procedures hemodialysis for patients on hemodialysis; intended to transfer patient measurements (e.g. blood pressure) into a computerized patient record system (cprs) in real-time. clinical procedures integrate cprs with the clinical instrument used for dialysis; at the time of this study, cp hemo was not widely implemented across [8] mandumpala & sebastian barriers to hospital pharmacy information system vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 86 vamcs cpoe computerized provider order entry incorporated into cprs; prescribers can order most medications and some labs [9] cprs computerized patient record system a component of vista; graphical user interface for some aspects of dhcp/vista framework; contains patient records, cpoe functionality, lab results, alerts, progress notes, reports and reminders, etc. [9] dhcp decentralized hospital computer program dos information framework implemented in va in 1982, later renamed to vista; currently used by va outpatient pharmacists to fill medication orders [9] mrms microsoft’s rights management services provides secure e-mail messaging; can set restrictions on document use: printing, forwarding, editing, etc.; often referred to as ‘rms’ but different from than rms system described below [10] pisces python implementation of the spki (simple public key infrastructure) certificate standard provides a secure system for va software [10] rms resource management system used for scheduling patient appointments and is integrated with vista [9] vista veterans health information systems and technology architecture information technology framework for ~1300 care sites [11] vistaweb (see vista) used by vamcs to view remote patient data from other vas; separate package from cprs [11] vpn one-va virtual private network allows remote veterans service organizations (i.e. clinics that have partnerships with va to care for veterans) to access va systems securely [11] these interviews aided in the analysis of the challenging aspects of electronic information systems. during the interviews, the employees highlighted the use of paper for maintaining records and the deficiency in maintaining an electronic information system. table 1 elaborates the software packages available for use in clinical setting. discussion our review gives a generalized perspective on pharmacy. it shows how pharmacy can be improvised and benefit from technology. we have learned that various hospital sites give a better understanding of how healthcare services can work collectively with pharmacies. the innovation of healthcare information systems gives better access to the hospital and pharmaceutical environment. a hospital pharmacist plays a huge role in a patient’s journey in the hospital. pharmacists deal with a variety of pharmaceutical services. they run different hospital units and deal with prescription drugs, they also give detailed information about the drugs and provide advice to patients, doctors, nurses, or other healthcare providers. pharmacists prevent errors that can take place in the prescribed medications. therefore pharmaceutical service is the core of patient care. our review has shown the improvement of pharmacy through information systems. an information system is fundamental in upgrading healthcare services. the pharmacy providers view their healthcare in an oriented manner. focusing on the pharmacy service also included laboratory staff, analysts, schedulers, etc. the optimization of information systems in pharmacy alone is not enough the overall optimization of the services is required. they have their greatest impact in organising and making available information, in identifying links between pieces of information, and in doing boring repetitive tasks, including checks for problems. the best medication processes will thus not replace people but will harness the strengths of information technology and allow people to do the things best done by people, such as making complex decisions and communicating with each other [12]. holler et al explained a study on the role of information systems in pharmacy improvement [13]. the study explained that information systems interest pharmacists. computerisation of ordering improves safety in several ways: firstly, all orders are structured, so that they must include a dose, route, and frequency; secondly, they are legible and the orderer can be identified in all instances; thirdly, information can be provided to the orderer during the process; and fourthly, all orders can be checked for a number of problems including allergies, drug interactions, overly high doses, drug-laboratory problems (giving a patient a drug when they have a known biochemical factor that predisposes them to risk), and whether the dose is appropriate for the patient's liver and kidney function [14]. health information technology poses a potential impact on the safety of the patient. this study conducted by holler et al., encouraged the use of information technology for improving mandumpala & sebastian barriers to hospital pharmacy information system vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 87 patient safety without any errors. in the upcoming future pharmacists will adopt the wider use of technology for drug administration and prescriptions. a study performed by pinder et al discovered an understanding that even though the clinical pathways have a structured approach for clinical practice the normal human nature to deliver healthcare often leads to errors and undocumented processes differing from organization to organization. pinder et al also evaluated the variation in patients in clinical care suffering from cataracts [15]. despite the variations in patients, there were also underlying similarities. this gives an understanding that clinical, patient, and management provide vital clues to the execution of the clinical care pathway. the tools that are now available should eventually be used in all hospitals. given the potential impact of these technologies, their diffusion has been surprisingly slow. one reason may be the lack of research showing how much of a difference the technologies make. funding for such research has been relatively limited, and relatively little support has come from the developers of the technologies. another, more important reason is lack of demand from the healthcare industry. safety has not been a high priority in medicine, in part because the problem of safety is generally undervalued. one reason for this lack of appreciation is that medical accidents occur in ones and twos rather than in large groups; moreover, many of those involved are ill and elderly. fortunately, public concern about the issue is substantial, and increasing, and the healthcare industry is beginning to take a more active interest. therefore the benefit of clinical care helps in examining clinical care pathways and identifying inefficiencies in pharmacy including patient safety and reduction in costs of medicines. conclusion in this review article, we gave an overview of the barriers to pharmacy service. we show how pharmacy information systems innovate the pharmacy service. technology has great potential to reduce medication errors in hospitals. we have reexamined the clinical care pathways along with the inefficiencies present in the pathway. staff resistance should be addressed through clear communication, identifying champions, emphasizing new information provided by the system, and facilitating collaboration among health professionals. pharmacy still has to go a long way to establish an information system in its management. references 1. mettler t, raptis da. what constitutes the field of health information systems? fostering a systematic framework and research agenda. in: health informatics journal. 2012. 10.1177/1460458212452496 2. sandra v. b. jardim. the electronic health record and its contribution to healthcare information systems interoperability. procedia technol. 2013, 9. 3. moriarty f, hardy c, bennett k, et al. trends and interaction of polypharmacy and potentially inappropriate prescribing in primary care over 15 years in ireland: a repeated cross-sectional study. bmj open. 2015, 5: 10.1136/bmjopen-2015-008656 4. o’sullivan d, o’mahony d, o’connor mn, et al. the impact of a structured pharmacist intervention on the appropriateness of prescribing in older hospitalized patients. drugs and aging. 2014, 31: 10.1007/s40266014-0172-6 5. powsner sm, wyatt jc, wright p. opportunities for and challenges of computerisation. lancet. 1998, 352: 10.1016/s01406736(98)08309-3 6. nanji kc, cina j, patel n, et al. overcoming barriers to the implementation of a pharmacy bar code scanning system for medication dispensing: a case study. j am med informatics assoc. 2009, 16: 10.1197/jamia.m3107 7. carroll n, richardson i. enablers and barriers for hospital pharmacy information systems. health informatics j. 2020, 26: 10.1177/1460458219832056 8. carroll jm, chin g, rosson mb, et al. development of cooperation: five years of participatory design in the virtual school. proc conf des interact syst process pract methods, tech dis. 2000. 9. nemeth c, nunnally m, o’connor m, et al. getting to the point: developing it for the sharp end of healthcare. j biomed inform. 2005, 38: 10.1016/j.jbi.2004.11.002 10. saleem jj, russ al, justice cf, et al. exploring the persistence of paper with the electronic health record. int j med inform. 2009, 78: 10.1016/j.ijmedinf.2009.04.001 11. sellen a, harper r. paper as an analytic resource for the design of new technologies. in: conference on human factors in computing systems proceedings. 1997. 10.1145/258549.258780 12. bates dw. using information technology to reduce rates of medication errors in hospitals. br. med. j. 2000, 320: 10.1136/bmj.320.7237.788 13. holler j. the role of information technology in advancing pharmacy practice models to improve patient safety. pharm. times. (2013). 14. bates dw, teich jm, lee j, et al. the impact of computerized physician order entry on medication error prevention. j am med informatics assoc. 1999, 6: 10.1136/jamia.1999.00660313 15. pinder r, petchey r, shaw s, et al. what’s in a care pathway? towards a cultural cartography of the new nhs. sociol. heal. illn. 2005, 27: 10.1111/j.1467-9566.2005.00473.x how to cite this article: janice jacson mandumpala, stephy sebastian. barriers to hospital pharmacy information system – an emerging concern. indian j pharm drug studies. 2022: 1(3) 84-87. funding: none conflict of interest: none stated sakhare g et al. pharmacovigilance of antibiotic usage vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 51 review article pharmacovigilance study on the increased use of antibiotics in pediatric treatment ghansham sakhare1, purva yelwande1, sandhya suryawanshi1, kiran ambhore1, rutuja pawar1, kishor jain1 from, 1 rjspm’s college of pharmacy, dudulgaon, pune, maharashtra, india. correspondence to: ghansham rajkumar sakhare, rjspm’s college of pharmacy, pune 412105, maharashtra, india. email: sham.sakhare@gmail.com abstract objective: to conduct the pharmacovigilance study on the increased use of antibiotics in pediatric treatment. materials and methods: a suitable, simple questionnaire associated with the study of pharmacovigilance assessment, for mainly the adverse drug reactions associated with the increased antibiotic use in pediatric treatment was designed and distributed among 200 families having children in the age groups of <1 to 8 year. this survey was done in pune region. amoxicillin, tetracycline, cephalexin, doxycycline were the antibiotics selected for the study. results: about selfmedication more than 50% groups said they did self-medication. and major source for it was medicines leftover from previous prescriptions. maximum response was obtained from 5–8year age group. adr were detected with the usage of antibiotics in 66 percent of groups. when it comes to the intensity of an adverse drug reaction, 74% of respondents said it was light, 22% said it was moderate, and 4% said it was severe. around 97 % groups said they went to doctor after occurrence of adverse drug reaction, 98 % percent groups said they were aware of the risks of self-medication. majority of groups knew about adverse drug reactions reporting system. when it comes to awareness of increased antibiotic use leading to antibiotic resistance, 98% of groups were found aware of the problem. discussion: survey showed that families having small children experienced adverse drug reactions as a result of using any of the antibiotics amoxicillin, tetracycline, cephalexin, or doxycycline. conclusion: in conclusion, our study strongly recommends that there is a great need to create awareness among the middleclass families having children to improve the reporting of adr’s. keywords: pharmacovigilance, pediatrics, anti-microbial resistance (amr), amoxicillin, tetracycline, cephalexin, doxycycline ntibiotics are medicines used to prevent and treat bacterial infections. antibiotic resistance is rising to dangerously high levels in all parts of the world. new resistance mechanisms are emerging and spreading globally, threatening our ability to treat common infectious diseases. a growing list of infections such as pneumonia, tuberculosis, gonorrhea, and foodborne diseases are becoming harder, and sometimes impossible, to treat as antibiotics become less effective [1, 2]. to understand the burden of threat posed by antimicrobial resistance, world health organisation (who) estimated that every year in the world, infections caused by multidrug resistant (mdr) bacteria result in 700,000 deaths across all ages, of which around 200,000 are newborns in europe, mdr infections in pediatric patients may represent up to 30% of the total cases in regions of the middle east, 90% of newborns with sepsis, hospitalized in icu, had resistant bacteria; in some areas of south east asia, 83% of children have e. coli resistant to first line antibiotics; in sub-saharan africa, 66% of neonatal sepsis and meningitis were found to be caused by bacteria resistant to antibiotics [3]. india contribution to the uppsala monitoring centre (umc) database is very little. this is essentially due to the absence of a vibrant adr monitoring system and also due to a lack of the reporting culture among the health care workers [4, 5]. quoting the joint who-unicef report of 2006: “children are not small adults when taking a drug.” the capacity of absorption, distribution, metabolism and elimination of a drug are very different between adults and children, and they continue to change during the stage of development. the risks resulting from the administration of a drug that has not been tested and proven in the pediatric population may therefore be due to overdose (increase in adverse reactions), ineffectiveness of the drug (for dosing) and use of a formulation which is not appropriate [6]. a sakhare g et al. pharmacovigilance of antibiotic usage vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 52 antibiotics are reported as the most troublesome of drugs contributing to approximately 15% 16% of cases of adverse drug reactions (adr’s), mostly related with the frequency of administration that accounts approximately for 23% of all adverse events recorded. children are more at risk of having adr’s because many drugs which are prescribed to this population have been marketed with limited or no experience of their efficacy and safety [7]. rational use of antimicrobials in the long run, therefore, demands continuous survey of antimicrobial use and related adr monitoring. [8]. rational use of antimicrobials in the long run, therefore, demands continuous survey of antimicrobial use and related adr monitoring. [9] as for antibiotic misuse in pediatrics, although critical, it has rarely been tackled in epidemiological studies. it could be related to several factors, such as the medication itself (e.g. taste acceptability, dilution and conservation), or the treating pediatrician (e.g. watchful waiting approach) or even the pharmacist (e.g. referral to pediatricians). the occurrence of adverse drug reactions (adr’s) is an-other problem with antibiotic use. adr’s in pediatric population may have relatively more severe effect than adults leading to significant morbidity among children [10]. inappropriate antibiotic-prescribing practices have been well described in developed nations [11]. therefore, using antibiotics to treat these viral infections is considered misuse or overuse of antibiotics. this misuse/overuse is common in children, and is currently considered to be one of the major public health issues worldwide [12]. materials and methods this was a cross-sectional, questionnaire-based survey which was conducted in pune region. this survey done in 200 families (groups); where in number of responses: 111 offline responses and 89 online responses were taken. it was conducted in middleclass families having children age group <1 to 8 years [13, 14]. a standardized questionnaire was made in order to identify the antibiotic prescription practices in common childhood diseases and attitudes towards pharmacovigilance [15]. the questionnaire was divided into several parts. the first part, included self-medication practices questions later part the names of antibiotics and adverse drug reactions observed. the last part include about awareness about pharmacovigilance program in india and antibiotic resistance. the families were requested to complete the questionnaire and to return it within 1 day to rjspm’s college of pharmacy students’ team. this survey deals with pharmacovigilance issues like adr’s due to antibiotic administration and the reporting rates of these. the method of self-administered questionnaire was implemented for data collection purposes [16, 17]. results figure 1 represents for question have you ever treated yourself (self-medicated) with antibiotics? 56% of group response was yes and 44% of groups response was no. fig. 1 have you ever treated yourself (self-medicated) with antibiotics? fig. 2 where did you usually obtain antibiotics for selfmedication? figure 2 represents for question, where did you usually obtain antibiotics from for self-medication? groups response 45% positive for leftover from previous prescription and for community pharmacies, it was 24%, while, 16% groups responses gone in favor of online shopping /e-pharmacies and responses for any other option were only 10%. 24 % 45 % 16 % 15 % community pharmacies leftover from previous prescription online shopping/e-pharmacies any other sakhare g et al. pharmacovigilance of antibiotic usage vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 53 fig. 3 what is age group of children? figure 3 represents in case of age group of children, maximum groups gave responses for age group 5-8 years i.e., 88 out of 200, for 1-3 years 50 responses were recorded and for 3-5 years age group 50 were there, and for 0–1-year age group 12 responses were recorded. figure 4 represents for question, any type of adverse drug reaction is observed with use of antibiotics 66% groups said yes and 34% family said no. figure 5 represents n case of severity of adverse drug reaction 74% responses were for mild adr, 22% were for moderate adr and 4% were for severe type of adr. figure 6 represents in case of what action taken by you to manage adr with antibiotics in pediatrics, maximum responses i.e., 97% groups said they went to doctor and only 3% were waited for subside adverse drug reaction. figure 7 represents for question are you aware of dangers of self-medication, 98% groups said yes and 2% said no. figure 8 represents in case of awareness of adverse drug reaction reporting system in india, 97% groups are aware of the adr reporting system and 3% are unaware of adr reporting system. figure 9 represents in case of awareness of increase use of antibiotics leading to resistance of antibiotics, 98% groups are aware of resistance and 2% are unaware. discussion 0 10 20 30 40 50 60 70 80 90 less than 1years i-3 years 3-5 years 5-8 years 12 50 50 88 number of childern fig. 4 any type of adverse drug reaction is observed with use of antibiotics? fig. 5 what is severity of adverse drug reaction? fig. 6 what action taken by you to manage adr with antibiotics in pediatrics? fig. 7 are you aware of dangers of self-medication? fig. 8 are you aware of adverse drug reaction reporting system in india? fig. 9 are you aware of awareness of increase use of antibiotics leading to resistance of antibiotics? sakhare g et al. pharmacovigilance of antibiotic usage vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 54 for fig 1 thus, it is seen that almost 50% of the surveyed sample have done self-medication at some point of time with antibiotics. verma et al indicates self-medication is very common problem in india [18]. fig. no.2 which discuss about where did you usually obtain antibiotics from for selfmedication? people are understandably unwilling to submit to the inconvenience of visiting a doctor for what they rightly feel they can manage for themselves. reasons for wide variations may be due to differences in education, socio-economic status, nonavailability of medical facilities and easy availability of drugs [19]. community pharmacy, e pharmacy is responsible factor for self-medication according to our study. fig. 3 tells us current study indicates majority population falls between 5 and 8 years of age. fig. no. 4 deals with question, any type of adverse drug reaction is observed with use of antibiotics 66% groups said yes and 34% family said no. in our study occurrence of adr is 34 % indicate higher incidence might be due to poor patient compliance. one of the cited research paper results reveal that the estimated average adr incidence in all selected studies was 9.52%. according to the study which was conducted in 38 different italian hospitals and recruited 1332 hospitalized patients reported that the onset of at least 1 adr [6]. fig. 5 talk about in case of severity of adverse drug reaction 74% responses were for mild adr, 22% were for moderate adr and 4% were for severe type of adr. only serious adrs are needed to be reported in pediatric care, seems to be the most significant factor of no reporting, according to mary et al. [20]. current study lowest score for severe adr shows awareness about reporting any type of adr. this might be due to survey done in urban part where majority of people are literate. the reasons for occurrence of adr in children may be due to immature drug metabolizing organ system in newborn and infants that put them at a higher risk of developing an adr [14]. fig. no. 6 tell about almost all groups went to doctor for adr treatment. fig. no. 7 almost all groups aware about dangers of self-medication which is a general indication. fig. 8 deals about in case of awareness of adverse drug reaction reporting system in india, 97% groups are aware of the adr reporting system and 3% are unaware of adr reporting system. in northern india, study done in year 2013 showed little awareness about the adr reporting system among the doctors in a tertiary care hospital [5]. due to increased use of social media past few years 2022 may resulted in increase in awareness about adr reporting in current study. fig. no. 9 indicates all groups aware about increase use of antibiotics leading to resistance of antibiotics which indicates people are well trained about use of antibiotics. table 1 shows us about many groups experienced adverse reaction with antibiotics indicates importance of adr monitoring system. in order to generalize our results, similar studies be done in other parts and paramedical colleges of the city and other states of the country. training sessions must clarify the roles of the various healthcare professional’s doctors, nurses, pharmacists and paramedical workers in pharmacovigilance. there should be closer relationship between the common public and the pharmacovigilance centers. results showed to be published and discussed in the public domain to spread awareness. to the best of our knowledge, this is one of the few attempts to explore antibiotics prescribing practices and attitudes on pharmacovigilance of hospital pediatricians in pune region. table 1 names of the group and the adverse drug reactions they experienced as a result of using a certain antibiotic group number antibiotic adverse drug reaction group 1 azithromycin diarrhoea group 2 azithromycin abdominal pain group 3 amoxicillin nausea group 4 amoxicillin fever group 5 tetracycline yellowing of teeth group 6 amoxicillin rashes, fever group 7 doxycycline nausea group 8 cephalexin rashes group 9 amoxicillin rashes, itching conclusion in conclusion, our study strongly recommends that there is a great need to create awareness among the middle-class families having children to improve the reporting of adr’s. the adr reporting should be made an integral part of the clinical activities in order to improve the patient care. references 1. https://www.who.int/news-room/factsheets/detail/antibioticresistance 2. tripathi k.d, essential of medical pharmacology, 6th edition: jaypee brothers medical publishers (p) ltd; 2008. p.688-849. 3. romandini a, pani a, schenardi p, et al. antibiotic resistance in pediatric infections: global emerging threats, predicting the near future. antibiotics. 2021; 10; 4: 393. 4. marc a, adrian m. adverse drug reactions: types and treatment options, am fam physician. 2003; 68; 9. 5. bajaj j, rakesh k. a survey on the knowledge, attitude and the practice of pharmacovigilance among the health care professionals in a teaching hospital in northern india. j clin diagnostic res. 2013; 7; 1: 9799. 6. zakir k, khayal m, yusuf k, et al. pharmacovigilance and incidence of adverse drug reactions in hospitalized pediatric patients: a mini systematic review. gaz egypt pediatric assoc. 2020; 23; 68: 24. 7. dattatreyo c, sukanta s, sabnam a, et al. a questionnaire-based survey to ascertain the views of clinicians regarding rational use of antibiotics in teaching hospitals of kolkata. indian j pharmacol. 2015; 47: 1 105. https://www.who.int/news-room/fact https://www.who.int/news-room/fact https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance sakhare g et al. pharmacovigilance of antibiotic usage vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 55 8. praveena g, yadav s, junapudi s. antibiotic use in pediatric infections; a study in tertiary care hospital. int j biomed res. 2018; 4(4): 26. 9. brian t, peter a, samir s, et al. short report: antibiotic use in pediatric patients admitted to a referral hospital in botswana. am j trop med hyg. 2009; 81(1): 129 131. 10. geitona m, toska a, latsou d, et al. antibiotics’ prescribing and pharmacovigilance attitudesamong pediatricians and pediatric residentsin. cyprus. j pharm pharmacol. 2014; 8: 75 84. 11. habarugira j, härmark l, figueras a. pharmacovigilance data as a trigger to identify antimicrobial resistance and inappropriate use of antibiotics: a study using reports from the netherlands pharmacovigilance centre. antibiotics. 2021; 10(12): 1512. 12. kapoor m, bajaj j, salwan s. prescription audit a tool to determine the effects of antibiotics in the pediatric inpatient department of a tertiary teaching care hospital in punjab. j pharmacovigilance drug res. 2021; 2(1): 27-32. 13. ettore n. children and adrs (adverse drug reactions). ital j pediatr. 2010, 36: 4. 14. sharma p, misra a, gupta n, et al. pediatric pharmacovigilance in an institute of national importance: journey has just begun. indian j pharmacol. 2017; 49: 390. 15. geitona m, toska a, latsou d, et al. antibiotics’ prescribing and pharmacovigilance attitudesamong pediatricians and pediatric residentsin. cyprus. j pharm pharmacol. 2014; 8: 75 84. 16. habarugira j, härmark l, figueras a. pharmacovigilance data as a trigger to identify antimicrobial resistance and inappropriate use of antibiotics: a study using reports from the netherlands pharmacovigilance centre. antibiotics. 2021; 10(12): 1512. 17. kapoor m, bajaj j, salwan s. prescription audit a tool to determine the effects of antibiotics in the pediatric inpatient department of a tertiary teaching care hospital in punjab. j pharmacovigilance drug res. 2021; 2(1): 27-32. 18. verma r, mohan l, pandey m. evaluation of selfmedication among professional students in north india: proper statutory drug control must be implemented. asian j phar clin res. 2010;3:60-4 19. phalke v, phalke d, durgawale p. self-medication practices in rural maharashtra. indian j community med. 2006; 31: 34-5 20. mary g, aikaterini t, dimitra l, et al. antibiotics’ prescribing and pharmacovigilance attitudes among pediatricians and pediatric residents in cyprus j pharm pharmacol. 2017; 8: 75 – 84 how to cite this article: sakhare g., yelwande p, suryawanshi s, ambhore k, pawar r, jain k. pharmacovigilance study on the increased use of antibiotics in pediatric treatment. indian j pharm drug studies. 2022;1(2):51-55. funding: none conflict of interest: none stated shanbhag et al. nelumbo nucifera flower extract in mice vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 108 original article screening of antidepressant activity of nelumbo nucifera flower extract in mice preeti shanbhag1, ramdas bhat2, sweta prabhu3, ar shabaraya4 from, 1pg scholar, department of pharmacology, 2assistant professor, department of pharmacology, 3pg scholar, department of pharmaceutics, 4principal and professor, srinivas college of pharmacy, valachil, mangalore, india corresponding to: ramdas bhat, assistant professor, department of pharmacology, srinivas college of pharmacy, valachil, mangalore, india -574143. tel.: +91 7795772463, email id: ramdas21@gmail.com abstract objective: to evaluate the anti-depressant activity of "nelumbo nucifera" in experimental mice. methods: the acute toxicity studies were conducted on fresh "nelumbo nucifera" flower extract. the extract (100–200 mg/kg) was able to induce the mice's immobility duration in the forced swimming and tail suspension tests in a dose-dependent manner; the effects are comparable to those of popular drugs, such as imipramine (10 mg/kg). result: both the lower dose (100mg/kg) and higher dose (200mg/kg) showed a dosedependent significant decrease in depression. in line with imipramine, our study found that the ethanolic extract of "nelumbo nucifera" significantly (p>0.005) decreased immobility in both the tail suspension test and the forced swimming model of depression. these findings showed that "nelumbo nucifera" exhibited in vivo effects that were selectively anti-depressant. conclusion: the results of this study, in summary, revealed that "nelumbo nucifera" ethanolic extract may have anti-depressant properties that make them potentially useful for treating patients with depressive disorders. however, more research is required to comprehend the mechanism of action and to pinpoint the key ingredient that produces anti-depressant-like activity. keywords: anti-depressant, nelumbo nucifera, imipramine, tail suspension test, force swimming test round 280 million individuals worldwide suffer from depression. depression is distinct from common mood swings and fleeting emotional reactions to problems in daily life. every year, around 700,000 people die by suicide a depressive episode lasts at least two weeks and is characterised by a depressed mood (sad, irritated, or empty feelings) or a loss of enjoyment or interest in activities for the majority of each day. depending on the frequency and severity of symptoms, the influence on the person's functioning, and the length of the episode, a depressive episode can be classified as mild, moderate, or severe. due to the negative side effects of synthetic pharmaceuticals, drugs of plant origin are becoming more and more popular. for the treatment of a variety of illnesses, ayurveda uses plants, plant derivatives, and the active compounds found in plants. despite the lack of scientific evidence supporting the antidepressant properties of "nelumbo nucifera," this study was chosen to examine the antidepressant effect of "nelumbo nucifera" in mice. the flower is chosen for the study as a result [1]. the current study's objective was to assess the “nelumbo nucifera” flower extract's anti-depressant effects. millions of people worldwide suffer from depression, a serious mental health problem, and the standard medications used to treat it have several side effects. therefore, there is a need for alternative therapies that are both safer and more potent. “nelumbo nucifera”, which has the potential to be an antidepressant, has been widely utilised in traditional medicine for a variety of reasons in this regard [2]. two commonly utilised animal models, tail suspension test and the forced swimming test, were used to assess extract's antidepressant potential. the findings demonstrated that the extract significantly reduced depressive symptoms in both tests.alkaloids, flavonoids, inositol, proteins, and saponins were all found in the “nelumbo nucifera” flower extract, according to the results of the phytochemical screening. it is known that these phytoconstituents have a range of pharmacological properties, including antidepressant effects. these substances may be a factor in the extract's apparent antidepressant action [3, 4]. although more research is required to pinpoint the precise mechanisms at play, the anti-depressant effects of “nelumbo nucifera” flower extract may be caused by the presence of these phytoconstituents. however, “nelumbo nucifera” may be used as an alternative treatment for depression, as shown by the study's findings. materials and methods the nectar of the "nelumbo nucifera" flower, which was verified by taxonomist mrs aparna upadhyaya, will be obtained from a pond close to mangalore. the department of pharmacology at srinivas college of pharmacy carried out the current investigation. a mailto:ramdas21@gmail.com shanbhag et al. nelumbo nucifera flower extract in mice vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 109 animal: from the indian institute of sciences, adult swiss albino rats (weighing 22–25g) of either sex were obtained. they will be kept in a standard environment with the following parameters: temperature 22°c, relative humidity 50%, and a 12-hour light/dark cycle. all investigations were approved by the institutional animal ethics committee. plant extract: after being chopped, the fresh lotus flowers were dried for 72 hours at 50°c in a hot air oven. for seven days, dried flowers were macerated in 95% ethanol. the extract was turned into a powder by being evaporated in a rotary evaporator and dried in a freeze-dryer. it was then kept at -20°c until it was needed. preliminary phytochemical screening: the ethanolic extract of "nelumbo nucifera" was screened for the presence of various phytoconstituents like alkaloids, flavonoids, inositol, proteins and saponins. acute toxicity: "nelumbo nucifera" flower ethanolic extract was tested for acute toxicity by the 2002 revision of oecd guideline no. 423. animals were monitored for fourteen days for any changes in behaviour, and 24 hours a day showed no toxicity in mice when administered in doses up to 2000 mg/kg orally, 100 and 200 mg/kg doses of the extract were utilised in subsequent investigations. experimental design tail suspension test: a total of 24 mice were divided into 4 groups of six. group 1: control (saline) group 2: standard (impiramine 10mg/kg) group 3: nnfe (low dose – 100mg/kg) group 4: nnfe (high dose – 200mg/kg) as a control group received a saline solution, group 2 received imipramine and groups 3& 4 received a low and high dose of “nelumbo nucifera” ethanolic extract, respectively. the medications were administered orally one hour before the trial. each mouse was held 150 cm off the ground, about 1 cm from the tail tip, using adhesive tape. the testing will take place in a silent, well-lit environment. six minutes in total were logged during the immobility time. the animal initially moved wildly in an attempt to escape, but when they were unable to do so, they became immobile. following that, the mice received medication for 21 days. following that, the immobility time was monitored for a total of 6 minutes. forced swim test: a total of 24 mice were divided into 4 groups of six. group 1: control (saline) group 2: standard (impiramine 10mg/kg) group 3: nnfe (low dose – 100mg/kg) group 4: nnfe (high dose – 200mg/kg) as a control group received a saline solution, group 2 received imipramine and groups 3& 4 received a low and high dose of “nelumbo nucifera” ethanolic extract, respectively. the medications were administered orally one hour before the trial. the animal was inserted into the model to begin the test. individual mice were made to swim for 15 minutes in an 11-centimetre-diameter glass beaker that was kept at a constant temperature of 27°c and filled with fresh water to a height of 6 centimetres. here the "per test" session was over. 24 hours later, each mouse was required to swim for 6 minutes in the same habitat once more as part of a "test session." the test session was conducted both before and after the medication therapy. a mouse is considered static if it floats stationary or moves just enough to keep its head above the water's surface. on average, the final four minutes of the six-minute exam were spent motionless. tail suspension test table 1: effect of “nelumbo nucifera” flower extract in tail suspension test dose duration of mobility in tst (in seconds) day 1 day 7 day 14 day 21 control (saline) 211.3±0.7350 218.4±0.855 209.9±0.4900 204.8±1.370 standard (imipramine) 123±1.555*** 129.8±0.2200*** 140.3±0.8500*** 125.4±1.060*** test 1 (nelumbo nucifera 100mg/kg) 141.1±1.055** 128.3±0.8200** 136.8±0.2050** 136.5±0.700** test 2 (nelumbo nucifera 200mg/kg) 130.4±0.9250*** 132.3±1.145*** 128.5±1.080*** 136.5±0.9700*** forced swimming test table 2: effect of nelumbo nucifera flower extract in forced swim test on mice dose duration of mobility in fst (in seconds) day 1 day 7 day 14 day 21 control (saline) 224.3±0.400 218.7±0.4000 227.1±0.0500 220.3±0.2500 standard (imipramine) 126.3±1.000*** 120.0±0.3500**** 130.3±0.4000*** 132.0±0.7500*** test 1 (nelumbo nucifera 100mg/kg) 171.7±1.800** 159.8±0.0500** 164.4±0.8500** 152.1±0.900** test 2 (nelumbo nucifera 200mg/kg) 141.7±0.8400*** 144.9±0.3350*** 142.9±0.3300*** 144.0±0.4900*** shanbhag et al. nelumbo nucifera flower extract in mice vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 110 d a y 1 d a y 7 d a y 1 4 d a y 2 1 d a y 1 d a y 7 d a y 1 4 d a y 2 1 d a y 1 d a y 7 d a y 1 4 d a y 2 1 d a y 1 d a y 7 d a y 1 4 d a y 2 1 0 50 100 150 200 250 tail suspension test treatment d u ra ti o n o f m o b il it y control impiramine test 1 (100mg/kg) test 2 (200mg/kg) graph 1: effect of “nelumbo nucifera” flower extract in tail suspension test d a y 1 d a y 7 d a y 1 4 d a y 2 1 d a y 1 d a y 7 d a y 1 4 d a y 2 1 d a y 1 d a y 7 d a y 1 4 d a y 2 1 d a y 1 d a y 7 d a y 1 4 d a y 2 1 0 50 100 150 200 250 forced swimming test treatment d u ra ti o n o f m o b il it y control standard test 1 (100mg/kg) test 2 (200mg/kg) graph 2: effect of nelumbo nucifera flower extract in forced swim test on mice results on preliminary phytochemical analysis of nnfe showed the presence of phytoconstituents like alkaloids, carbohydrates, flavonoids, glycosides, saponins, steroids, tannins, protein, inositol. acute toxicity of nnfe showed no behavioural changes nor mortality at a dose of 2000mg/kg. by examining the variations in the period of inactivity in the two models, the anti-depressant effects of the ethanolic extract of “nelumbo nucifera” (100mg/kg and 200mg/kg) and standard medications imipramine were investigated. discussion animal models like the forced swimming test (fst) and the tail suspension test (tst) are frequently used to examine the effectiveness of possible antidepressant medications. a tiny rodent (such as a mouse or rat) is used in the fst, and the duration of time the animal remains motionless is tracked. the duration of immobility is used to gauge depressive-like behaviour. the animals are given antidepressant medication before the test, and the difference in immobility time is used to determine the effectiveness of the medication. similar to this, the tst involves suspending a small rat by its tail and timing how long it remains still. the immobility time, like the fst, is used as an indicator of depressive-like behaviour, and the change in immobility time following the administration of antidepressant medications is used to assess the efficacy of those medications. the fst and tst have both been extensively utilised in preclinical investigations and have been proven to be accurate measures of a drug's antidepressant effectiveness. these models have significant drawbacks as well, though. for instance, some have claimed that the immobility duration in the fst and tst might not truly reflect animal behaviour that resembles depression because the immobility could be caused by other things, such as exhaustion or hopelessness. the fst and tst are nevertheless often employed in preclinical research despite these critiques because of their simplicity of use and the close relationship between the outcomes of these tests and the clinical effectiveness of medications in humans. the fst and tst are still useful methods for researching the effectiveness of possible antidepressant medication as a result. the forced swimming test and the tail suspension test were used in the study to evaluate the “nelumbo nucifera” flower extract's antidepressant potential. “nelumbo nucifera” may be used as an alternative treatment for depression because the results demonstrated considerable anti-depressant effects.alkaloids, flavonoids, inositol, proteins, and saponins were found in the extract, indicating the possibility that these phytoconstituents contributed to the anti-depressant effect that was reported. the study did, however, have certain drawbacks, including a small sample size, a lack of human trials, and a limited understanding of the mechanism of action. it will take more investigation with larger sample sizes and human studies to validate these findings and pinpoint the precise mechanisms at play [5-10]. hence the present study showed “nelumbo nucifera” might be useful in depression, as it increases dopamine levels in the brain increase decrease monoamine oxidase. our results confirm the traditional use of plants as antidepressants. conclusion in summary, this study's findings suggest that “nelumbo nucifera" flower extract may have promise as an antidepressant. because of the considerable anti-depressant action seen in both animal models, there is reason to believe it could be used as an alternative to traditional depression treatments. more investigation is needed to identify the precise mechanisms at play and assess their safety and effectiveness in human studies. however, these findings add to the expanding body of research that shows traditional medicine is effective in treating mental health illnesses like depression. shanbhag et al. nelumbo nucifera flower extract in mice vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 111 references 1. ahmed a, ramesh j, ganguly s, et al. investigating the feasibility of assessing depression severity and valencearousal with wearable sensors using discrete wavelet transforms and machine learning. information. 2022 aug 27; 13(9):406. 2. ohayon mm, schatzberg af. chronic pain and major depressive disorder in the general population. journal of psychiatric research. 2010 may 1; 44(7):454-61. 3. kroenke k, spitzer rl, williams jb. the phq‐9: validity of a brief depression severity measure. journal of general internal medicine. 2001 sep; 16(9):606-13. 4. samy rp, pushparaj pn, gopalakrishnakone p. a compilation of bioactive compounds from ayurveda. bioinformation. 2008; 3(3):100-110. 5. bourin m, chenu f, ripoll n, david dj. a proposal for a decision tree to screen putative antidepressants using forced swim and tail suspension tests. behavioural brain research. 2005 nov 7; 164(2):266-9. 6. mineur ys, belzung c, crusio we. effects of unpredictable chronic mild stress on anxiety and depression-like behaviour in mice. behavioural brain research. 2006 nov 25; 175(1):43-50. 7. cryan jf, mombereau c. in search of a depressed mouse: utility of models for studying depression-related behaviour in genetically modified mice. molecular psychiatry. 2004 apr; 9(4):326-57. 8. cryan jf, markou a, lucki i. assessing antidepressant activity in rodents: recent developments and future needs. trends in pharmacological sciences. 2002 may 1; 23(5):238-45. 9. unal g, canbeyli r. psychomotor retardation in depression: a critical measure of the forced swim test. behavioural brain research. 2019 oct 17; 372:112047. 10. can a, dao dt, arad m, terrillion ce, piantadosi sc, gould td. the mouse forced swim test. jove (journal of visualized experiments). 2012 jan 29(59): e3638. how to cite this article: shanbhag p, bhat r, prabhu s, ar shabaraya. screening of antidepressant activity of nelumbo nucifera flower extract in mice. indian j pharm drug studies. 2022; 1(3) 108-111. funding: none conflict of interest: none stated baby et al over-the-counter medicine abuse vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 92 review article over-the-counter medicine abuse – a pharmacist’s emerging responsibility martin baby john, alasandra rose ms, janice jacson mandumpala from, pharm d intern, department of pharmacy practice, nirmala college of pharmacy, muvattupuzha, kerala, india. correspondence to: janice jacson mandumpala, pharm d intern, department of pharmacy practice, nirmala college of pharmacy, muvattupuzha, kerala, india, pin – 686661 email: janice.jacson@gmail.com abstract self-medication is an essential component of the healthcare system. over-thecounter medications (otc) are components of selfmedication. the administration of otc medications in patients increases the risk of abuse of these drugs. with pharmacists being as accessible as they are, pharmacists are the first line of contact in purchasing otc medications and have an upper hand in ensuring safety, and efficacy and preventing abuse in patients. this review aims to give brief information on otc medications along with their abuse. the importance of patient and pharmacist interaction has been discussed. in addition, the potential for a behind-the-counter drug category and its prospective role in improving pharmacistpatient relations have been elaborated. this review gives knowledge on the barriers faced by pharmacists in otc medication abuse and how it can be controlled and managed effectively. the current health system should work more effectively to provide a striking balance between the purchase of otc medications and their abuse. key words: over-the-counter, abuse, misuse, pharmacists edications provided by pharmacists without the prescription of a doctor are known as overthecounter (otc) medications. it is defined as ‘medications that can be purchased without a prescription and are safe and effective when used according to the directions on the label, and as directed by a health care professional.’ in recent years there is an expanding trend of self-medication with otc medicines and is becoming an increasingly popular practice around the world. as per recent estimates, the global prevalence of self-medication ranges from 11.2% to 93.7% [1]. otc medications are employed for common ailments and provide a cure for a wide range of conditions but not limited to headache, fever, muscular pain, heart-burn, common cold, and allergies [2]. otc drugs have an advantage for the healthcare system as it boosts pharmacists’ skills along with the reduction of prescribed drug costs for the public. pharmacists are the first point of care while dispensing otc medications. however, the increasing availability of otc medicines increases the patient’s risk of subjecting to interactions and adverse effects of drugs. patients are encouraged to self-treatment which may lead to potential misuse of the medications [3]. the risk of otc medicines may include self-diagnosis, incorrect treatment, addiction in the long run, drug interactions, and poly-pharmacy. this may be particularly more dangerous in vulnerable populations such as the elderly [4]. the majority of patients don’t discuss the consumption of otc medicines with their treating physicians therefore they tend to be unaware of the potential risks of the medications. the increased administration of drugs by patients has increased drastically in the absence of professional help. otc medicines are consumed by customers and are not even recorded in many pharmacies [5]. the abuse of otc drugs is not often intentional and it can arise due to incorrect information about the drugs such as improper dosage form, lack of knowledge, drug-drug or drugfood interactions, and improper duration of the dose. there is a constant rising concern about the harms that can be associated with the use of otc medications. the objective of this review is to give a brief understanding of the potential abuse caused by otc medications. this review elaborates on the role of pharmacists in managing otc medications along with future steps that need to be taken to restrain the issue of otc medications worldwide. this review gives a perspective to the pharmacists to reduce otc medication abuse and strategies to implement betterment in the future. abuse of otc medications: the abuse of otc medications is a cause of a significant rate of morbidity and mortality [6]. most commonly abused otc medications include caffeine, antihistamine, antitussives, expectorants, ephedrine, pseudoephedrine, steroids, etc. tinsley ja et al reported a case study of a thirty-three-year-old woman who was admitted for inpatient treatment of ephedrine abuse [7]. it was reported that the patient took 25 mg of a capsule every day for appetite suppression. this otc ephedrine medication was lifted by the patient from the place where she worked which also led to her termination. the patient consumed the drug for 18 months. m baby et al over-the-counter medicine abuse vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 93 even after the patient’s numerous attempts to discontinue the drug she failed due to constant rebound. this addiction also interfered with her daily chores and children’s duties. the findings on the patients’ data were unremarkable. this implicates the need to address the lingering threat of otc abuse within the public domain. from the worldwide abuse of otc medications cold or cough products consisting of dextromethorphan, analgesics, antihistamines, and hypnotics are widely highlighted for their potential abuse [8]. a rising number of cases have been reported with the abuse and misuse of propylhexedrine (benzedrex), and otc nasal decongestants. misuse of propylhexedrine is associated with cardiac and psychiatric adverse effects [9]. cough and antihistamines are the most highly abused otc medication and sometimes the patients aren’t even aware of the abuse until they find the uncontrollable urge to consume medications. similarly, dimenhydrinate is an anti-histamine otc indicated for nausea and vomiting. it is more often misused for its psychotropic effects, including hallucinations and euphoria. in addition to frank abuse, there is also a potential for drug dependence upon long-term misuse [10]. although, there is a general trend in otc abuse of drugs from many different therapeutic classes and numerous dosage forms and drug delivery systems are implicated in otc drug abuse. one of the main notable reasons for otc medication abuse is an increase in access to medications [11,12]. medications are made accessible by converting from prescription to otc medications. these switches are done when a firm’s patent expires. however, in the case of prescription antihistamines like claritin, zyrtec, and allegra, blue cross was petitioned to be switched to otc by the parent companies [13]. patient autonomy is increased by switching medications which eventually increases the decisions of healthcare. a record of total of 12.9 billion dollars in profit was generated for the switch of cold, allergy, analgesics, and dermal products. the exact reason for this switch to otc medications has led to the abuse of medications. table 1list of most commonly abused otc medications class of drugs examples references antihistamines diphenhydramine and coricidin [14] cough medicines cough medicines containing dextromethorphan [15] codeinecontaining products compound analgesics (codeine with ibuprofen or paracetamol) and cough medicines [15] analgesics aspirin and acetaminophen [16] hypnotics sominex and nytol [14] laxatives (oral and rectal) sodium phosphate laxatives and laxatives containing bisacodyl [17] decongestants pseudoephedrine [18] the role of a pharmacist in otc medications: a patient has easier access to the pharmacist when compared to a general physician. numerous issues that are faced by the patients can be easily resolved such as indecision in the brand name of otc medication, proper medication use, and the exact duration of consumption of medications. there are many marketing strategies for different products which tend to confuse patients. one of the common marketing strategies is line extension [19]. a huge amount of capital is invested in otc medication advertisements and in-line extensions. once a manufacturer claims a brand name, other products under the manufacturer are sold under the same brand name which is called a line extension. for example, the brand name tylenol has many extensions under the same brand name as tylenol pm, tylenol cold, and tylenol cough. line extensions of the same brand can sometimes be confusing for patients due to multiple ingredients in the formulations. interaction of the patient with a pharmacist before buying any otc medications or even prescription medications can help in better decision-making for the administration of the drug. advertisements of otc medication can usually be misleading as the advertisement focuses more on the advantages of the drug with minimal information on drug interactions and safety concerns [20]. this advertisement may also be a cause of polypharmacy in patients. a pharmacist can give a better insight into the drug along with information on the side effects and safety concerns. in the recent years, there have been an evolving trend in the role of clinical pharmacists as a patient safety agent. as the pharmacists have a significant impact on public health and on improving patient's quality of life, there is need to incorporate minute details such as otc medications, medication adherence and many others to ensure positive patient outcomes [21]. the role of a pharmacist in otc medication abuse: since ancient times pharmacists have always focussed on the drug distribution system. over the past few years, there is an increasing number of incidences of chronic illness which has led to the prevention of the complex utilization of drugs and this has increased responsibility on the shoulders of pharmacists in providing broader services [22]. pharmacists have demonstrated a productive impact on hospitals by obtaining medical history and medical reconciliation of patients [23]. there is an increasing demand for healthcare to increase a pharmacist’s role. pharmacists are the initial point of contact while purchasing otc medications. pharmacists require regular monitoring of otc medication in a specific population [8]. for baby et al over-the-counter medicine abuse vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 94 example, for elderly patients pharmacists should be on a better lookout and also for patients who ask for frequent refills [24]. pharmacists can manage the abuse of otc medications by proactively improving their clinical skills and by giving oral and written information about the otc medications to the patients. there are various programs for the management of prescription drugs along with refills of prescription drugs such programs should also be formulated for otc medications. various methods were used by pharmacists in the past to control otc medication abuse, the most common three methods are keeping the compromised medications out of sight, questioning pharmacists upon refills of the medication, and refusing to sell any compromised products [4]. it was also proved that 62% of the pharmacists take measures to reduce otc medication abuse by not displaying implicated medications and refusing the sale of such medications. in a study conducted by frank et al to demonstrate the clinical management of dextromethorphan abuse [25]. it was found that dextromethorphan is an antitussive, inexpensive otc medication and that the abuse of this drug was reported by dissociative effect. huge amounts of ingestion of the drug can cause hypertension, tachycardia, and respiratory depression. therefore, pharmacists should be well aware of the dissociative effects of the abuse of dextromethorphan. pharmacists should also interview the patients before dispensing such medications with potential abuse. other techniques used by pharmacists are reporting otc medication abuse to a physician. similarly, programs like prescription drug monitoring log information about a patient upon visiting the pharmacy for refills. table 2common strategies by pharmacists to control otc medication abuse [8, 26] specific locations strategies initiated by pharmacists pharmacy 1. declining sales of implicated otc medications 2. immediately contact other pharmacies of the suspicious behaviour of the patient abusing otc medication 3. declaring products were out of stock 4. prevent medication by hiding the supplies 5. giving only a few amounts of medication patient participation 1. providing counselling to the patients on the potential abuse of the otc medications 2. raising awareness through the internet and support groups for advising patients 3. giving out information leaflets involvement of the doctor and other services 1. providing consultation and 2. engagement by the doctor 3. giving referral to the physician utilizing the services of private clinics 4. using special services of drug and alcohol abuse barriers to preventing otc medication abuse: there are many challenges faced by pharmacists in monitoring otc medication abuse. pharmacists are never in the practice of keeping records of otc medications and this creates a barrier to the necessary information for proper counselling decisions. in 2005 a survey was conducted by adrea et al to evaluate drugrelated problems [25]. the survey was conducted on community pharmacists and the pharmacists were constructed to record basic statistics of the patients such as drug interactions, prescription, and non-prescription drugs along with patient data. the results of the pharmacists showed 10,427 drug-related problems. most of the drugrelated problems were associated with drug-drug interactions. the conclusion of this study was that pharmacists are responsible for the proper use of prescription and otc medications however the role of pharmacists in healthcare still needs to be recognized. keeping in mind the potential abuse of otc medications the us federal law government passed the combat methamphetamine epidemic act of 2005 (cmea) [27]. this act was enacted to minter the amount the pseudoephedrine a person can purchase from a pharmacy. the main focus of this act was to decrease the illegal use of methamphetamine which can be produced from medications like ephedrine and pseudoephedrine which are commonly used cough and cold otc medications. the cmea has kept a limit on the purchase of nine grams of pseudoephedrine in a period of one month. the lack of pharmacists in monitoring the use of otc medications has led to further open doors for abuse of otc medications. moreover, the legal laws of otc medication distribution are not maintained and the laws are also not revised to keep up with pharmacists’ needs [25]. for example, a study conducted by tommy et al proved that in community pharmacies the pharmacists were overworked. the increased workload of pharmacists caused reduced attention for the patient’s otc medication abuse. pharmacists are present in limited numbers and pharmacy technicians play an important role in preventing otc medication abuse as well, they give an extra layer to patient safety [28]. behind the counter [btc] medications – a change maker: btc medications are medications that are the third category of drugs that are available after consultation with the pharmacist. the pharmacist performs screening, necessary tests, and counselling before dispensing the btc medication to the baby et al over-the-counter medicine abuse vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 95 patient. patients consuming btc medications could be suffering from increased blood pressure (bp), allergies, inflammation, and pain. otc medication with the potential risk of high abuse can be included in the btc medications list. if the pharmacists work in a collaborative manner with the patients they can lead to a safer use of medications. btc medications serve as a bridge between otc medications and prescription drugs. conclusion this review concluded the essential role of pharmacists in otc medication abuse. pharmacists need to be well informed about the otc medications along with their abuse. pharmacist vigilance can help reduce otc medication abuse. a better communication practice between pharmacists and patients can help in understanding the core of otc medication abuse. above all, the current health care system calls for more regulation on otc medications, particularly the ones with identified potential abuse. there needs to be a striking balance between access to otc medication and their risk of abuse. table 3potential drugs that can be shifted from otc medications to btc medications. medications example reference high cholesterol statins [29] hyperglycaemia insulin [30] cold remedies pseudoephedrine and ephedrine [7,27] schedule v cough syrups certain codeinecontaining products [30] emergency contraceptives plan b [29,30] painkillers painkillers with small amounts of codeine (up to 12.8 mg per tablet) and aspirin [23] sleep aid/allergy diphenhydramine [31,32] references 1. chautrakarn s, khumros w, phutrakool p: self-medication with over-the-counter medicines among the working age population in metropolitan areas of thailand. front pharmacol. 2021, 12: 10.3389/fphar.2021.726643 2. anderson jg: demographic factors affecting health services utilization: a causal model. med care. 1973, 11: 10.1097/00005650-197303000-00003 3. wazaify m, shields e, hughes cm, et al. societal perspectives on over-the-counter (otc) medicines. fam pract. 2005, 22: 10.1093/fampra/cmh723 4. hughes cm, mcelnay jc, fleming g. benefits and risks of selfmedication. drug saf. 2001, 24: 10.2165/0000201820012414000002 5. bond c, hannaford p. issues related to monitoring the safety of over-the-counter (otc) medicines. drug saf. 2003, 26: 10.2165/00002018-200326150-00001 6. williams jf, kokotailo pk. abuse of proprietary (overthecounter) drugs. adolesc. med. clin. 2006, 17: 10.1016/j.admecli.2006.06.006 7. tinsley ja, watkins dd. over-the-counter stimulants: abuse and addiction. mayo clin. proc. 1998, 73: 10.4065/73.10.977 8. cooper rj: ‘i can’t be an addict. i am.’ over-the-counter medicine abuse: a qualitative study. bmj open. 2013, 3: 10.1136/bmjopen-2013-002913 9. aschenbrenner ds. abuse or misuse of otc decongestant produces serious adverse effects. am. j. nurs. 2021, 121: 10.1097/01.naj.0000758484.08733.6f 10. schifano f, chiappini s, miuli a, et al. focus on over-thecounter drugs’ misuse: a systematic review on antihistamines, cough medicines, and decongestants. front. psychiatry. 2021, 12: 10.3389/fpsyt.2021.657397 11. albsoul-younes a, wazaify m, yousef am, et al. abuse and misuse of prescription and nonprescription drugs sold in community pharmacies in jordan. subst use misuse. 2010, 45: 10.3109/10826080802490683 12. soller rw. evolution of self-care with over-the-counter medications. clin ther. 1998, 20: 10.1016/s01492918(98)800180 13. eickhoff c, hämmerlein a, griese n, et al. nature and frequency of drug-related problems in self-medication (over-the-counter drugs) in daily community pharmacy practice in germany. pharmacoepidemiol drug saf. 2012, 21: 10.1002/pds.2241 14. griffiths rr, johnson mw. relative abuse liability of hypnotic drugs: a conceptual framework and algorithm for differentiating among compounds. j. clin. psychiatry. 2005, 66. 15. baker sd, borys dj. a possible trend suggesting increased abuse from coricidin® exposures reported to the texas poison network: comparing 1998 to 1999. vet hum toxicol. 2002, 44. 16. johnston ld, miech ra, o’malley pm, et al. monitoring the future: national survey results on drug use 1975-2018. 2018 overview: key findings on adolescent drug use. natl inst drug abus natl institutes heal. 2019. 17. lessenger je, feinberg sd. abuse of prescription and over-thecounter medications. j. am. board fam. med. 2008, 21: 10.3122/jabfm.2008.01.070071 18. pomeranz jl, taylor lm, bryn austin s. over-the-counter and out-of-control: legal strategies to protect youths from abusing products for weight control. am j public health. 2013, 103: 10.2105/ajph.2012.300962 19. encyclopedia of pharmaceutical science and technology, fourth edition. 2013. 10.1081/e-ept4 20. covington tr. nonprescription drug therapy: issues and opportunities. am. j. pharm. educ. 2006, 70: 10.5688/aj7006137 21. badmanaban r, mandumpala jj, krupamol joy, et al. a study on pharm d students present views and future plans. world j curr med pharm res. published online first: 2020. 10.37022/wjcmpr.vi.135 22. derry s, moore ra, mcquay hj. single dose oral codeine, as a single agent, for acute postoperative pain in adults. cochrane database syst rev. published online first: 2010. 10.1002/14651858.cd008099.pub2 baby et al over-the-counter medicine abuse vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 96 23. mcbride aj, pates r, ramadan r, et al. delphi survey of experts’ opinions on strategies used by community pharmacists to reduce over-the-counter drug misuse. addiction. 2003, 98: 10.1046/j.1360-0443.2003.00345.x 24. saljoughian m. avoiding medication errors. u.s. pharm. 2020, 45: 10.1201/9781420071764-c10 25. hämmerlein a, griese n, schulz m. survey of drug-related problems identified by community pharmacies. ann pharmacother. 2007, 41: 10.1345/aph.1k207 26. schmitges fw, radovani e, najafabadi hs, et al. attachment-1 copy 2.jpeg.pdf. skelet muscle. 2016, 6. 27. murty s, sansgiry ss. pseudoephedrine laws in the us are we doing enough? [2]. ann. pharmacother. 2006, 40: 10.1345/aph.1h032 28. westerlund t, almarsdóttir ab, melander a. drug-related problems and pharmacy interventions in community practice. int j pharm pract. 1999, 7: 10.1111/j.2042-7174.1999.tb00947.x 29. ried ld, huston sa, kucukarslan sn, et al. risks, benefits, and issues in creating a behind-the-counter category of medications. j am pharm assoc. 2011, 51: 10.1331/japha.2011.10134 30. steven pray w, pray ge. behind-the-counter products: a third class of drugs. u.s. pharm. 2011, 36. 31. garcía-sáiz mm, aldea-perona a, rodríguez-jiménez c, et al. presentation of the book ‘guide to drug use during pregnancy’. basic clin pharmacol toxicol. 2011, 109. 32. disler rt, gallagher rd, davidson pm, et al. factors impairing the postural balance in copd patients and its influence upon activities of daily living. eur respir j. 2019, 15. how to cite this article: martin baby john, alasandra rose ms, janice jacson mandumpala. over-the-counter medicine abuse – a pharmacist’s emerging responsibility. indian j pharm drug studies. 2022: 1(3) 92-96. funding: none conflict of interest: none stated microsoft word ijpds_3688-galley mandumpala medication reconciliation vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 1 review article medication reconciliation – a patient safety strategy janice jacson mandumpala from, pharm d intern, department of pharmacy practice, nirmala college of pharmacy, muvattupuzha,kerala, india. correspondence to: janice jacson mandumpala, pharm d intern, department of pharmacy practice, nirmala college of pharmacy, muvattupuzha, kerala, india – 686661. email: janice.jacson@gmail.com abstract patient safety is an international concern and mandates the attention of healthcare professionals. recently, it has been recognized that multiple strategies and members of the healthcare team from the treating physician to the dispensing pharmacist have a key role to play in assuring patient safety. one such role is that of the clinical pharmacist and the medication reconciliation conducted by them. it is done to get a thorough understanding of the patient medical history, medication history, drug allergy history and many others that could be a loophole for patient harm. this review summarizes a brief review about what is patient safety, and the patterns of employing medication reconciliation in different clinical scenarios that is the emergency department, in transitions of care and in geriatric care. it is specifically important to address the role of growing technology in the same field and so we have also enlisted the ongoing efforts done in the same domain. this review also addresses the role of clinical pharmacists and their emerging responsibility in maintaining a patient safety culture. key words: medication reconciliation, patient safety, emergency department, geriatric care, clinical pharmacists edication reconciliation has the potential to reduce significant morbidity and mortality. this in turn causes financial strain on the healthcare system and is a source of preventable costs. it is an important piece of the medication puzzle. medication reconciliation is an inevitable aspect while ensuring patient safety and quality. it is a major intervention that tackles the issue of medication discrepancies and avoids patient harm, particularly during care transitions. medication reconciliation clarifies, corrects and specifies the medications the patients are consuming at different durations of their hospitalization, followed by the necessary corrections on the medical records. a medication reconciliation program also reduces confusion regarding the medications that the patient is using and has a major contribution in preventing unintentional medication changes, all of which cause a major resource strain on the hospital. this paves the way to maintain a standardized pattern of patient care in a care facility [1,2]. according to the joint commission, the steps involved in medication reconciliation are as follows [3]: ‘(1) develop a list of current medications; (2) develop a list of medications to be prescribed; (3) compare the medications on the two lists; (4) make clinical decisions based on the comparison; and (5) communicate the new list to appropriate caregivers and to the patient.’ patient safety culture nurturing a culture of safety is the core aspect of promoting quality care among patients. safety culture within healthcare institutions has proven to reduce errors, and mortality and reduce adverse drug events [4–6]. it influences clinicians, clinical pharmacists, nurses and other healthcare professionals by providing cues about the importance of ensuring patient safety along with other patient outcomes. the promotion of patient safety culture is a constellation of interventions targeted at minimizing patient harm. the various existing patient safety strategies are team training, interdisciplinary rounding or executive walk rounds, and unit-based strategies [7]. one among them is the medication reconciliation process. medication reconciliation can be performed by clinical pharmacists or pharmacy technicians, electronic medical record tools, and patient-centered strategies. studies have implied the emerging role of clinical pharmacists in a hospital setting where there have been instances in which patient medication histories are frequently recorded inaccurately by physicians during admission which results in medication-related errors and compromised patient safety [8]. contributions from a clinical pharmacist to improvise patient outcomes can have a huge impact in the m mandumpala medication reconciliation vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 2 healthcare field. clinical pharmacists have ventured out of the pharmacy to ward round participation and in intensive care units [9]. therefore, patient safety culture can be developed at the hands of a clinical pharmacists through the medication reconciliation process. medication reconciliation in transitions of care transitions in care including admission and discharge from a hospital put a patient at risk for various aspects related to treatment from miscommunication to inadvertent information loss. deficits in information transfer at hospital discharge can adversely affect patient safety. possible interventions include computer-generated summaries and standardized formats that facilitate the timely and precise transfer of reliable patient information to physicians and make error-free discharge summaries [10]. most of the transitions are unplanned, and often result from unanticipated or emergency medical problems that occur in during nights, weekends or any time of the day, involving physicians who may not have an ongoing relationship with the patient. such transitions happen quickly and in a very short time frame which creates a barrier to a timely response from the formal and informal support systems [11]. a study conducted by forster et al reported that among the adverse events that occurred after the hospital half of them were preventable or ameliorable. this study important implications for quality improvement at the time of discharge. there is a need to follow patients more closely after discharge [12]. in alliance with the findings of the above study, a longitudinal multi-centre study conducted in a hospital examining the effectiveness of medication reconciliation at admission, discharge and post-discharge has come to similar conclusions. at admission, discharge and postdischarge, changes in medication regimens were necessary for 66.5%, 62.9% and 52.8% of patients, respectively during the medication reconciliation process [13]. a recent meta-analysis conducted by alemayehu et al has conclusively shown that pharmacist-led medication reconciliation programs have positive clinical outcomes at hospital transitions. they found a reduction in the rate of all cause readmissions (19%), all-cause emergency visits (28%) and ade-related hospital visits (67%) [14]. this implies that a pharmacist can effectively target fragments of services across the entire spectrum of care transitions. therefore, with consistent time and effort patient safety is achievable. medication reconciliation in emergency department clinical pharmacy services such as medication reconciliation have immense benefits while identifying medication discrepancies and potential adverse drug events in the emergency departments. in a meta-analysis conducted by chou et al the researchers concluded that a pharmacy-led medication reconciliation reduced the medication discrepancies by 68% in the emergency department. similarly, patients with poly-pharmacy and comorbidities received marked benefits with a reduction in medical discrepancies at the hands of a pharmacy-led medication reconciliation [15]. in another randomized controlled trial conducted in 2017, the authors analysed the improvement in admission medication reconciliation with pharmacists or pharmacy technicians in the emergency departments. as per the findings of the trial, it was found that pharmacists and pharmacy technician-led medication reconciliation in the emergency department reduced the admission medication history errors and admission medication order errors by over 80% [16]. accurately documenting a patient’s home medications and allergies at the time of admission in an emergency department improves the efficiency and quality of patient care. the multiple benefits of employing a pharmacist-led medication reconciliation in the emergency department are as follows: reduction in length of hospital stay, decreased future emergency room visits, reduction in drug-related admissions (such as adverse drug reaction, drug-related complication, hypersensitivity), decreased medication errors (especially the preventable ones), reduction in adverse events following drug administration, proper documentation of patient allergies, improved accuracy and completeness of patient profiles and most importantly costeffectiveness [17]. medication reconciliation and geriatric care medical care of the elderly is challenging work and is an essential part of the daily routine of a general practitioner. the older population is most often accompanied by multiple comorbidities, heavy prescriptions, complex medication regimens and above all polypharmacy. moreover, decreased organ function and physiological reserves pose an exaggerated challenge in elderly care. while prescribing the clinician has to be extra cautious about the compromised pharmacokinetics due to ageing and the associated drug interactions. implementation of clinical pharmacy services such as medication reconciliation, medication order review and discharge summary review minimizes the chance of exposing vulnerable population stratum to threats of medication errors [18]. medication reconciliation involving therapeutic optimization is essential to ensure the safety of geriatric patients. however, its impact post discharge is hampered due to less or no recognition by general practitioners [19]. mandumpala medication reconciliation vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 3 similarly, iatrogenic effects contribute largely to emergency admissions among elderly people. clinical pharmacists play a key role in reviewing and monitoring a patient's medication chart, this is an inevitable fragment of the medication reconciliation process. in a study conducted among geriatric patients by beckett et al, there was a 23% improvement in the medication profile appropriateness when a pharmacist-led medication reconciliation was conducted within 24 hours of admission [20]. therefore, targeting geriatric patients for medication reconciliation is of substantial benefit to both the healthcare system and the individual. medication reconciliation and technology electronic support can be a useful tool for improving the present scenario of the medication reconciliation process. to implement electronic support in the medication reconciliation process there is a need to have properly designed tools and a proper context for implementation [21]. however, there is a lack of scientific evidence for the impact of technology on identifying medication discrepancies during the medication reconciliation process. in a recent meta-analysis conducted in 2016, it was shown that electronic tools can reduce the incidence of medication discrepancies. as per the findings of this study, there was a reduction of 45% in medication discrepancies after the implementation of electronic tools for medication reconciliation [22]. in another randomized controlled trial, it was found that electronic medication reconciliation reduced adverse drug events, medication discrepancies and other adverse outcomes compared with usual care [23]. therefore, it can be understood that enhanced electronic medication reconciliation systems within the hospital setting can be a useful strategy for improvising clinical pharmacy services. role of clinical pharmacists in medication reconciliation pharmacists are the most ideal candidates to conduct medication history interviews and reconcile medications as they are more aware of the drug names, characteristics, effects, dosage forms, administration, adverse drug profile, side effects and toxicity profile of drugs. an incomplete or inaccurate medication history may lead to compromised patient safety and pharmacists have the expertise and experience to scrutinize such errors and optimize a patient’s drug therapy through clinical interventions (figure 1). the biomedical literature also indicates that pharmacist conducted medication reconciliation is more accurate, saves money, and increases patient safety [24–26]. a study conducted in a tertiary care hospital in china has also revealed that medication reconciliation performed by pharmacist trainees can minimize unintentional medication discrepancies [27]. in another observational study conducted in croatia, it was evidenced that clinical pharmacist-led medication reconciliation was an important tool in detecting medication discrepancies and preventing adverse patient outcomes [28]. similarly, in a study conducted in saudi arabia, the researchers have concluded that medication reconciliation is crucial in reducing medication errors and pharmacists are the ideal resources to avoid medicationrelated errors and the associated risks and complications [8]. in alliance with the above findings, it can be suggested that not only pharmacists/ clinical pharmacists/ pharmacy technicians have a positive role in the medication reconciliation process but also pharmacy students. in a recent study conducted to analyse the benefits of employing pharmacy students in the medication reconciliation process, it was found that pharmacy students are a potential workforce solution. a particular finding from the study was that the fourth pharmacy students completed the best possible medication histories and identified discrepancies with prescribed medications for patients. also, the pharmacy students were able to identify around 70% of medication discrepancies in the admitted patients during the medication reconciliation [29]. therefore, pharmacy students can provide a beneficial service to the hospital by extrapolating their skills in clinical pharmacy services. conclusion medication reconciliation is an emerging tool to ensure patient safety in the hospital. it requires tremendous efforts from each healthcare professional to recognize treatment related harm. in such scenarios, a clinical pharmacist can contribute immensely and recognize errors or certain contributions in a way that can help improve patient outcomes. medication reconciliation is particularly important when a geriatric patient is being admitted, with multiple comorbidities and the complex medication regimen, poly-pharmacy and non-adherence become an alarming threat. a similar situation arises in the emergency department and in the mandumpala medication reconciliation vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 4 transitions of care where medications may go omitted or missed. therefore, it is imperative to introduce timely patient safety tools to maintain the ongoing effort to achieve patient outcomes. references 1. rozich jd, howard rj, justeson jm, et al standardization as a mechanism to improve safety in health care. jt comm j qual saf. 2004; 30: 10.1016/s1549-3741(04)30001-8 2. howard rl, avery aj, howard pd, et al. investigation into the reasons for preventable drug related admissions to a medical admissions unit: observational study. qual saf heal care. 2003, 12:. 10.1136/qhc.12.4.280 3. sentinel event the joint commission. accessed: 2022; https://www.jointcommission.org/resources/sentinelevent 4. braithwaite j, westbrook mt, travaglia jf, et al. cultural and associated enablers of, and barriers to, adverse incident reporting. qual 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de winter s, spriet i, indevuyst c, et al. pharmacistversus physician-acquired medication history: a prospective study at the emergency department. qshc. 2010; 19: 10.1136/qshc.2009.035014 27. guo q, guo h, song j, et al. the role of clinical pharmacist trainees in medication reconciliation process at hospital admission. ijcp. 2020; 42: 10.1007/s11096-020-01015-2 28. marinović i, marušić s, mucalo i, et al. clinical pharmacist-led program on medication reconciliation implementation at hospital admission: experience of a single university hospital in croatia. cmj. 2016; 57: 10.3325/cmj.2016.57.572. 29. deep l, schneider cr, moles r, et al. pharmacy student-assisted medication reconciliation: number and types of medication discrepancies identified by pharmacy students. pp. 2021; 19:10.18549/pharmpract.2021.3.2471. how to cite this article: janice jacson mandumpala. medication reconciliation – a patient safety strategy. indian j pharm drug studies. 2023; 2(1) 1-4. funding: none conflict of interest: none stated https://www.jointcommission.org/resources/sentinelevent tahilani et al artificial intelligence in pharmaceutical industries vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 47 review article the era of artificial intelligence in pharmaceutical industries a review praveen tahilani1, hemant swami2, gaurav goyanar2, shivani tiwari1 from, 1 sagar institute of research and technology pharmacy, bhopal, mp, 2 school of pharmaceutical science, sage university, indore m.p. corresponding to: mr. praveen tahilani, sagar institute of research and technologypharmacy, bhopal, m.p. email: tahilanipraveen@gmail.com, tel: 8770501549 abstract as a growing sector, the era of artificial intelligence, machine learning and data science in the pharmaceutical industry contributes in the drug discovery process, giving emphasis on how new technologies have improved effectiveness. as in the current scenario artificial intelligence including machine learning may be considered the future for a wide range of disciplines and industries specially the pharmaceutical industry. as we know today pharmaceutical industries producing a single approved drug cost the company millions with many years of rigorous testing prior to its approval, reducing costs and time is of high interest. the involvement of artificial intelligence will be useful to the pharmaceutical industry and also be of interest to anyone doing research in chemical biology, computational chemistry, medicinal chemistry and bioinformatics. key words: artificial intelligence, pharmaceutical, machine learning, research, chemistry rtificial intelligence (ai) is a branch of study that combines intelligent machine learning, particularly intelligent computer programmes that produce outcomes like human attention processes [1]. this procedure typically entails gathering data, creating effective mechanisms for using that data, illuminating precise or approximative conclusions, and making oneself modifications or adjustments [2]. ai is typically used to analyse machine learning to mimic human cognitive functions [2, 3]. ai is used to conduct analyses that are more accurate and to get helpful interpretation [3]. according to this the development and invention of ai applications are frequently linked to the concern about the threat of unemployment. however, practically all developments in the use of ai technology are being applauded because of the industry's massive reliance on its effectiveness. the practical uses of ai technology in numerous technological and scientific disciplines have recently made it a highly vital component of industry [3, 4]. the emerging movement to accept ai applications in pharmacy, including drug discovery, formulation creation for drug administration, and other healthcare applications, has already moved from hype to hope [5, 6]. predicting in vivo reactions, therapeutic drug pharmacokinetic characteristics, appropriate dose, etc. is also made possible by the use of ai models [2, 7]. according to the importance of pharmacokinetic prediction of drugs, the uses of in silico models facilitate their effectiveness and inexpensiveness in the drug research [8]. artificial intelligence (ai) has become more prevalent in a number of societal fields, most notably the pharmaceutical industry. in this review, we focus on how ai is being used in a variety of pharmaceutical industry fields, such as drug discovery and development, drug repurposing, increasing pharmaceutical productivity, and clinical trials, among others. this use of ai lessens workload of human workers while also achieving goals quickly. we also talk about how various ai tools and methodologies interact, current problems and solutions, and the potential applications of ai in the pharmaceutical sector. the pharmaceutical industry has dramatically increased its data digitization during the last few years. the difficulty of gathering, examining, and applying that knowledge to address challenging healthcare situations is a challenge that comes along with digitalization [9]. because ai can handle massive amounts of data with improved automation, this encourages its usage [10]. technology-based artificial intelligence (ai) systems can replicate human intelligence by using a variety of cutting-edge tools and networks. however, it does not pose a danger to totally replace human physical presence [11, 12]. ai makes use of hardware and software that can analyse and learn from input data to make independent judgements for achieving predetermined goals. as this review describes, its uses in the pharmaceutical industry are constantly expanding. according to the mckinsey global institute, the rapid advances in aia mailto:tahilanipraveen@gmail.com tahilani et al artificial intelligence in pharmaceutical industries vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 48 guided automation will be likely to completely change the work culture of society [13, 14]. ai: networks and tools: ai encompasses a number of approach fields, including machine learning as its core paradigm as well as reasoning, knowledge representation, and solution search (ml). in machine learning (ml), algorithms are used to find patterns in a set of data that has been further categorised. deep learning (dl), a branch of machine learning that uses artificial neural networks (anns). these are a collection of intricately connected computing components that simulate the electrical impulse transmission in the human brain by using "perceptons" that are similar to biological human neurons [15]. every node in an ann receives a different input, and they all work together or alone to solve issues by converting inputs to outputs using algorithms [16]. anns involve various types, including multilayer perceptron (mlp) networks, recurrent neural networks (rnns), and convolutional neural networks (cnns), which utilize either supervised or unsupervised training procedures [17, 18]. classification of ai according to their ability, ai can be categorized as i) artificial narrow intelligence (ani) or weak ai: it performs a narrow range task, i.e., facial identification, steering a car, practicing chess, traffic signalling, etc. ii) artificial general intelligence (agi) or strong ai: it performs all the things as humans and also known as human level ai. it can simplify human intellectual abilities and able to do unfamiliar task. iii) artificial super intelligence (asi): it is smarter than humans and has much more activity than humans drawing, mathematics, space, etc. according to their presence and not yet present, ai can be classified as follows i) type 1: it is used for narrow purpose applications, which cannot use past experiences as it has no memory system. it is known as reactive machine. there are some examples of this memory, such as a ibm chess program, which can recognize the checkers on the chess playing board and capable of making predictions. ii) type 2: it has limited memory system, which can apply the previous experiences for solving different problems. in automatic vehicles, this system is capable of making decisions there are some recorded observations, which are used to record further actions, but these records are not stored permanently. iii) type 3: it is based upon “theory of mind”. it means that the decisions that human beings make are impinged by their individual thinking, intentions and desires. this system is nonexisting ai. iv) type 4: it has self-awareness, i.e., the sense of self and consciousness. this system is also non-existing ai. artificial intelligence and robotics: robotics and artificial intelligence share a shared origin and a long history of interaction and scholarly debate. it might be argued that not all machines are robots, and that artificial intelligence likewise has concerns for virtual agents. robots are produced as hardware and artificial intelligence is a hypothesis. the two are related because a software agent that controls the robot examines data from these sensors, decides what to do next, and then directs the actions to be taken in the real environment. it has numerous robotics applications [20]. patients will also look into potential drug options as they become more involved in their healthcare decision. through target audience marketing, pharmaceutical companies can further assure the right information is presented at the right time to facilitate informed patent and provider discussions [19]. “it is time for connected pharma”: however, progress is far from uniform and progress is likely to be “lumpy” at best. ai technology is well on its way to becoming ubiquitous and has huge scope, enhancing technology at many levels, leading to much better, faster patient outcomes. pharmaceutical automation: assisted by artificial intelligence industrialization produced automation because it was necessary to boost output, produce items of consistently high quality, and free people from dangerous and taxing tasks. technology advancements today provide the fundamental foundation of automation. the majority of pharma players are aware of the advantages of implementing new technology, but there is still a persistent and alarming gap between strategy and an organization's capacity to implement a workable data analytics solution [21]. the adoption of ai allows for learning from real time data.  identifying the right candidates for clinical trials.  processing real time patient feedback.  integrating data exchanges with partners.  distributors and caregivers. there are just few examples on how to improve drug discovery outcomes, while aligning operational efficiencies to deliver better care to the patients, often getting the right medication to the right patient at right time is really about getting right information in front of healthcare provider. armed with complete real-time drug insights, doctors are able to choose right prescription for the best possible outcome. automation applications continue to grow with enabling technologies such as [22]: 1. wireless 2. nanotechnology tahilani et al artificial intelligence in pharmaceutical industries vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 49 3. advance storage and memory 4. sensors and analyzers 5. advance software algorithms 6. artificial intelligence. ai in advancing pharmaceutical drug devlopment: the subsequent inclusion of a novel therapeutic molecule into an appropriate dosage form with the requisite delivery properties is necessary. the traditional method of trial and error can be replaced in this area by ai [23]. with the use of qspr, a variety of computational methods can be used to overcome challenges with stability, dissolution, porosity, and other aspects of formulation design [24]. decision-support tools operate through a feedback mechanism to monitor the entire process and sporadically adjust it [25]. they employ rulebased systems to choose the type, nature, and quantity of the excipients based on the physicochemical properties of the medicine. based on the input parameters, the model expert system (mes) decides and gives suggestions for formulation development. ann, in contrast, ensures hassle-free formulation development by using backpropagation learning to link formulation parameters to the intended response, which is jointly regulated by the control module [26]. the influence of the powder's flow behaviour on the die-filling and tablet compression process has been studied using a variety of mathematical tools, including computational fluid dynamics (cfd), discrete element modelling (dem), and the finite element method [27, 28]. the effect of tablet geometry on its disintegration profile can also be studied using cfd [29]. the quick manufacture of pharmaceutical items may benefit greatly from the integration of these mathematical models with ai. ai in pharmaceutical marketing: modern manufacturing systems are attempting to impart human knowledge to robots as a result of the growing complexity of production processes, as well as the growing desire for efficiency and greater product quality [30]. the pharmaceutical business may benefit from the application of ai in manufacturing. utilizing the automation of many pharmaceutical activities, tools like cfd use reynolds-averaged navier-stokes solvers technology to examine the effects of agitation and stress levels in various pieces of equipment (such stirred tanks). similar methods, including big eddy simulations and direct numerical simulations, use sophisticated techniques to address challenging flow problems in manufacturing [31]. the innovative chapter platform, which uses a scripting language called chemical assembly and several chemical codes, aids digital automation for the synthesis and manufacture of molecules [32]. sildenafil, diphenhydramine hydrochloride, and rufinamide have all been successfully manufactured using this method, and the yield and purity are noticeably similar to those obtained through manual synthesis [33]. ai technology can effectively complete granulation in granulators with capacities ranging from 25 to 600 l [34]. neuro-fuzzy logic and technology were used to correlate key factors with their answers. in order to anticipate the proportion of granulation fluid to be supplied, the necessary speed, and the diameter of the impeller in both geometrically identical and dissimilar granulators, they developed a polynomial equation [35]. ai in quality control and quality assurance: a balance of different factors must be achieved throughout the production of the desired product from raw materials [36]. it takes human intervention to maintain batch-to-batch consistency and conduct quality control testing on the products. this illustrates the need for ai deployment at this time and may not be the optimal strategy in every situation [37]. by implementing a "quality by design" approach, the fda modified current good manufacturing practices (cgmp) in order to better understand the crucial process and precise standards that determine the ultimate quality of the pharmaceutical product [38]. ai in clinical filed: clinical trials take about 6-7 years to complete and include a substantial financial outlay in order to determine the safety and effectiveness of a medicinal product in people for a specific illness condition. only one out of every ten compounds that undergo these trials, however, receive successful clearance, which represents a significant loss for the industry [39]. these failures may be the result of bad infrastructure, poor technical requirements, and poor patient selection. with the use of ai, these problems can be minimised thanks to the abundance of digital medical data that is currently available [40]. conclusion as a result of the ai technological approaches' belief that humans can imagine knowledge, solve problems, and make decisions, there has been an increase in interest in using ai technology for analysing and interpreting some critical areas of pharmacy, such as drug discovery, dosage form design, poly pharmacology, hospital pharmacy, etc. it has been found to be beneficial to use automated workflows and databases for efficient studies that apply ai techniques. the construction of novel hypotheses, strategies, predictions, and assessments of many connected elements can be done with the ease of less time consumption and affordability thanks to the usage of ai technologies. references 1. mak kk, pichika mr. artificial intelligence in drug development: present status and future prospects. drug discov today. 2019; 24(3):773-80. 2. hassanzadeh p, atyabi f, dinarvand r. the significance of artificial intelligence in drug delivery system design. adv drug deliv rev. 2019; 151:169-90. 3. russel s, dewey d, tegmark m. research priorities for robust and beneficial artificial intelligence. ai mag. 2015;36(4):105-14. tahilani et al artificial intelligence in pharmaceutical industries vol 1 | issue 2 | 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research on marketing and sales in the artificial intelligence age. mwais; 2018. 12. wirtz b.w. artificial intelligence and the public sector— applications and challenges. int. j. public adm. 2019; 42:596– 615. 13. smith r.g., farquhar a. the road ahead for knowledge management: an ai perspective. ai mag. 2000; 21 171–178. 14. lamberti m.j. a study on the application and use of artificial intelligence to support drug development. clin. ther. 2019; 41:1414–1426. 15. beneke f., mackenrodt m.-o. artificial intelligence and collusion. iic int. rev. intellectual property competition law. 2019; 50:109–134. 16. steels l., brooks r. routledge; 2018. the artificial life route to artificial intelligence: building embodied, situated agents. 17. bielecki a., bielecki a. foundations of artificial neural networks. in: kacprzyk janusz., editor. models of neurons and perceptrons: selected problems and challenges. springer international publishing; polish academy of sciences, warsaw, poland. 2019. pp. 15–28. 18. kalyane d. artificial intelligence in the pharmaceutical sector: current scene and future prospect. in: tekade rakesh k., editor. the future of pharmaceutical product development and research. elsevier; 2020. pp. 73–107. 19. russell s, dewey d, tegmark m. research priorities for robust and beneficial artificial intelligence. ai magazine. 2015 dec 31; 36(4):105-14. 20. lakshmi teja t, keerthi p, debarshi datta nb. recent trends in the usage of robotics in pharmacy, indian journal of research in pharmacy and biotechnology, 2(1), 1038-1043. 21. yussupova n, kovács g, boyko m, bogdanova d. models and methods for quality management based on artificial intelligence applications. acta polytechnica hungarica. 2016; 13(3):45-60 22. brady m. artificial intelligence and robotics. springer, berlin, heidelberg. in robotics and artificial intelligence 1984, 47-63. 23. guo m. a prototype intelligent hybrid system for hard gelatin capsule formulation development. pharm technol 2002;6:44–52. 24. mehta c.h. computational modeling for formulation design. drug discovery today. 2019; 24:781–788. 25. zhao c. toward intelligent decision support for pharmaceutical product development. j. pharm. innovation. 2006; 1:23–35. 26. rantanen j., khinast j. the future of pharmaceutical manufacturing sciences. j. pharm. sci. 2015; 104:3612–3638. 27. ketterhagen w.r. process modeling in the pharmaceutical industry using the discrete element method. j. pharm. sci. 2009; 98:442–470. 28. chen w. mathematical model-based accelerated development of extended-release metformin hydrochloride tablet formulation. aaps pharmscitech. 2016; 17:1007–1013. 29. meziane f. intelligent systems in manufacturing: current developments and future prospects. integr. manuf. syst. 2000; 11:218–238. 30. steiner s. organic synthesis in a modular robotic system driven by a chemical programming language. science. 2019; 363:eaav2211. 31. faure a. process control and scale-up of pharmaceutical wet granulation processes: 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2013; 31:72–81. 38. reklaitis r. pharmahub; 2008. towards intelligent decision support for pharmaceutical product development. 39. wang x. 2009 international conference on computational intelligence and software engineering. intelligent quality management using knowledge discovery in databases; ieee; 2009. 1–4. 40. hay m. clinical development success rates for investigational drugs. nat. biotechnol. 2014; 32:40–51. how to cite this article: tahilani & swami. the era of artificial intelligence in pharmaceutical industriesa review indian j pharm drug studies. 2022: 1(2); 47-50. funding: none conflict of interest: none stated tahilani et al. nutraceuticals and fortified foods supplements in india vol 1 | issue 1 | apr jun 2020 indian j pharm drug stud | 1 review article nutraceuticals and fortified foods supplements in india: challenges and opportunities – a comprehensive review praveen tahilani1, jitendra banweer1, d p chatterjee2, gaurav goyanar2, abhishek sharma1 from 1sagar institute of research technology &science pharmacy, bhopal, 2department of pharmaceutical science, sage university, indore m.p correspondence to: prof. praveen tahilani, sagar institute of research technology & science pharmacy bhopal, india, email: tahilanipraveen@gmail.com abstract background: as the growing interest in the overall health of the community the term nutraceuticals, functional foo d ingredients, and dietary supplements are gaining importance worldwide. objective: the study aimed to identify and study th e importance of food fortification and the importance of nutraceuticals for their efficacy and effectiveness in india. methods pubmed and google scholar were searched and only article and information relevant to the topic was selected for review. results: a total of 53 articles were selected for review which included information about the nutraceuticals and fortifie d foods supplements. conclusion: there are no scientifically proven functional foods for improving public health. there is a requirement to change the platform of a balanced and nutritious diet required for healthy people. key words: fortification, nutraceuticals, functional foods, public health, dietary supplements unctional foods are termed as products that resemble traditional foods but possess different physiological benefits. however, nutraceuticals are those derived from foods, but are used in the medicinal form of pills, capsules or liquids and again have demonstrated physiological benefits. in other parts of the world, the latter group has now been termed under a new category as natural health products that promote health. food fortification (ff) is defined as the addition of one or more essential nutrients to a food, whether or not it is normally contained in the food, to prevent or correct a demonstrated deficiency of one or more nutrients in the population or specific population groups [1]. fortification therefore differs from enrichment, which is the process of maintaining the nutrients to a food removed during the process or production. ff includes many process like bio fortification, microbial bio fortification and synthetic biology, commercial and industrial fortification, and home fortification. as we know that bio fortification involves creating micronutrient crops using traditional breeding techniques and biotechnology, the term biotechnology (genetic engineering) is to bio fortify staple crops is more modern and has gained much attention in recent years. example of this approach is the transgenic ‘golden rice containing twice the normal levels of iron and significant amounts of beta-carotene [2]. microbial bio fortification involves method using probiotic bacteria (mostly lactic acid bacteria), which ferment to produce carotene either in the foods we eat or directly in the human intestine [3]. rice and wheat are the main staple foods in india; especially the eastern and southern parts mostly depend on rice while the northern and western part of the country depends on wheat. from the world scenario, on average 30% of calories come from rice and it could reach up to more than 70% in some low-income country [4]. according to the gain report, the domestic consumption of milled rice is shown at about 97.6 mmt in 2017/18 and wheat consumption has been given at about 93 million. moreover, the milling process causes the loss of almost all vitamins and minerals of nutritional importance [5]. as we know that the principle reason for the growth of the functional food market is necessity of the current population and health trends. as the populations are aging, includes life expectancy continues to rise, as does the contribution made by older individuals to the total population. people today are more nutrition-concern than ever before, their interest in healthrelated information being met by many courses of information. [6] f mailto:tahilanipraveen@gmail.com tahilani et al. nutraceuticals and fortified foods supplements in india vol 1 | issue 1 | apr jun 2020 indian j pharm drug stud | 2 technical information: as we know that all foods are functional to some extent because all foods provide taste, aroma and nutritive value. as the foods are now being examined intensively for added physiologic benefits, which may reduce chronic disease risk or otherwise optimize health. it is these research efforts that have led to the worldwide interest in the growing food category and now recognized as “functional foods.” [7] another term often used as a synonym with functional foods is “nutraceuticals,” several factors are responsible for the fact that this is one of the most promising areas of research in the nutrition sciences today which gives an emphasis in nutritional and medical research on associations between diet and dietary constituents and health benefits, and a favorable regulatory environment including the consumer selfcare phenomenon, and rapid growth in the market for health and wellness products is observed [8]. according to the department of health and human services, diet plays a role in 5 of 10 of the leading causes of death. an accumulating body of research now suggests that consumption of certain foods or their associated physiologically active components may be linked to disease risk reduction [9]. functional foods of animal origin: recently the most popular class of physiologically-active components derived from animal products are the (n-3) fatty acids, predominantly found in fatty fish such as salmon, tuna, mackerel, sardines and herring [10]. as we know that dha is a vital component of the phospholipids of cellular membranes, especially in the brain and retina of the eye, and is necessary for their proper functioning [11]. functional foods of plant origin: many plant foods or physiologically active ingredients derived from plants have been investigated for their role in disease prevention and health. however, only a small number of these have had substantive clinical documentation of their health benefits. an even smaller number have surpassed the rigorous standard of “significant scientific agreement” required by the fda for authorization of a health claim. these plant foods currently eligible to bear an fda-approved health claim include oat soluble fiber [12], soluble fiber from psyllium seed husk [13] soy protein [14] and steroland stanol-ester–fortified margarine [15]. nutraceuticals and functional food: the term nutraceuticals is a combination of a nutrition and medicine, this term was introduced in 1989 [16]. nutraceuticals are defined as “any substance that may be considered a food or part of a food and provides medical or health benefits, including the prevention and treatment of disease [17]. according to international food information council (ific) “foods or dietary components that may provide a health benefit beyond basic nutrition” [18]. another body that is international life sciences institute of north america (ilsi) given “foods that by virtue of physiologically active food components provide health benefits beyond basic nutrition” [19]. finally the nutrition business journal classified functional food as “food fortified with added or concentrated ingredients to functional levels, which improves health or performance [20]. plant foods as nutraceuticals: plant foods are a rich source of phenolic and poly phenolic compounds, there are blueberry leaves that are excellent sources of antioxidants [21]. the study shows that the leaves are useful to suppress the expression of hepatitis c virus rna [22]. in cereals and legumes, the bran portion are also rich in phenolics as compared to the endosperm [23]. in recent studies, scientists had focused on marine algae and their constituents as nutraceuticals and functional foods for health-promotion [19,20]. as we know that marine algae are primarily used for the production of single-cell oil rich in dha, and other pufa [21], long-chain omega three pufa are chosen as their effectiveness in prevention and treatment of coronary heart disease [22], hypertension [23], diabetes [24], arthritis and other inflammations [25], autoimmune disorders [26] mental health and neural function as in depression and schizophrenia and cancers [27] and are essential for maintenance and development of normal growth, especially for the brain and retina [28]. food fortification: not a new approach ff or enrichment is the term used for addition of essential vitamins and minerals to the basic foods improve the nutritional value and could overcome nutritional gaps in human beings. as we know that adding iodine to salt has been started simultaneously in many parts of the world [29]. another is addition of vitamin d and vitamin a to the dairy products by 1932 [30]. as the time passes the addition of iron and folic acid to flour become common in the many western countries [31]. in this growing world the very important aspect of human life is health benefits of functional foods, it is not surprising that major companies are interested in developing such foods for the health and well-being of human race. a recent survey of 38 chief research officers of major food companies conducted by the institute of food technologists ranked research efforts into the development of foods considered to be healthful well ahead of research efforts directed toward food safety, or toward the development of either organic or reduced fat foods. after several studies over the last few years the tendency for consumers to view the “kitchen cabinet as the medicine cabinet” was initially identified as a leading trend in the food industry in 1994. this “self-care” phenomenon remains a leading consumer trend today. the tahilani et al. nutraceuticals and fortified foods supplements in india vol 1 | issue 1 | apr jun 2020 indian j pharm drug stud | 3 10th annual consumer trend report from the food marketing institute and prevention magazine found that that 76% of consumers strongly or mostly agree that eating healthy food is a better way to manage illness than medication [32]. the need of research is currently directed toward increasing our understanding of “functional foods.” efforts to identifying how functional foods and food ingredients might help prevent chronic disease or optimizes health, thereby reducing healthcare costs and improving the quality of life for many consumers. an emerging discipline that will have a profound effect on future functional foods research and development efforts is nutria genomics, which investigates the interaction between diet and development of diseases based on an individual’s genetic profile [33]. other fields that will greatly influence the future of functional foods is biotechnology, and bioinformatics. recent examples of biotechnology-derived crops which have tremendous potential to improve the health of millions worldwide include golden rice and iron-enriched rice [34]. the wide range of fortified foods has been justified by the fact that given dietary allowances for many nutrients are commonly not met through the normal diet. however, the virtual elimination of micronutrient deficiencies in developed countries has been attributed in large part to fortification [35]. although it is well identified that ff is one of the preferred and cost-effective approaches in overcoming micronutrient malnutrition, its effectiveness in developing countries is yet to be demonstrated. one of the factors which have to be taken an account is the lack of simple and affordable technology to fortify foods with stable and bio available nutrients without compromising commonly accepted taste and appearance [36]. global issues of fortification: the who issued guidance for the fortification of maize and wheat flour in 2009 based not on the distribution of intake across different segments of the population but on national per capita in takes and assuming that no other source provided the referred micronutrients [37]. as the fortification is implemented, there is often reluctance on the part of policy makers to reduce the content of the nutrient being fortified, even in the face of evidence that higher doses are not needed to demonstrate benefit. folic acid fortification of grain products is well established as a means to reduce the incidence of neural tube defects [38]. the major group of nutraceuticals is endogenous in origin, being natural products responsible for eliciting activity in healthy humans. a number of plant constituents are increasingly becoming available, some individual entities e.g., resveratrol from grapes and wine and some complex combinations of constituents e.g., pycnogenol. the two glycol samino glycans (gags), glucosamine and chondroitin are derived from various animal materials. [39] recent advances in nutraceuticals & fortified food in india: numerous studies conducted over the last few years have concluded that there are increasing numbers of consumers taking responsibility for well-being of their family, they are knowingly being aware regarding the current need for personnel health [40]. the “selfcare” concept of an individual is gaining a vast popularity in the world. as studied the 10th annual consumer trend report given by the food marketing institute and prevention magazine summarized that 76% of humans are strongly or mostly agree on the concept that eating healthy food is a better way to overcome illness than medication. as the age is a very important factor as it affects the immunity of a individual so the humans of above 50 years are more prone to get ill in comparison to others. conclusion as many functional foods may have promised for improving public health, but still claims are not scientifically proven. there are many pitfalls where studies need to be conducted to get the best results in every human body. nutrition deficiency is a serious problem in india as well as many different parts of the world. ff can be expected to play a wide role in the improvement of this condition. so far, the studies conducted with ff in india found having positive results to improve condition in pregnant and lactating women, the elderly population, as well as a population of different socioeconomic categories. to fulfill a balanced and nutritious diet required for the healthy people ff approach is gaining par importance for the betterment of society. references 1. codex alimentarius. 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nutraceuticals and diabetes-an update; planta indica; octoberdecember, 2008; 4(4); 45-47. 36. sloan ae. top 10 trends to watch and work on: the more things change, the more they stay the same. food technol. 1994;48:89100. 37. pandav cs, yadav k, srivastava r, et al. iodine deficiency disorders (idd) control in india. indian j med res. 2013;138:418-33. 38. food marketing institute & prevention magazine. shopping for health: reaching out to the whole health consumer. food marketing institute, washington, dc. 2013. 39. dychtwald, k. (1999) age power. in: age power: how the 21st century will be ruled by the new old, pp. 1– 30. jeremy p. tarcher/putnam, new york, ny. 40. moldovan op. study quality parameters obtained from bakers flour fortified with legumes. phd thesis. usamv-cn; 2016. how to cite this article: tahilani p, banweer j, chatterjee dp, goyanar g, sharma a. nutraceuticals and fortified foods supplements in india: challenges and opportunities a comprehensive review. ind j pharm drug stud 2020; 1(1):1-4. funding: none; conflict of interest: none stated bhat r et al. protective effects of ruta chalepensis linn. extracts on drug-induced seizures vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 113 original article exploring the protective effects of aqueous extracts of ruta chalepensis linn. on drug induced seizures in animal models ramdas bhat1, hemalatha c h2, a r shabaraya3 from, 1assistant professor, 2pg scholar, department of pharmacology, 3principle and head of the department of pharmaceutics, srinivas college of pharmacy, valachil, post farangipete, mangalore, karnataka, india 574143. abstract background and objective: convulsion is a sudden, involuntary muscle contraction causing rapid and repetitive movements of the body. ruta chalepensis linn. in is a medicinal plant used to treat a wide range of disorders including seizure. but, the anticonvulsant activity of this plant has not been studied in deep. we therefore sought to evaluate the anticonvulsant activity of an aqueous leaf extract of ruta chalepensis linn. on convulsing induced in mice. method: the anticonvulsant activity of an aqueous leaf extract of ruta chalepensis linn. on convulsing induced in mice, by strychnine nitrate (2.0 mg/kg) and pentylenetetrazole (ptz) (80 mg/kg) in mice. chronic study was conducted using low, medium and high doses of arc (250, 500 and 750mg/kg respectively). the above-mentioned doses were administered daily once for a period of 7 consecutive days. aqueous extract (250, 500 and 750 mg/kg), diazepam (10mg/kg intraperitoneally or oral.), and distilled water (10 ml/kg, i.p) were administered before induction of seizures. results: in strychnine induced convulsion test the onset of seizure was significantly delayed in the animals treated with ruta chalepensis linn leaf aqueous extract at 750mg/kg. in ptz induced convulsion model the onset of seizure was significantly delayed in the animals treated with ruta chalepensis linn leaf aqueous extract at a dose of 750mg/kg. conclusion: as a result of foregoing ruta chalepensis linn leaf aqueous extract has considerable diuretic activity at higher doses. key words: aqueous extracts of ruta chalepensis linn (aerc), strychnine-induced convulsion test, pentylenetetrazole (ptz) induced seizures, epilepsy, glycine, gabanergic. pilepsy is a neurological disorder characterized by recurrent and unpredictable seizures caused by sudden bursts of abnormal electrical activity in the brain. seizures can be of different types, including tonicclonic, myoclonic, absence, and others, and are often treated with anticonvulsant medications to reduce the risk of seizures [1]. despite the availability of different medications, there are still a considerable number of patients who experience inadequate seizure control or suffer from medication side effects. this has led to a search for alternative treatment options, including herbal remedies that have been used for centuries to treat seizures [2]. ruta chalepensis linn. is a medicinal plant that has been traditionally used for various ailments, including seizures. access this article online received – 05th april 2023 initial review – 08th april 2023 accepted – 25th april 2023 quick response code the plant contains several bioactive compounds, such as alkaloids, flavonoids, and coumarins, which have various pharmacological activities, including anticonvulsant, anti-inflammatory, and analgesic effects [3]. pentylenetetrazole (ptz), strychnine are commonly used compounds to induce convulsions in animal models [4]. strychnine is a potent convulsant that acts by blocking the inhibitory neurotransmitter glycine in the spinal cord, leading to increased excitability and ultimately, convulsions [5]. while ptz-induced convulsions are mainly mediated by the inhibition of the neurotransmitter gaba, strychnine -induced convulsions are not mediated by gaba [6], making them a useful model for investigating alternative pathways and mechanisms involved in seizures. the use of animal models in the study of convulsions and seizures has been widely accepted and employed in the field of epilepsy research [7]. _______________________________________ correspondence to: ramdas bhat, assistant professor, department of pharmacology, srinivas college of pharmacy, valachil, post farangipete, mangalore, karnataka, india-574143. email: ramdas21@gmail.com e mailto:ramdas21@gmail.com bhat r et al. protective effects of ruta chalepensis linn. extracts on drug-induced seizures vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 114 animal models allow for the investigation of various factors that contribute to the development of convulsions, such as age, sex, species, diet, water, day/light cycle, temperature, preparation dose, and route of administration. furthermore, animal models allow for the evaluation of the efficacy and safety of potential anticonvulsant treatments [8, 9]. the aqueous extracts of ruta chalepensis linn. have been reported to have several pharmacological activities, including anticonvulsant effects, which makes them a promising candidate for further investigation. the evaluation of the potential protective effects of ruta chalepensis linn. against both ptz and strychnine induced convulsions in animal models may provide new insights into the mechanisms of action involved in these seizures and identify new therapeutic strategies for the treatment of epilepsy. methodology animals: the following study used mice of both sexes weighing around 18-22g. the animals were fed a standard pelleted diet (lipton india ltd., mumbai) and distilled water ad libitum under a constant 12 hours light and dark cycle. prior to the experiment, all animals were housed in laboratory conditions for 5 days. the animals are now divided into 5 groups containing 6 animals each. all experiments were carried out in accordance with the ethical standards. plant extract: plant was collected in mangalore and authentication was done by botanist, pilikula nisargadhama, vamanjoor. the leaves are separated shade dried for one day and the aqueous drug extract was prepared by steam distillation. the extract was suspended in 1% tween 80, and was administered orally to the animals by gastric intubation using force feeding needle. dose was selected as 250mg/kg and 500mg/kg and 750mg/kg as lower, intermediate and higher dose respectively based on the acute toxicity study (ld50). study type: screening of ruta chalepensis linn leaf aqueous extract for its anticonvulsant activity using experimental animals. screening models used: a) strychnine-induced convulsions test b) pentylenetetrazole (ptz) -induced seizures. study site: department of pharmacology, srinivas college of pharmacy, valachil, mangalore. sample size: 60 mice were selected for the entire study study duration: 7 days inclusion criteria: mice of both sex having weight of 1822g was included in study exclusion criteria: mice above 25g was excluded from the study. experimental design acute toxicity studies: acute toxicity study was performed in accordance with oecd guidelines. no adverse effect or mortality was detected in mice up to 2 gm/kg, p.o of ruta chalepensis linn during the 24 to 72 hrs observation periods. the rats were continually monitored for 5 hours during this time for any obvious behavioral, neurological, or autonomic toxic effects, mortality after 24 to 72 hrs (table 1 and 2). strychnine induced convulsion: the mice utilized for this study were groups of six of either sex with a weight of between 18-22 respectively. they were now treated with diazepam 10 mg/kg which is a standard drug. one hour after the administration of the standard drug the mice were injected with 2 mg/kg strychnine nitrate i.p the time until occurrence of tonic extensor convulsions and death noted during a 1 h period. with this dose of strychnine convulsions were observed in 80% of the controls. the animals employed are mice of either sex that weigh between 18 and 22 g. the animals are divided into 6 groups and 6 groups receive either the test substance or the reference medication through injection, intravenously, or orally. as a control, six mice from another group are used. thirty minutes following an intravenous injection, sixty minutes following a subcutaneous injection ptz is subcutaneously given at an 80 mg/kg dose. each animal is housed in its own plastic cage for a one-hour observation period. there are records of seizures and tonic-clonic convulsions. convulsions must be present in at least 80% of the animals in the control group. table 1: grouping of animals in strychnine induced convulsion group treatment dose no. of animals parameters to be assessed group i (control) saline + strychnine 10ml/kg + 2mg/kg 6 percentage inhibition of seizures relative to control group ii (standard) diazepam + strychnine 10mg/kg + 2mg/kg 6 group iii (low dose) aerc + strychnine 250mg/kg + 2mg/kg 6 group iv (moderate dose) aerc + strychnine 500mg/kg + 2mg/kg 6 group v (high dose) aerc + strychnine 750mg/kg + 2mg/kg 6 bhat r et al. protective effects of ruta chalepensis linn. extracts on drug-induced seizures vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 115 table 2 pentylenetetrazole-induced seizures (ptz) statistical analysis: all the data were expressed in mean ± sem. the significance of differences in mean between control and treated animals for different parameters determined by one way anova followed by dunnett’s multiple comparison test. significance for difference between groups were evaluated for student’s t-test to come to final conclusion. result and discussion aerc leaves were screened for anticonvulsant activity using strychnine and ptz induced convulsion model in mice. chronic study was conducted using low, medium and high doses of arc (250,500& 750mg/kg respectively). the above-mentioned doses were administered daily once for a period of 7 consecutive days. it was observed that lower dose (250mg/kg) of aerc did not produce significant anticonvulsant effect as compared to control. but medium and high doses (500&750mg/ kg respectively) exhibited a significant anticonvulsant effect by increasing onset time of seizures and reducing the duration of tonic-clonic seizures (figure 1 – 3). strychnine induced convulsion: the mice were given ruta chalepensis leaf extract in three different doses considerably delayed the onset of seizures. of the 6 animals tested, 4 survived at the higher dose (750 mg/kg), 2 survived at the intermediate level (500 mg/kg), and none survived at the lower dose. for higher and moderate doses, the percentage protection was 66.66 and 33.33 respectively (table 3). table 3: effect of aerc on strychnine induced convulsion. all values are expressed as (mean ± s.e.m), n=6, **p≤0.01, ***p≤0.001 when compared with control. (statistically analyzed by anova allowed by dunnett’s-test). contro l diazepam aerc (2 50mg/kg) aerc (5 00 m g/kg) aerc (7 50 m g/kg) 0 100 200 300 400 500 effect of aerc on strychnine induced convulsion treatment on se t o f t on ic -c lo ni c co nv ul si on (s ec ) control diazepam aerc (250mg/kg) aerc (500 mg/kg) aerc (750 mg/kg) fig. 1: effect of aerc on strychnine induced convulsion. aerc leaves were tested for their ability to prevent convulsions induced by strychnine in mice. a chronic study was conducted using three different doses of aerc (250mg/kg, 500mg/kg, and 750mg/kg), administered once daily for seven consecutive days. the lower dose did not show a significant anticonvulsant effect compared to the control group, but the medium and high doses (600mg/kg and 800mg/kg) demonstrated a significant effect by group treatment dose no. of animals parameters to be assessed group i (control) saline + pentylenetetrazole 10ml/kg + 80mg/kg 6 percentage inhibition of seizures relative to control group ii (standard) diazepam + pentylenetetrazole 10mg/kg + 80mg/kg 6 group iii (low dose) aerc + pentylenetetrazole 250mg/kg + 80mg/kg 6 group iv (moderate dose) aerc + pentylenetetrazole 500mg/kg + 80mg/kg 6 group v (high dose) aerc + pentylenetetrazole 750mg/kg + 80mg/kg 6 treatment onset of tonic clonic convulsion mean ± sem status of animal (1hr) (no. of animals alive) % protection control 254.50 ± 0.29 0 0 diazepam 481.72 ± 0.87*** all 100 aerc (250mg/kg) 252.83 ± 0.57 0 0 aerc (500mg/kg) 276.55 ± 0.89 2 33.33 aerc (750mg/kg) 276.50 ± 1.52** 4 66.66 bhat r et al. protective effects of ruta chalepensis linn. extracts on drug-induced seizures vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 116 delaying the onset of seizures and reducing the duration of tonic-clonic seizures. the protective effect of the medium and high doses of arc was 33.33% and 66.66% for up to one hour after administration, respectively, while the lower dose showed no protective effect. the standard drug diazepam (5mg/kg) exhibited a significant anticonvulsant activity and provided 100% protection (table 4). ptz induced convulsion: ruta chalepensis leaf extract, 750 mg/kg, considerably delayed the onset of seizures in mice treated in the ptz-induced convulsion model, and it significantly decreased the length of tonic-clonic seizures for both the moderate and higher dosages of test as well as standard. in the test with the higher dose, only two of the six animals showed signs of survival, whereas one animal did so for the test with the moderate level and none at the low amount. the percentage of protection was 33.33 for higher doses and 16.66 for moderate doses, respectively. contro l dia ze pam aerc (2 50m g/k g) aerc (5 00 m g/k g) aerc (7 50 m g/k g) 0 200 400 600 800 1000 effect of arc on ptz induced convulsion treatment o ns et o f s ei zu re (s ec ) control diazepam aerc (250mg/kg) aerc (500 mg/kg) aerc (750 mg/kg) fig. 2: effect of aerc on ptz induced convulsion. contro l diaze pam aerc (2 50m g/k g) aerc (5 00 m g/k g) aerc (7 50 m g/k g) 0 10 20 30 40 50 effect of arc on ptz induced convulsion treatment d ur at io n of to ni ccl on ic s ei zu re (s ec ) control diazepam aerc (250mg/kg) aerc (500 mg/kg) aerc (750 mg/kg) fig. 3: effect of aerc on ptz induced convulsion table 4: effect of aerc on ptz induced convulsion. treatment onset of seizure (sec) mean± sem duration of tonic clonic seizure (sec) mean± sem status of animal (no. of animals alive) % protection(1hr) control 660.17 ± 4.01 38.83 ± 1.85 0 0 diazepam 924.67 ± 2.65*** 6.50 ± 0.76*** all 100 aerc (250mg/kg) 663.33± 2.30 34.00 ± 1.39 0 0 aerc (500mg/kg) 671.67 ± 4.02 28.5 ± 0.99*** 1 16.66 aerc (750mg/kg) 689.83 ± 4.40*** 23.50 ± 1.05*** 2 33.33 all values are expressed as (mean ± s.e.m), n=6, ***p≤0.001 when compared with control. (statistically analyzed by anova allowed by dunnett’s-test). aerc leaves were tested for anticonvulsant activity in mice using the ptz induced convulsion model. three different doses of arc (250mg/kg, 500mg/kg, and 750mg/kg) were administered once daily for seven bhat r et al. protective effects of ruta chalepensis linn. extracts on drug-induced seizures vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 117 consecutive days. the lower dose did not show significant anticonvulsant activity compared to the control group, but the medium and high doses showed a significant effect by delaying the onset of seizures and reducing the duration of tonic-clonic seizures. the protective effect of the medium and high doses of aerc was 83.33% and the lower dose was 33.33% for up to one hour after administration. the standard drug, diazepam (1mg/kg), exhibited significant anticonvulsant activity and provided 100% protection. ruta chalepensis is an ancient medicinal plant still used in the traditional medicine of many countries as a laxative, anti-inflammatory, analgesic, anti-spasmodic, abortifacient, antiepileptic, emmenagogue and for dermatopathy treatment. the main aim of this work is to investigate the antiepileptic activity of ruta chalepensis aqueous leaf extracts in experimental animals [10]. the antiepileptic activity of the ruta chalepensis leaf extracts has not yet been studied for anti-convulsant activity. he exact mechanism underlying antiepileptic activity of ruta chalepensis linn. leaf extract is not clear but it may be apparently related to active compounds present in ruta chalepensis are reviewed. chemical studies have reported the presence of several flavonoids, alkaloids, phenols, amino acids, furanocoumarins and saponins in the leaves of ruta chalepensis linn [11]. the results indicate that aqueous leaf extract of ruta chalepensis linn. may have an antiepileptic like effect. however further experiments are necessary to confirm this hypothesis. future prospectives research in the field of plant-based anticonvulsants has yielded promising results in recent years. however, it is important to note that this research is still in its early stages, and more studies are needed to determine the safety and efficacy of these treatments. nonetheless, the discovery of new plant-based anticonvulsants has the potential to lead to the development of new and more effective therapies for convulsions. further research and clinical trials will be necessary to fully evaluate the therapeutic potential of these plant-based treatments. conclusion in six meticulously planned studies involving laboratory animals, the aqueous leaf extract of ruta chalepensis linn. showed strong anticonvulsant action, indicating the possibility of using it to treat different kinds of epilepsy. by using actual measurement and statistical validation, the results were validated and observer bias was removed. also, the chemical components of the extract were determined. these findings give a scientific basis for the extract's therapeutic efficacy even if more research on human patients is required. acknowledgements: we are thankful to research guide, principal and management of srinivas college of pharmacy, mangalore for providing all the necessary facilities to carry out this research work. references 1. stafstrom ce, carmant l. seizures and epilepsy: an overview for neuroscientists. cold spring harbor perspectives in medicine. 2015 jun 1; 5(6): a022426. 2. ventola cl. epilepsy management: newer agents, unmet needs, and future treatment strategies. pharmacy and therapeutics. 2014 nov; 39(11): 776-77. 3. al-ghamdi ay, fadlelmula aa, abdalla mo. total phenolic content, antioxidant and antimicrobial activity of ruta chalepensis l. leaf extract in al-baha area, saudi arabia. jordan j biol sci. 2020; 13: 675-80. 4. annafi os, umukoro s, eduviere at. evaluation of the anticonvulsant and anxiolytic potentials of methyl jasmonate in mice. scientia pharmaceutica. 2014 sep; 82(3): 643-54. 5. salim m. a, ammoo am, ali mh, et al. antiepileptic effect of neuroaid® on strychnine-induced convulsions in mice. pharmaceuticals. 2022 dec; 15(12): 1468-69. 6. shimada t, yamagata k. pentylenetetrazole-induced kindling mouse model. jove (j of visualized experiments). 2018 jun 12(136): e56573. 7. kandratavicius l, balista pa, lopes-aguiar c, et al. animal models of epilepsy: use and limitations. neuropsychiatric dis. and treat. 2014 sep 9: 1693-705. 8. wang y, wei p, yan f, et al. animal models of epilepsy: a phenotype-oriented review. aging and disease. 2022 feb; 13(1): 215-16. 9. reddy ds, kuruba r. experimental models of status epilepticus and neuronal injury for evaluation of therapeutic interventions. international j of molecular sciences. 2013 sep 5; 14(9): 18284-318. 10. althaher ar, oran sa, bustanji yk. phytochemical analysis, in vitro assessment of antioxidant properties and cytotoxic potential of ruta chalepensis l. essential oil. j of essential oil bearing plants. 2020 nov 1; 23(6): 1409-21. 11. szewczyk a, grabowski m, zych d. ruta chalepensis l. in vitro cultures as a source of bioactive furanocoumarins and furoquinoline alkaloids. life 2023 feb 6; 13(2): 457-8. how to cite this article: ramdas bhat, hemalatha c h, a r shabaraya. exploring the protective effects of aqueous extracts of ruta chalepensis linn. on drug induced seizures in animal models. indian j pharm drug studies. 2023: 2(3) 113-117. funding: none conflict of interest: none stated mandumpala depression among cancer survivors vol 1 | issue 3 | oct dec 2022 indian j pharm drug studies | 79 review article depression among cancer survivors – an untold story janice jacson mandumpala from, department of pharmacy practice, nirmala college of pharmacy, kerala, india correspondence to: janice jacson mandumpala, pharm d intern, department of pharmacy practice, nirmala college of pharmacy, muvattupuzha, ernakulam, kerala, india. email: janice.jacson@gmail.com abstract mental health is an important area in the treatment of patients. cancer not only burdens the individual during the treatment phase but also during the survivorship period. multi-morbidities, being female, pre-existing psychological illness are few among risk factors that intensify the course of depression among cancer survivors. depression can seep in through various means from the effects of treatment to accepting the reality of survivorship and it may vary depending on the type of cancer. screening and as sessing for depression, patient self-management strategies, internet based mindfulness therapy and psychoeducational interventions are few among the possible ways to tackle the threat of depression in cancer survivors. this review summarizes the causes of depression, depression among various cancer types [thyroid, prostate, breast and rectal/ colorectal cancer] and applications of evidence to practice to overcome depression in cancer survivors. key words: depression, cancer survivors, breast cancer, prostate cancer, thyroid cancer, rectal cancer he psychological impact of diseases is an important aspect of the clinical cure of patients. “mental illness is like any other medical illness” is an undeniable truth and necessitates immediate action in the healthcare sector [1]. psychological problems are persistent among cancer patients, depression and anxiety being very common. this can make the treatment more challenging with additional difficulty in the management and cure of cancer. depressive symptomatology is evident during the advanced stages of the disease and in the inpatient settings [2]. studies have elaborated on the need for developing a management strategy to address the increasing burden of depression symptomatology among cancer patient [3]. the psychological impact of diseases is an important aspect of the clinical cure of patients. “mental illness is like any other medical illness” is an undeniable truth and necessitates immediate action in the healthcare sector [1]. psychological problems are persistent among cancer patients, depression and anxiety being very common. this can make the treatment more challenging with additional difficulty in the management and cure of cancer. depressive symptomatology is evident during the advanced stages of the disease and in the inpatient settings [2]. studies have elaborated on the need for developing a management strategy to address the increasing burden of depression symptomatology among cancer patients [3]. causes the psychological impact of diseases is an important aspect of the clinical cure of patients. “mental illness is like any other medical illness” is an undeniable truth and necessitates immediate action in the healthcare sector [1]. psychological problems are persistent among cancer patients, depression and anxiety being very common. this can make the treatment more challenging with additional difficulty in the management and cure of cancer. depressive symptomatology is evident during the advanced stages of the disease and in the inpatient settings [2]. studies have elaborated on the need for developing a management strategy to address the increasing burden of depression symptomatology among cancer patients [3]. a variety of factors can steer depression among cancer survivors which may or may not interact with one another. individual characteristics such as age, gender, ethnicity, sexuality, religion, disability, co-morbid health conditions and marital or co-habitation status influence development of depression. psychological distress, coping behaviour, hopelessness, denial, anger, fear, grief, resilience, concern for others, change in self-image are psychological response to diagnosis and in the journey thereafter that impacts a cancer patient’s mental health. social and contextual factors that has an undeniable affect are educational level, employment status, healthcare system, social and family support and stressful life events such as loss of a loved one. prior psychological behaviour is also another less enquired history for cancer patients and it includes preexisting psychiatric disorders, previous suicidal behaviour and prior coping behaviour. finally, treatment related modalities have a huge impact on the current and future status of cancer patients. they t mandumpala depression among cancer survivors vol 1 | issue 3 | oct dec 2022 indian j pharm drug studies | 80 include long term complications associated with treatment such as development of infertility and secondary cancers that can have adverse mental effects [7]. multiple comorbidities among cancer survivors also can influence their treatment outcomes and further research can prevent the development of adverse events due to comorbid conditions in these patients [8]. screening for depression is crucial in the web of multi-morbidities. approaches for managing multi-morbidities should also include effective ways to tackle the impending threat of depression [9]. depression among various types of cancer survivors thyroid cancer multiple comorbidities among cancer survivors also can influence their treatment outcomes and further research can prevent the development of adverse events due to comorbid conditions in these patients [8]. screening for depression is crucial in the web of multi-morbidities. approaches for managing multi-morbidities should also include effective ways to tackle the impending threat of depression [9]. prostate cancer prostate cancer is the second most prevalent cancer after lung cancer among men. treatments tailored include radical prostatectomy, radiotherapy, observational or watchful waiting and chemotherapy. they pose adverse risk of side effects such as incontinence of urine, bowel problems, erectile dysfunction, pain, insomnia and fatigue that persists for a long time. gynecomastia and hot flashes are the two prominent side effects of androgen deprivation therapy. they are associated with shame, loss of masculinity, stigma and followed by psychological distress. urine incontinence may trigger fear of smelling, leakage of urine and an embarrassment to use incontinence pads, together culminating in social isolation with aggravated depressive symptoms [12]. physical side effects are positively associated with sleep disturbances and increases the burden of cancer-related fatigue. thus, the pleiades of consequences post treatment among cancer survivors can pose a significant risk for depression in this cohort [13]. lack of food security, racial discrimination, housing instability and other consequent effects of racial differences can elevate the incidence of depression among prostate cancer survivors belonging to specific communities. racial disparities can have an impending burden on the existing situation of a prostate cancer survivor. in a study conducted by parikh et al, it has been concluded that incident depression is common among african american cohort of prostate cancer survivors in comparison to the whites [14]. although evidence is nascent, future studies, should examine the impact of race on the development of depression among cancer survivors. breast cancer breast cancer is the most prevalent cancers in the world. emotional distress among breast cancer patients can reduce their medication adherence, and lead to poor quality of life and intense fear of their disease. breast cancer patients often experience a spectrum of depressive symptomatology [15]. the prevalence of depression among breast cancer survivors ranges from 13% to 45% [16]. breast cancer survivors include those who have received treatment and are on long term follow up to reduce the recurrence of cancer and to improve the quality of life [17]. breast cancer survivors are much-ignored section of the population who are at an increased risk of developing depression in comparison to breast cancer patients. they experience a multitude of symptoms such as impaired sleep, fatigue, arthralgia, stress, impaired sleep, hot flashes, vaginal dryness, decreased sexual interest, problems during urination, weight gain, anxiety and depression. there is an impending need to address these issues and timely deliver interventions to help them [18]. screening for depressive symptoms is needed but without assessing the need for intervention, the practicality of the issue remains unmet. shreds of evidence reflect a greater incidence of depression among the early survivors [within 5 years of treatment] than late survivors [within 10 years of treatment]. the preceding group of survivors tend to experience depression due to fear of recurrence and treatment related side effects [19]. a recent study conducted among the black breast cancer survivors reiterates the need for a good patient-provider relationship and patient empowerment as the key components to develop during the breast cancer survivorship program. incidence of depression can be largely decreased among the black breast cancer survivors with the incorporation of special patient-centered communication [20]. rectal/ colorectal cancer the different types of surgery instituted for rectal cancer are permanent ostomy, temporary ostomy followed by anastomosis and initial anastomosis. a study has found that the prevalence of depression among rectal cancer survivors to be 24.7% among long term survivors and more prevalent among long term survivors who have undergone permanent ostomy. as per the findings of the study conducted by chongpison et al. females face greater burden of depression when undergoing permanent ostomy in comparison to anastomosis [21]. colorectal cancer survivors also develop persisting symptoms of depression, approximately 1 in 5 survivors post treatment. risk factors such as multi-morbidities, being single or divorced, lower educational status and high disease stage can contribute to the genesis of depression in this vulnerable cohort. hence, it is vital to know whether the survivors need psychological help to reduce their depressive symptoms [22]. mandumpala depression among cancer survivors vol 1 | issue 3 | oct dec 2022 indian j pharm drug studies | 81 application of evidence in clinical practice patient reported outcomes is the corner stone of cancer survivorship. therefore, it is necessary to also assess the symptoms associated with mental health among cancer survivors during their routine health check-up. screening and assessing for depression in the oncology setting can improve and help achieve better patient outcomes [23]. the screening will help detect patients who have a major depressive disorder but are not undergoing any treatment or patients in whom depression is not recognized [24]. certain cancer treatments are prone to induce neurotoxicity and this aspect should be considered while screening for psychiatric conditions. certain symptoms that are associated with poor outcomes and examples include pain and anxiety among depressed patients. targeting these symptoms can be an inestimable strategy to cope with depression. it is also necessary to engage in depth interviews that can extract the exact symptoms that are known to interfere with the function and quality of life. fatigue, hopelessness, worthlessness, negative self-image and helplessness are a few of them. hence, the importance of identifying the symptoms and their associated burden on the patients cannot be neglected and should be incorporated during the screening and counselling process [6]. it is necessary to raise awareness across various strata of the healthcare system about mental health symptoms during cancer survivorship. patient preferences are another unexplored area when considering screening for depression. evidence suggests that cancer patients prefer screening services during follow up through face to face interviews at regular intervals with a nurse within their cancer services. the patient perspective remains an unquestionable area of importance, as patient centered care is the aim of medical care [25]. oncology health professionals are better suited for screening and can initiate the process of screening than wait for any adverse event from occurring. routine screening can be applied for depression screening in cancer patients and among survivors, than selectively providing psycho-oncology services [26]. the patient when visiting the primary health care centres can seek self-management techniques. good eating habits, engaging in daily exercise and hygienic sleeping practices are few to follow. patients are also encouraged to engage in community reconnection, make interpersonal changes and function optimally [27]. evidence suggests incorporation of physical activity as a part of daily routine to prevent developing depression among cancer survivors. participating in physical activity and meeting weekly goals of exercise duration and intensity, is a low cost, nonpharmacological intervention that can reduce the severity and incidence of depression during cancer survivorship [28]. as per consensus statements from an international multidisciplinary roundtable meeting, it is concluded that cancer survivors should participate in exercise training, improve their physical fitness and restore their normal functioning which can help them mitigate the overwhelming effects of depression post-treatment. aerobic training, aerobic plus resistance training and resistance training alone in specific doses can prevent the advent of depression among cancer survivors [29]. it is advocated that every survivor develops a ‘culture of wellness’ and proactively works to maintain a healthy mental state. internet based mindfulness therapy is another novel and patient friendly approach practiced actively in the developed nations. this is a particularly useful technique among the breast and prostate cancer cohort as they have documented long term survival. this method not only addresses the acute symptom care but also teaches skills that can help patients prevent symptom aggravation and reversal. old age is a significant barrier to internet based therapy attributed to their inadequate knowledge about internet and lack of motivation. internet based therapy is useful for individuals as they can participate from their homes. this is a promising method for patients who do not seek special psychological treatment [30]. online mindfulness therapy can be employed for various groups of patients such as those in whom mindfulness skills will be difficult to reach out traditionally and for those who are on waitlist for face to face interview [31]. endorsement of such techniques through the clinician or healthcare professionals can drive patient uptake and engagement [32]. although internet based mindfulness therapy is in its infancy, in the context of public health it is an emerging strategy to alleviate depressive symptomatology and reduce the prevalence of depression among cancer survivors. clinical trials have shed light on the novel methods that can be incorporated in healthcare practice for managing depression among cancer survivors. a study conducted in quebec and ontario has discussed about the usefulness of telephonedelivered depression self-care intervention for cancer survivors. as per the findings of their study candirect, a telephonedelivered intervention produced greater reduction in depressive symptoms and greater quality of life. thus, instilling hope in newer directions such as candirect can satisfy the unmet needs of cancer survivors in the society [33]. evidence also suggests the role of a nurse-delivered intervention in addition to the usual care that can substantially improve the clinical outcomes in patients with cancer. nursedelivered interventions include education about depression, conducting a problem-solving session to develop a positive attitude among patients while dealing with testing situations, helping them communicate their concerns to the clinicians and also gaining the cooperation of clinicians [34]. nursing interventions targeted to alleviate depression among breast cancer survivors should accommodate the psychological needs of the patient and their care takers [35]. mandumpala depression among cancer survivors vol 1 | issue 3 | oct dec 2022 indian j pharm drug studies | 82 screening for depression can be implemented through the phq-2 item questionnaire, further diagnosis can be established where screening results necessitate treatment initiation. although the rate of false positive detection for depression remains high, nurse based interviews and screening should be considered. it should be conducted to improve symptoms and prevent relapse, rather than increasing the duration or burden of treatment [36]. inculcating a strong evidence based practice from the point of care itself can be the genesis of improved mental health among cancer survivors. there is an unmet need for psycho-educational interventions among cancer survivors which needs to be addressed. psychological needs have been categorized as one of the eight unmet needs in a cancer patient’/survivor’ journey, especially among breast cancer survivors. studies from the united states highlight a higher preference among patients for individual professional counselling over received pharmacological treatment for depression. psychiatric medications are least preferred and are only desired by patients when diagnosed with depression. therefore, it can be understood that psychoeducational counselling is well recognized by the patients and can be readily adopted within the cancer care setting [37]. conclusion depression is a leading cause of concern in the healthcare sector. the scientific community has addressed the importance of identifying major depressive disorder among cancer patients. there is still a lack of strong evidence and very less attention is paid to the mental health vulnerabilities faced by cancer survivors. it is more pronounced among females who carry a higher risk for mental health diseases. from the given review, it is understood that individuals diagnosed with cancer undergo varying levels of trauma during their treatment. not only are the treatment effects a causative factor for the development of depression, but the future concerns associated with cancer cure are equally contributory. cancer itself and a cure from it pose the risk of depression, but the situation is even grim when the needs of a survivor remains unaddressed. future studies are warranted to assess the prevalence of depression across various cohorts. such scientific and epidemiological shreds of evidence can help tailor the right strategies for addressing health concerns prevalent in society. references 1. malla a, joober r, garcia a. “mental illness is like any other medical illness”: a critical examination of the statement and its impact on patient care and society. vol. 40, journal of psychiatry and neuroscience. 2015. 2. naser ay, hameed an, mustafa n, et al. depression and anxiety in patients with cancer: a cross-sectional study. front psychol. 2021; 12. 3. strong v, waters r, hibberd c, et al. management of depression for people with cancer [smart oncology 1]: a randomised trial. lancet. 2008; 372 [9632]. 4. de moor js, mariotto ab, parry c, et al. cancer survivors in the united states: prevalence across the survivorship trajectory and implications for care. cancer epidemiol biomarkers prev. 2013; 22 [4]. 5. maddams j, utley m, møller h. projections of cancer prevalence in the united kingdom, 2010-2040. br j cancer. 2012; 107 [7]. 6. götze h, friedrich m, taubenheim s, et al. depression and anxiety in long-term survivors 5 and 10 years after cancer diagnosis. support care cancer. 2020; 28 [1]. 7. niedzwiedz cl, knifton l, robb ka, et al. depression and anxiety among people living with and beyond cancer: a growing clinical and res. priority. vol. 19, bmc cancer. 2019. 8. fowler h, belot a, ellis l, et al. comorbidity prevalence among cancer patients: a population-based cohort 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long-term depression incidence and associated mortality among african american and white prostate cancer survivors. cancer. 2021; 127 [18]. 15. tsaras k, papathanasiou i v., mitsi d, et al. assessment of depression and anxiety in breast cancer patients: prevalence and associated factors. asian pacific j cancer prev. 2018; 19 [6]. 16. lyons ma, ph d, prescott b, et al. women’s experiences during the first year following diagnosis of breast cancer. south online j nurs res. 2002; 3 [1]. 17. breast cancer: survivorship | cancer.net [internet]. [cited 2022 jul 18]. available from: https://www.cancer.net/cancertypes/breastcancer/survivorship 18. palmer sc, demichele a, schapira m, et al. symptoms, unmet need, and quality of life among recent breast cancer survivors. j community support oncol. 2016; 14 [7]. 19. cho oh, hwang kh. association between sleep quality, anxiety and depression among korean breast cancer survivors. nurs open. 2021; 8 [3]. 20. royak-schaler r, passmore sr, gadalla s, et al. exploring patientphysician communication in breast cancer care for african american women following primary treatment. oncol nurs forum. 2008; 35 [5]. 21. chongpison y, hornbrook mc, harris rb, et al. self-reported depression and perceived financial burden among long-term rectal cancer survivors. psychooncology. 2016; 25 [11]. 22. mols f, schoormans d, de hingh i, et al. symptoms of anxiety and depression among colorectal cancer survivors from the populationmandumpala depression among cancer survivors vol 1 | issue 3 | oct dec 2022 indian j pharm drug studies | 83 based, longitudinal profiles registry: prevalence, predictors, and impact on quality of life. cancer. 2018; 124 [12]. 23. decker vb, tofthagen c. depression: screening, assessment, and interventions in oncology nursing. clin j oncol nurs. 2021; 25 [4]. 24. meijer a, roseman m, milette k, et al. depression screening and patient outcomes in cancer: a systematic review. vol. 6, plos one. 2011. 25. yim j, arora s, shaw j, et al. patient preferences for anxiety and depression screening in cancer care: a discrete choice experiment. value heal. 2021; 24 [12]. 26. tu ch, hsu mc, chi sc, et al. routine depression screening and diagnosing strategy for cancer inpatients. psychooncology. 2014; 23 [9]. 27. mcallister-williams rh, arango c, blier p, et al. the identification, assessment and management of difficultto-treat depression: an international consensus statement. vol. 267, journal of affective disorders. 2020. 28. d’errico m, fessele kl. a retrospective, cross-sectional study of self-reported physical activity and depression among breast, gynecologic, and thoracic cancer survivors. oncol nurs forum. 2021; 48 [1]. 29. campbell kl, winters-stone km, wiskemann j, et al. exercise guidelines for cancer survivors: consensus statement from international multidisciplinary roundtable. med sci sports exerc. 2019; 51 [11]. 30. nissen er, o’connor m, kaldo v, et al. internet-delivered mindfulness-based cognitive therapy for anxiety and depression in cancer survivors: a randomized controlled trial. psychooncology. 2020; 29 [1]. 31. spijkerman mpj, pots wtm, bohlmeijer et. effectiveness of online mindfulness-based interventions in improving mental health: a review and meta-analysis of randomised controlled trials. vol. 45, clinical psychology review. 2016. 32. segal z v, dimidjian s, beck a, et al. outcomes of online mindfulness-based cognitive therapy for patients with residual depressive symptoms: a randomized clinical trial. jama psychiatry. 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2022: 1(3) 79-83. funding: none conflict of interest: none stated suthar and mashru advanced uv spectrophotometric method development and validation vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 27 research article advanced uv spectrophotometric method development and validation for simultaneous estimation of azelnidipine and telmisartan in pharmaceutical dosage form pallavi suthar1, rajashree mashru2 from, 1 assistant professor, department of chemistry, shree swaminarayan sanskar pharmacy college, gujarat, 2 professor, hod of pharmaceutical quality assurance department, maharaja sayajirao university of baroda, india. correspondence to: pallavi suthar, assistant professor, department of chemistry, shree swaminarayan sanskar pharmacy college, near zundal circle, s. p. ring road, between chandkheda-adalaj, zundal, gandhinagar382421, gujarat, india. email: pallaveemali12@gmail.com abstract the presented research work aims to develop and validate three advanced uv spectrophotometric methods for the simultaneous estimation of azelnidipine (azl) and telmisartan (tel). these methods offer a higher degree of sensitivity than already-existing methods of analysis. by implementing advanced spectroscopic techniques such as the simultaneous method, q-ratio method, and first derivative spectroscopy method. these methods offer a higher degree of sensitivity than already-existing methods of analysis. by implementing advanced spectroscopic techniques such as the simultaneous method, q-ratio method, and first derivative spectroscopy method. the linearity of the three methods was in the range of 2 µg/ml to 12 µg/ml for azl and 10 µg/ml to 50 µg/ml for tel. the correlation coefficients for simultaneous estimation were 0.999 and 0.998 for azl and tel, respectively, and 0.9992 and 0.9989 for azl and tel, respectively, for the first derivative method, whereas the correlation coefficients for the q-ratio method were 0.999 and 0.9988, respectively. the lod values obtained by the simultaneo us estimation method were found to be lower as compared to those obtained by the first derivative method and the q-ratio method, proving that the sensitivity of the simultaneous estimation method is high. key words: method development, validation, azelnidipine, telmisartan, concentration. he chemical formula for azelnidipine (azl) is 3(1diphenylmethylazetidin3-yl)-5-isopropyl-12amino1,4-dihydro-6-methyl-4-(3-nitrophenyl)-3,5pyridine dicarboxylate. it is a dihydropyridine (dhp) type calcium channel blocker (ccb) used for the treatment of hypertension [1, 2]. azl has two enantiomers due to an asymmetric carbon at the 4-position of the dhp ring. the pharmacological action of azl resides in the (r) enantiomer. this is in marked contrast to other ccbs in which the (s) enantiomer is responsible for the biological activity (figure 1) [3, 4]. the peculiar three-dimensional structure of the active enantiomer of azl may be related to its unique pharmacological features that are not shared by other dhps, such as a long-lasting reduction in blood pressure, decreased heart rate, and anti-atherosclerosis effect [6, 8]. azl also shows a diuretic effect by increasing urine volume and thus reducing the retention of ions [9, 10]. telmisartan [tel] is a chemical compound that is 2-(4[4methyl-6-(1-methyl-1h-1, 3-benzodiazol-2-yl)-2-propyl1h-1, 3-benzodiazol-1-yl] methyl phenyl) benzoic acid [1113]. it is an angiotensin ii receptor antagonist used in the management of hypertension. generally, angiotensin ii receptor blockers (arbs) such as telmisartan bind to the angiotensin ii type 1 (at1) receptors with high affinity, causing inhibition of the action of angiotensin ii on vascular smooth muscle and ultimately leading to a reduction in arterial blood pressure [14– 17]. recent studies suggest that telmisartan may also have ppar-gamma agonistic properties that could potentially confer beneficial metabolic effects [17–18]. after a literature review, it was known that numerous methods had already been described for the estimation of telmisartan, such as visible spectrophotometric methods, stability-indicating uv spectrophotometric methods, and rp-hplc methods. for the determination of telmisartan in human plasma, advanced techniques such as lc-ms were also found [19-20]. figure 1 – chemical structure of azelnidipine t suthar and mashru advanced uv spectrophotometric method development and validation vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 28 figure 2 – chemical structure of telmisartan material and methods apparatus and software: a shimadzu uv-1700 doublebeam spectrophotometer connected to a computer with shimadzu uv-probe 2.10 software installed was used for all the spectrophotometric measurements. the samples were weighed on an electronic balance (a120) by shimadzu. chemicals and reagents: all the dilutions were made using methanol as the diluent. preparation of standard stock solution: the standard stock solutions of both azl and tel are prepared by taking 10 mg of the standard drug in a 10 ml volumetric flask and making up the volume using methanol as the diluent to achieve a concentration of 1000 µg/ml. preparation of working standard solution: the working stock solution of azl is prepared by taking 1 ml of the standard stock solution and transferring it to a 10 ml volumetric flask. the volume is made up to the mark using methanol to get a concentration of 100 µg/ml. similarly, the standard working solution of tel is prepared by taking 1 ml of the standard stock solution and transferring it to a 10 ml volumetric flask. the volume is made up to the mark using methanol to get a concentration of 100 µg/ml. preparation of series for calibration curves for simultaneous method, first derivative method, and qratio method: for preparing the solutions used in obtaining the calibration curve, a series of previously calibrated volumetric flasks were used. to prepare the linearity of azelnidipine, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, 1.0 ml, and 1.2 ml were withdrawn from the working standard solution of azl and taken into separate volumetric flasks, and the volume was made up with methanol to prepare a series of solutions having concentrations in the range of 2 µg/ml to 12µg/ml. similarly, to prepare the linearity of telmisartan, 1 ml, 2 ml, 3 ml, 4 ml, and 5 ml were withdrawn from the working standard solution of tel and taken into separate volumetric flasks, and the volume was made up with methanol to prepare a series of solutions having concentrations in the range of 10 µg/ml to 50 µg/ml. preparation of sample solutions (test solutions): various pharmaceutical dosage forms are available for this particular drug combination, including the azova-t40 tablet dosage form. the dosage of each of these forms would vary according to the conditions of the patient. for the estimation of the azova-t40 formulation, one just needs to empty the content of azova-t40 into a 100ml volumetric flask and make up the volume using methanol. then 2.5 ml of this solution is taken into another volumetric flask, and the volume is made up using methanol again. this solution is then placed in the uv spectrophotometer for quantitative analysis against a methanol blank. simultaneous equation method: this method uses the absorbance at two selected wavelengths, one at λ max of one drug where other drug also shows considerab le absorbance (λ2) and other being the wavelength at which the first drug has practically nil absorbance (λ1). absorptivity of azelnidipine and telmisartan were calculated at both the wavelengths. the concentration of azelnidipine and telmisartan can be calculated from following equations: cy = a1ax2-a2ax1/ax2ay1 –ax1 ay2 … (1) cx = a2 ay1-a1ay2/ax2ay1ax1ay2 … (2) where, a1 and a2 are the absorbance of mixture at λ1 and λ2 respectively, ay1 and ay2 are absorptivity of y at λ1 and λ2 respectively, ax1 is absorptivity of x at λ2, cx is concentration of x, cy is concentration of y. figure 3 – overlay spectra of azelnidipine and telmisartan calculation for azelnidipine cx = a2 ay1-a1ay2/ax2ay1ax1ay2 = 1.477*0.0282-1.466*0.356/0.084*0.282-0.366*0.356 = 7.92ppm calculation for telmisartan cy = a1ax2-a2ax1/ax2ay1 –ax1 ay2 = 1.466*0.084-1.477*0.366/0.282*0.084-0.366*0.356 = 39.54ppm q-absorbance ratio method: this method, also called the "absorption ratio method," is a modification of the simultaneous equation’s method. according to this method, the ratio of absorbance at any two wavelengths for a substance, which obeys beer's law, is a constant value independent of the concentration and path length. this constant is termed "hufner's quotient" or qvalue. the method involves the measurement of absorbance at two wavelengths, one being the λ max of one of the components (λ suthar and mashru advanced uv spectrophotometric method development and validation vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 29 2) and the other being a wavelength of equal absorptivity of the two components (λ 1), called the isoabsorptive point. cx = (qm qy/qx qy) *a/a1….(3) cy=(qm-qy/qy-qx) *a/a2….(4) where, cx and cy are the concentrations of x and y respectively, a is absorbance of sample at iso-absorpitive wavelength and a1 and a2 are the absorptivity of x and y respectively at iso-absorpitive wavelength. figure 4 – overlay spectrum of azelnidipine and telmisartan first derivative zero crossing point method: derivative spectroscopy involves the conversion of a normal spectrum (a fundamental or zero-order spectrum) to its first, second, or higher derivative spectra by differentiating the absorbance of the sample with respect to the wavelength. the advantages of using derivative spectroscopy are that it leads to the separation of overlapped signals, the elimination of background caused by the presence of other compounds in a sample, and an improvement in the resolution of mixtures. sensitivity and specificity: if the measured height of derivative peak of analyte is performed at those wavelengths at which the spectra of other components are undergoing zeroing (cross through the zero line), the measured amplitude is proportional only to concentration of the analyte in consideration – zero crossing technique. figure 5 – zero crossing point of azelnidipine figure 6 – zero crossing point of telmisartan validation parameters linearity: in three of the developed methods, three sets of calibration curves were plotted between the absorbance and concentration. the calibration curve that showed the best values is represented below in figures 7–8. the linearity of the simultaneous equation method was determined by the zeroorder spectra of both drugs individually in the range of 200–400 nm. thus, at 255 nm (the λ max of azl), whereas at 297 nm (the λ max of tel), the linearity of the simultaneous equation method was found in the range of 2 µg/ml to 12 µg/ml for azl (r2 = 0.9996) and 10 µg/ml to 50µ g/ml for tel (r2 = 0.9989). figure 7 – calibration curve of azelnidipine figure 8 – calibration curve of telmisartan figure 9 – calibration curve of azelnidipine figure 10 – calibration curve of telmisartan suthar and mashru advanced uv spectrophotometric method development and validation vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 30 figure 11 – calibration curve of azelnidipine figure 12 – calibration curve of telmisartan the absorbance ratio method compares absorbances at two different wavelengths, one being an isoabsorptive point and the other being the λ max of one of the two components. from the overlay spectra of two drugs, it is evident that azl and tel show an isoabsorptive point at 274.4 nm. the second wavelength employed is 297 nm, which corresponds to the λ max of tel. the linearity shown in figure 9 & 10. the qabsorbance ratio method was found in the range of 10 µg/ml to 50 µg/ml for tel (r2 = 0.999) and 2 µg/ml to 12 µg/ml for azl (r2 = 0.998). then these zero-order spectra were converted into their respective first derivative spectra using the uv probe software itself. a ∆ = 5 nm and a scaling factor of 10 were used to convert zero-order spectra to first derivative spectra. thus, here at 234 nm (the zero crossing point of tel), the estimation of azl is possible, whereas at 274 nm (the zero crossing point of azl), the estimation of tel is done as shown in figures 11 and 12. the linearity of the first order derivative method was found in the range of 10 µg/ml to 50 µg/ml for tel (r2 = 0.9989) and 2 µg/ml to 12 µg/ml for azl (r2 = 0.9989). limit of detection and limit of quantification the limit of detection and limit of quantification was calculated using the series of calibration curves plotted. the lod and loq values were determined using the following equations and the data is represented in below table 1. table 1 – limit of detection and limit of quantification for azelnidipine and telmisartan parameter drag simultaneous method q-absorbtion method first derivative lod azl 0.35 0.16 1.8 tel 0.73 0.77 1.71 loq azl 0.927 0.048 5.4 tel 2.16 2.34 5.16 precision the precision of an analytical method expresses the closeness of agreement between a series of measurements which are obtained by performing multiple samplings of the same homogenous sample under the given conditions of the method. in this section of the article the two developed methods have been analyzed for precision at two levels: 1. repeatability (precision under the same operating conditions over a short interval of time) 2. intermediate precision (variations in the results obtained at different intervals) from the results of precision, it may be concluded that both the methods developed are precise as the %rsd values are less than 2. it also may be concluded here that the dual wavelength method is more precise than the 1st derivative method. in the data below represents the repeatability data, whereas table2 (a) and table2 (b) present the data for intra-day and inter-day precision respectively. precision data are represented in terms of %rsd and the nominal concentration of azelnidipine (tel) was kept 8µg/ml and 10µ (azl) and telmisartan g/ml respectively. accuracy: recovery studies for the uv-spectrophotometric methods were conducted using the standard addition method by taking a nominal concentration of 10µg/ml for azelnidipine and 20µg/ml for telmisartan from the formulation (test sample) and then spiking this solution by 80%, 100% and 120% of standard drug (api) (table 3). assay of marketed formulation; 20 tablets of formulation (azova-t40) containing 8 mg of azelnidipine and 40 mg of telmisartan were weighed accurately. the average weight of tablets was found and tablets were powdered. the tablet powder equivalents to 40 mg of telmisartan was weighed and transferred into 100ml volumetric flask and volume is made upto the mark using methanol to get 400 μg/ml solution. the content was filtered through the whatman filter paper to get clear solution. from the clear sample stock solution dilutions 1 ml was withdrawn and taken into 10 ml volumetric suthar and mashru advanced uv spectrophotometric method development and validation vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 31 flask and volume is made upto the mark using methanol to obtain 40 μg/ml of telmisartan and 8μg/ml of azelnidipine. the resulting solutions were analyzed for drug content by spectrophotometric method at 255 nm and 297 nm for azl and tel, respectively. assay was repeated 6 times and standard deviation was calculated. the drug content in azl and tel was found by three methods and results are mention in below tables (table 4). results the two methods discussed above may be compared with each other by comparing their limits of detections and limits of quantifications as shown in the table 5. anova and the ttest were used to obtain the assay results from the simultaneous equation method and the qabsorbance ratio method, and the results are shown in table 6. table 2 – precision (a) repeatability (b) interday precision repeatability azelnidipine telmisartan concentration parameter simultaneous method q-ratio first derivative concentration parameter simultaneous method q ratio first derivative 8 µg/ml mean 0.0285 0.2803 0.0135 10 µg/ml mean 0.0155 0.1816 0.0155 sd 0.00164 0.00136 0.0016 sd 0.00055 0.0013 0.0005 %rds 0.001 0.4872 0.0010 %rsd 0.001 0.7519 0.001 table 3 – accuracy of azelnidipine and telmisartan simultaneous q-ratio first derivative interday – azelnidipine conc. µg/ml mean sd (n=3) %rsd mean sd (n=3) %rsd mean sd (n=3) %rsd 6 0.217333 0.00152 0.702 0.142 0.0017 1.219 0.0106 0.0011 0.0010 8 0.284667 0.00115 0.405 0.181 0.0015 0.840 0.049 0.0615 0.010 10 0.344 0.002 0.581 0.22 0.001 0.454 0.016 0.0017 0.001 interday –tel misartan conc. µg/ml mean sd (n=3) %rsd mean sd (n=3) %rsd mean sd (n=3) %rsd 10 0.354 0.0015 0.283 0.280 0.0015 0.544 0.016 0 0 20 0.714 0.0020 0.280 0.537 0.0025 0.468 0.0303 0.0005 1.903 30 1.075 0.002 0.186 0.802 0.0011 0.143 0.044 0.0005 1.29 table 4 – assay results drug concentration taken (μg/ml) concentration of μg/ml %recovery %rsd assay result-simultaneous method azl 8 7.93 99.12 0.0144 tel 40 39.93 99.98 0.0724 assay result –q absorption ratio method azl 8 7.98 99.75 0.0147 tel 40 39.24 98.1 0.0723 assa y result –first derivative method azl 8 7.8 97.5 0.147 tel 40 39.65 99.12 0.0145 table 5 – comparison of methods para-meter drug simultaneous method q-absorption ratio method first derivative method lod azl 0.35 0.16 1.8 tel 0.73 0.77 1.71 loq azl 0.927 0.048 5.4 tel 2.16 2.34 5.16 table 6 – two way anova anova source of variation ss df ms f p-value f crit sample 0.59535 3 0.19845 0.710972 0.55952 3.238872 suthar and mashru advanced uv spectrophotometric method development and validation vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 32 columns 0.464816667 1 0.464816669 1.665263 0.215232 4.493998 interaction 1.100816667 3 0.366938889 1.314604 0.304208 3.238872 within 4.466 16 0.279125 total 6.626983333 23 conclusion the simple, rapid, accurate, and precise simultaneous methods, q absorbance ratio and first derivative methods of uv spectroscopy have been developed and validated for the routine analysis of azl and tel in api and pharmaceutical dosage form. this method was validated as per the ich guidelines. the values of the standard deviation and coefficient of variation calculated were satisfactory, which indicates the suitability of the proposed methods for routine estimation of azl and tel. according to lod, loq, and assay results, the simultaneous method is more accurate and precise than the q-ratio method and the first derivative method. there was a significant difference between the three methods for azl and tel. references 1. raskapur kd, patel mm, captain ad. uvspectrophotometric method development and validation for determination of azelnidipine in pharmaceutical dosage form. toxicology. 2010; 106:135-43. 2. rele rv, patil sp. ultra-violet spectrophotometric method for estimation of azelnidipine from bulk drug and pharma ceutical formulation. asian j res chem. 2010; 3(4): 1077-9. 3. kumar m, chandra u, garg a, et al. impurity profiling of azelnidipine and telmisartan in fixed dose combination using gradient rp-hplc method. annals of rscb [internet]. 2021 may 6 [cited 2022 dec. 6]: 15050-67. 4. raskapur kd, patel mm, anandkumari d. quality assurance. inte j pharm pharmaceu sci. 2012; 4: 1. 5. modi j, patel sk, parikh n, et al. stability indicating analytical method development and validation for estimation of azelnidipine. wjpr. 2016; 5 (2): 831-847. 6. gore m, dabhade ps. international j pharmaceutical sciences research. 7. prabhakar d, sreekanth, j jayaveera kn. method development and validation of azelnidipine by rp-hplc int j chem tech research. 2018; 11(1): 07-12 8. patel n, patel jk. scholars research library der pharmacia lettre, 2012; 4 (4): 1080-1084 9. thakare l, ahmad s, shastry vm. development and validation of uv-visible spectrophotometric method for estimation of cilnidipine and telmisartan in bulk and dosage form. indo-am j pharmaceu res. 2017; 7(04) 10. yuvasri s, murugan s, vetrichelvan t. firstorder derivative and uv-spectrophotometric methods for simultaneous determination of telmisartan and azelnidipine in bulk and tablet dosage form. eur j biomed pharmaceu sci. 2021; 8(5): 290-294. 11. mohite pb, pandhare rb, bhaskar vh. simultaneous estimation of ramipril and telmisartan in tablet dosage form by spectrophotometry. eurasian j analytical chem. 2010;5(1):89-94. 12. hirpara kp, dave vm, faldu ds, et al. uv spectrophotometric determination for simultaneous estimation of amlodipine besylate and telmisartan in combination. j pharm sci bio res. 2012; 2: 133. 13. bais s, singh c, singhvi i, et al. novel method for quantitative estimation of telmisartan from tablet formulation by colorimetric method. asian j pharmaceutical analysis. 2014; 4(2): 54-6. 14. patil up, gandhi sv, sengar mr, et al. simultaneous determination of atorvastatin calcium and telmisartan in tablet dosage form by spectrophotometry. int j chem tech res. 2009; 9(1): 970-973. 15. golher hk, pillai s. simultaneous estimation of amlodipine besylate and telmisartan by uv-spectrophotometry. analyst. 2010: 1218-1221 16. sasidhar rl, vidyadhara s, deepti b, et al. development and validation of rp-hplc method for the simultaneous determination of hydrochlorothiazide, amlodipine besylate and telmisartan in bulk and pharmaceutical formulation. oriental j chem. 2014; 30(4): 1815-1822. 17. naim ma, ahmed ae, gj kh. stability indicating reversephase high-performance liquid chromatography method development and validation for simultaneous estimation of telmisartan and benidipine hydrochloride in pharmaceutical dosage form. asian j pharm clin res. 2018; 11(5): 342-350. 18. kurade vp, pai mg, gude r. rp-hplc estimation of ramipril and telmisartan in tablets. ijps. 2009;71(2):148-151. 19. rupareliya rh, joshi hs. stability indicating simultaneous validation of telmisartan and cilnidipine with forced degradation behavior study by rp-hplc in tablet dosage form. int scholarly res notices. 2013; 2013: 1-6. 20. kumar m, jupally vr. development and validation of a stability indicating rp-hplc method for simultaneous determination of telmisartan and amlodipine in combined dosage form. asian j pharm clin res. 2014; 7(1): 32-35. how to cite this article: pallavi suthar, rajashree mashru. advanced uv spectrophotometric method development and validation for simultaneous estimation of azelnidipine and telmisartan in pharmaceutical dosage form. indian j pharm drug studies. 2023; 2(1) 27-32. funding: none conflict of interest: none stated sonwani et al. role of adenosine receptor agonists in inflammatory conditions vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 69 original article adenosine receptor agonists suppress macrophage inflammatory protein -1α production and collagen induced arthritis hari sonwani, virendra kumar sharma, steffi thomas, akhlesh kumar from, department of pharmacology, school of pharmacy, lnct university, kolar road ,bhopal 462042 [m.p] india. correspondence to: hari sonwani, department of pharmacology, school of pharmacy, lnct university, kolar road, bhopal462042 madhya pradesh, india. email: harisonwani10@gmail.com abstract the production of proand anti-inflammatory cytokines is modulated by ligands of various adenosine receptor subtypes. in this study, we looked at how adenosine and different ligands for the adenosine receptor subtypes (a1, a2, a3) affected the production of the chemokine macrophage inflammatory protein (mip) 1a in immune-stimulated raw macrophages in vitro. in addition, we investigated whether an a3 adenosine receptor agonist reduces mip-1α production and influences the course of inflammation in collagen-induced arthritis. the a3 receptor agonist n6-(3-iodobenzyl)-adenosine-5'-n-methyluronamide (ib-meca) and, less potently, the a2 receptor agonist 2-p-(2-carboxyethyl) phenethylamino-5'-n-ethylamino-5'-n-ethylamino-5'-nethylamino-5'-n adenosine was a mild inhibitor, and the selective a1 receptor agonist 2-chloron-cyclopentyladenosine (ccpa, 1 200 m) proved ineffective. mip-1α production is inhibited by suppression of its steady-state mrna levels and was linked to the a3 and a2 agonists. we concluded that stimulation of a3 and, to a lesser extent, a2 adenosine receptors decreases mip-1α expression based on our in vitro findings. we studied whether ib-meca, as the most powerful inhibitor of mip-1α expression, also influences the production of other pro-inflammatory mediators. ibmeca (1 300 m) reduced the production of il-12, il-, and, to a lesser extent, nitric oxide in immune-stimulated cultured macrophages in a dose-dependent manner. we explored whether the a3 agonist ib-meca alters the course of inflammation, mip-a production, and the degree of neutrophil recruitment in arthritis because mip-α is a chemokine that increases neutrophil recruitment into inflammatory areas. in the case of ibmeca (0.5 mg/kg/day) decreased the degree of joint inflammation in a mouse model of collagen-induced arthritis. ib-meca decreased neutrophil infiltration and inhibited the production of mip-1α, il-12, and nitrotyrosine (a marker of reactive nitrogen species) in the paws. we conclude that adenosine receptor agonists, particularly the a3 agonist ib-meca, inhibit mip-α synthesis and have anti-inflammatory properties. as a result, stimulating adenosine receptor subtypes a3 and a2 may be a promising technique for the treatment of acute and chronic inflammatory diseases. key words: inflammation, cytokines, adenosine receptor, arthritis denosine is a purine nucleoside generated by a number of cells in response to metabolic stress adenosine receptor ligands a1, a2a, a2b, and a3 have been found to inhibit the synthesis of pro-inflammatory mediators, diminish the recruitment of polymorphonuclear leukocytes (pmns), and exhibit anti-inflammatory properties [1]. furthermore, the ability to release adenosine is related to the anti-inflammatory activitie of medicines such methotrexate, sulphasalazine, and adenosine kinase inhibitors both a1 and a2 receptor agonists were reported to decrease tnfα production in raw 24.7 cells in recent research macrophages or human monocytes, and the rank order of agonist potency was not typical of either a1 or a2 receptors implying that the a3 receptor may be involved. furthermore, adenosine, but not adenosine, in human monocytes, selective a1 and a2 receptor agonists increased il-10 production it was further proven that suppression of tnf-α generation by lps-stimulated u937 (human monocyte) cells was primarily an a3 receptor mediated mechanism using particular a1, a2 and a3 receptor-mediated mechanism using particular a1, a2, and a3 receptor agonists and antagonists in the murine j774.1 macrophage cell line, ado receptor agonists prevented endotoxin activation of the tnfα gene and protein expression in a dose-dependent manner similar to the a3 receptor [2]. although the above research showed that several adenosine receptor agonists can affect the production of pro and anti-inflammatory cytokines no information on the modification of the production of chemokines by adenosine receptor ligands was previously published. mip-1 α (macrophage inflammatory protein) is a low-molecular-weight cc chemokine that has significant inflammatory effects [3]. the enhancement of neutrophil recruitment is the key mechanism for inflammatory sites. we investigated whether adenosine receptor ligands affect the production of mip-1 α a mailto:harisonwani10@gmail.com sonwani et al. role of adenosine receptor agonists in inflammatory conditions vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 70 protein and mrna in macrophages because mip-1 α is an important mediator of acute inflammation anadenosine receptor ligands can exert potent anti-inflammatory effects. we investigated whether the a3 agonist ib-meca impacts mip-1α production, the course of inflammation, and neutrophil recruitment in a model of collagen-induced arthritis after finding that adenosine receptor agonists, most notably the a3 agonist, suppress mip-1α expression. methods adenosine agonists have an effect on mip-la production in ran 264.3 cells: the mouse macrophage cell line raw 24.7 was grown. 1 ml of 1300 mm adenosine, the a1 receptor agonist 2-chloro-n-cyclopentyladenosine (ccpa), the a2 receptor agonist 2-p-(2-carboxyethyl) phenethylamino-5'-nethyl-carboxamidoadenosine (cgs2180), and the a3 receptor agonist n-(3-iodobenzyl)-adeno. research biochemicals inc. provided adenosine agonists. after 30 minutes, cells were stimulated with lps, and supernatants or cells were collected 1–3 hours later for mip-1α protein or mrna assays. the effect of a 1-h pretreatment with the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (ehna, 50 m) on the inhibitory effect of adenosine on mip-1α production was investigated in a second set of tests. mip-1α is a protein that was discovered in the human body as previously described, using murine elisa kits bought from genzyme ) (detection limit: 1.5 pg/ml) the experiment was not hampered by any test chemicals. adenosine agonists' effects on mip-la steady-state mrna levels: a guanidinium isothiocyanate/chloroform-based approach (trizol) was used to extract total rna from each well, followed by isopropanol precipitation. on a 1% formaldehyde gel, cytoplas mic rna (15 g) was separated and transferred to a nylon membrane. the rat mip-1α cdna sequence is 92 percent identical to the mouse sequence, indicating cross-hybridization between the two species. by random priming, this cdna was radiolabeled with 32p-dctp (specific activity, 3,000. the radioactivity of probes was measured. the northern blot was hybridized at 42°c for 1 hour after scintillation counting and 1.5107 c.p.m. were applied. the hybridized filters were washed serially at 53°c with a solution of 2 sodium citrate, sodium chloride, and 0.1 percent sds (2ssc). autoradiography was performed using hodak -omat-ar film at —70°c. membranes were stripped with boiling 5 mm edta and rehybridized with a 32pradiolabeled oligonucleotide probe for 18s ribosomal rna after probing for mip-1α. in ran 264.3 cells, the effect of ib meca on cytokine and no production. in the instance of the a3 agonist ib-meca, we wanted to see if it affected the production of il12, il-, and no in raw 24.7 macrophages as well. the cells were pretreated with 1 300 mib-meca in these tests. after 30 minutes, cells were stimulated with either lps (10 g ml—1) or murine ifn(200 u ml—1; genzyme, boston, ma, usa). this lps concentration is higher than that employed in investigations on the effects of adenosine receptor agonists on mip-1α generation. our early research has shown that greater lps concentrations are required to reach optimal no generation levels. only the combination of a greater dose of lps and ifnresulted in detectable il-12 production in this cell type. after a 24-hour incubation period at 37°c, the culture supernatant fluids were collected and kept at —70°c. elisa kits specifically for murine cytokines was used to determine cytokines. elisa kits from genzyme were used to quantify il-12 (p40 and p70) and ila spectramax 250 microplate reader was used to read the plates at 450 nm. il12 (p40) had detection limits of 10 pg ml—1, il-12 (p70) had detection limits of 5 pg ml—1, and ilhad detection limits of 5 pg ml—1. assays were carried out as previously described. and mg/ml by stirring at 4°c overnight. cii was dissolved and stored at —70°c until needed. the addition of mycobacteuium tuberculosis h37ra at a dose of 2 mg ml—1 yielded complete freund's adjuvant (cfa). cii was emulsified with an equal volume of cfa before injection. as previously described (szabo' et al., 1998), collageninduced arthritis was developed. on day 1, mice were given 100 l of the emulsion (containing 100 g cii) intradermally at the base of the tail. a second injection of cii in cfa was given on day 21. animals were given ib-meca at a dose of 0.5 mg kg—1 to stimulate a3 adenosine receptors. this dose was chosen based on prior in vivo investigations auchampach et al., 1997; tracey et al., 1994; tracey et al., 1997). starting on day 18, animals were given either vehicle (n=18) or ib meca (n=18; 0.5 mg kg—1, i.p.) every 24 hours. the mice were checked for arthritis on a daily basis. employing a macroscopic scoring system that ranges from 0 to 4 (0=no indications of arthritis, 1=swelling and/or redness of the paw or one digit, 2=two joints involved, 3=more than two joints involved, and 4=severe arthritis of the entire paw and digits). each mouse's arthritic index was computed by adding the four unique paw scores. no animals were removed from the calculations, and severity indices were determined for entire groups of mice (vehicle-treated or ibmeca-treated). nitrotyrosine immunohistochemistry and histology: animals were sacrificed under anesthesia at the end of the experiments (day 35), and their paws and knees were taken and preserved for histological analysis by an investigator who was blinded to the treatment regimen. joints were embedded for nitrotyrosine immunohistochemistry. snap frozen in liquid nitrogen in m1 medium. a microtome equipped with a carbide steel knife was used to cut cryostat pieces (m). by immunohistochemistry, joint slices were examined for the presence of nitrotyrosine, a peroxynitrite indicator for 15 minutes, endogenous peroxidase was quenched with 0.3 sonwani et al. role of adenosine receptor agonists in inflammatory conditions vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 71 percent h2o2 in pbs. by incubating the slice in 2 percent normal goat serum in phosphate-buffered saline for 0 minutes, non-specific adsorption was reduced. after that, the sections were treated with a 1: 500 dilution of primary antinitrotyrosine antibody overnight biotin-conjugated goat antirabbit igg, and an avidin-biotin-peroxidase complex was used to identify specific labeling. anti-nitrotyrosine antibodies were used to incubate sections in control studies. in the presence of 10 mm nitrotyrosine, an antibody was produced. the nitrotyrosine staining in the figures was removed as a result of this intervention. in paw extracts, cytokines, chemokines, and nitrotyrosine were detected: aqueous joint extracts were prepared from control animals and animals with 35 days of arthritis as described by homogenization in a lysis buffer in the presence of a mixture of protease inhibitors (10 g ml—1 aprotinin, 20 g ml—1 leupeptin, 10 g ml—1 pepstatin a, and 1 mm pmsf, ph 7.25) elisa ) was used to determine the presence of il12, tnf-α, and mip-1α in the extracts, as well as the presence of nitrated proteins using western blotting each sample weighed thirty gram heated to 95°c for 3 minutes after being diluted in an equal volume of treatment buffer. after that, the samples were put into a 1% tris-glycine solution. gels were operated for 2 hours at 120 volts, then transferred to 0.45 m nitrocellulose at 30 volts for 0 minutes using the 1/2towbin buffer system (1.45 g tris, 7.2 g glycine, 800 ml di h2o and 200 ml meoh). the membrane was blocked in 1 percent bsa: 1 percent nonfat milk in pbs-tween (phosphate-buffered saline with 0.05 percent tween 20) for 1 hour before being probed with rabbit anti-nitrotyrosine (1 g/ ml in pbstween) overnight at 4°c. the blot was washed three times with pbs-t and once with h2o before being incubated with the secondary antibody, goat anti-rabbit-hrp, for 1.5 hours (1 : 3000). after washing the blot three times in pbs-t and once in di h2o, 1.5 ml mixed ecl chemiluminescence reagent was applied for 1 minute. after that, the blot was exposed to x-ray film for 0 seconds. measurements of myeloperoxidase in paw homogenates: in addition, the activity of myeloperoxidase, a marker of neutrophil infiltration, was evaluated in the paws as described paws were homogenized in a solution of 0.5 percent hexadecyl-trimethylammonium bromide mixed in 10 mm potassium phosphate buffer (ph 7) and centrifuged at 20,000g for 30 minutes at 4°c. an aliquot of the supernatant was allowed to react with tetramethyl-benzidine (1. mm) and 0.1 mm h2o2 in a solution. at a wavelength of 50 nm, spectrophotometry was used to determine the rate of change in absorbance. myeloperoxidase. the amount of enzyme that degraded 1 mol of hydrogen peroxide per minute at 37°c was measured in milliunits per mg protein. analyzing and presenting data: all figures and text data are expressed as the mean of n observations, where n is the number of wells (9 wells from two to three independent experiments) or the number of animals analyzed. oneand two-way analysis of variance were used to investigate the data sets, and individual group averages were compared using dunnett's test. the statistical differences in the arthritic indices were tested using the mann-whitney u-test (2-tailed, independent) in the arthritis studies. statistical significance was defined as a p-value of less than 0.05. results mip-l α is suppressed by adenosine agonists in immunestimulated macrophages.mip-1α was produced in considerable amounts in response to lps (10 ng/ml). the a3 agonist ib-meca and, to a lesser extent, the a2 agonist cgs-2180 inhibited mip-1α production, whereas the a1 receptor agonist ccpa had no impact (figure 1). at 200 m, adenosine had a minor inhibitory effect, resulting in a 213 percent inhibition (p<0.01). the mtt experiment revealed that the adenosine agonists tested had no effect on mitochondrial respiration. because adenosine deaminase degrades adenosine quickly, we wanted to see if inhibiting adenosine deaminase changed the degree of inhibition. there was no increase in the inhibitory action of adenosine in the presence of the adenosine deaminase inhibitor ehna (50 m) (245% inhibition at 200 mmip-1α steady-state mrna levels rise in a time-dependent manner ib-meca caused a significant reduction in mip-1α production, which was accompanied by a significant, dose-dependent reduction in mip-1α mrna (figure 2). mip-1α mrna expression was unaffected by adenosine or the a1 agonist, however, steadystate mrna levels were decreased by the a2 agonist cgs2180 (figure 2). overall, the degree of inhibition of mip-1α protein and mip-1α mrna expression by adenosine agonists had a fairly high association. the degree of suppression of mip-1α mrna levels and protein levels, on the other hand, did not always match. using the elisa approach, we discovered that cgs (at 200 m) suppressed mip-1α protein synthesis by roughly 50% whereas the inhibition of mip-1α protein production by nearly 50% (figure 1) lps elicited the synthesis of mrna was virtually completely eliminated differences in measurement duration (protein was tested 3 hours after lps, mrna 2 hours after lps) and/or sensitivity of the two assays. these disparities could be due to the methods utilized (elisa versus northern blotting). ib-meca inhibited mip-1α production the most strongly of all the adenosine agonists tested. as a result, we examined the effect of this agonist on the generation of other inflammatory mediators (il-12, il-, no) by activated macrophages and in an in vivo model of inflammation in the following experiments. lps (10 g ml—1) or ifn(200 u ml—1) did not elicit measurable levels of il-12 (p40 or p70) sonwani et al. role of adenosine receptor agonists in inflammatory conditions vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 72 in raw 24.7 macrophages after a 24-hour treatment even though the combination of lps (10 g ml—1) and ifn(200 u ml—1) promoted the production of il-12 p40 (figure 4a), il-12 p70 was not generated in measurable amounts. pretreatment of cells with ib-meca 30 minutes before lps + ifnresulted in a concentrationdependent reduction of il-12 p40 production, as measured at 24 hours (figure 4a). as determined 24 hours after stimulation, lps (10 g ml—1) stimulated the release of iland nitrite (the breakdown product of no) in the culture supernatants of raw 24.7 cells. ib-meca, administered 30 minutes before lps, reduced the generation of iland nitrite (figure 4b and c). in all of these trials, ib-meca had no effect on cell viability as assessed by the mtt assay (not shown). ibstronger meca's inhibition of il-12 and mip-1α and less reduction of no and ilproduction could be due to two distinct a3 receptor subtypes. the recent research, on the other hand. ib-meca therapy protects mice from collagen-induced arthritis: the majority of the vehicle-treated mice developed arthritis between days 2 and 35 after the first collagen immunization treatment with ib-meca (0.5 mg/kg once day i.p.) reduced the occurrence of arthritis and lowered the disease's severity. the paws of the vehicletreated arthritic animals showed evidence of severe suppurative arthritis at day 35, with extensive neutrophil, macrophage, and lymphocyte infiltration. in addition, there was severe or moderate necrosis, hyperplasia, and sloughing of the synovium, as well as inflammation spreading into the neighboring musculature the degree of arthritis was dramatically reduced in the ib-meca treated mice, with moderate cell infiltration, mild to moderate necrosis, and synovial hyperplasia the reduction in neutrophil infiltration was measured by paw myeloperoxidase. arthritis induced a 991 mu/mg protein increase in myeloperoxidase levels (n=12). there was a significant increase in the number of ib-meca-treated animals. at the end of arthritis, there was a substantially decreased degree of rise in the paw myeloperoxidase concentration (3 mu/mg protein; n=12, p0.01). at 35 days, there was a considerable increase in mip-1α and il-12 p40 levels in the aqueous extracts of the arthritic paws, but no detectable tnf-α or il12 p70 levels in the extracts ibmeca therapy reduced the formation of lps-stimulated cells or lps-challenged animals, according to the results in lps-stimulated cells or lps-challenged animals. increased levels of il12 p40 and mip-1α in the joints (figure 7). we detected the emergence of nitrotyrosine-positive staining in the inflamed joints using immunohistochemistry and western blotting of proteins in aqueous joint extracts, but not in healthy control animals. the degree of nitrotyrosine staining was lowered after ibmeca therapy (figure 3, 4 & 5). figure 1 (a): effect of adenosine [ado], the a1 agonist ccpa, the a2 agonist cgs, and the a3 agonist ibmeca on the production of mip-1α mrna in response to stimulation with bacterial lipopoly-saccharide lps (10 ng ml—1) in raw macrophages. (a) dose-response showing the effect of 0.5, 5, 50, and 100 μm ib-meca on the production of mip-1α mrna at 3 h after stimulation. (b) effect of adenosine, ccpa, cgs, and ib-meca (200 μm each) on the production of mip-1α mrna at 3 h after stimulation. representative blots of n=3 − 4 blots are shown; 18s mrna was unaffected by any of the treatments were. this possibility was not intended to be explored. figure 2: effect of adenosine [ado], the a1 agonist ccpa, the a2 agonist cgs, and the a3 agonist ibmeca on the production of mip-1α at 3 h after stimulation with bacterial lipopolysaccharide lps (10 ng ml—1) in raw macrophages. mip-1α production in the absence of the inhibitors amounted to б.б±1.3 ng ml—1, and was considered 100%. n=б − 9 wells from two to three independent experiments. *p<0.05 and **p<0.01 indicate significant inhibition of the production of mip1α. sonwani et al. role of adenosine receptor agonists in inflammatory conditions vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 73 figure 3 – (a) ib-meca suppresses collagen-induced arthritis in mice. the percentage of arthritic mice (mice showing clinical scores of arthritis >1) is represented. (b) effect of ib-meca on the severity of collagen-induced arthritis. the median arthritic score during collageninduced arthritis. n=10 − 12. there was a significant increase in the arthritic score from day 28 (*p<0.01), and there was significant suppression of the arthritic score by ib-meca from day 30 (¢p<0.05). figure 4: ib-meca suppresses the production of inflammatory mediators in immune-stimulated cultured raw macrophages. cells were stimulated with lps (10 μg ml—1) and ifn-ц (200 mu/ml) for 24 h for the measurement of il-12 (p40), with lps (10 μg ml—1) for 24 h for il-б and nitrite measurements, or with lps (10 ng/ml) and ifn-ц(200 mu ml—1) for 3 h for the measurement of mip-1α absolute values of il-12, il-б, and nitrite in the absence of ibmeca treatment amounted to 378±23 pg ml—1, 7б±4 ng ml—1 and 49±5 μm respectively, and were considered 100%. n=б − 9 wells from two to three independent experiments. *p<0.05 and **p<0.01 indicate significant inhibition by ib-meca treatment. figure 5 ib-meca suppresses the production of inflammatory mediators in aqueous paw extracts of mice subjected to collagen-induced arthritis. levels of mip-1α and il-12 are shown in samples from control animals (c), from collagen-induced arthritis at 35 days (a), and ib-meca-treated mice subjected to collageninduced arthritis (m) at 35 days. n=10 − 12. *p<0.05 indicates suppression of il-12 and mip-1α production in arthritis by ibmeca. discussion different adenosine receptor subtypes activate different signal transduction processes in different cells. martin and dorf (1991) in macrophages, however, a3 receptor stimulation does not require cyclic amp, protein kinase a, or the transcription factor nf-kappa b, despite the fact that stimulation of this receptor subtype might change the makeup of the ap-1 transcription complex further research is needed to determine the cellular signaling pathway by which a3 activation reduces cytokine or mip-1α production [3-5]. in our settings, the a1 agonist had no substantial inhibitory effect on mip-1α generation, but adenosine was a weak inhibitor. adenosine is primarily an a1 receptor agonist, with minor a2 and a3 agonist properties [6]. our research with the adenosine deaminase inhibitor showed that adenosine deaminase, which is present in the fetal calf, degrades adenosine quickly. the low amount of inhibition is not attributable to serum. the lower eacacy of adenosine in mip-1α production may be due to its reduced potency on a2 and a3 receptors, according to our findings [7]. in addition to inhibiting mip-1α production, ib-meca decreased il-12, il-, and no production in immune stimulated cultured macrophages, according to the current study. adenosine receptor agonists have recently been shown to prevent endotoxin-mediated stimulation of the tnf-α gene and protein expression in the murine j774.1 macrophage cell line in a dose-dependent manner characteristic of the a3 receptor [9, 10]. we recently demonstrated that stimulating a3 receptors with the selective a3 receptor agonist ib-meca reduced plasma tnf-α while sonwani et al. role of adenosine receptor agonists in inflammatory conditions vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 74 increasing il-10 in lps-treated mice. the current study's findings add to these earlier findings. the raw 24.7 cell studies are merely a rough approximation of the properties of primary or resident macrophages, or the in vivo scenario in arthritis, where resident macrophages play a role in the production of chemokines, cytokines, and no [11]. despite this, the control of chemokine, cytokine, and no production by adrenergic, purinergic receptors in monocytic/macrophage cells and primary monocytes/ macrophages displays striking parallels the in vivo assessments of no, il-12, and mip-1α production in joint extracts in the current investigation were similarly in good agreement with our in vitro findings in raw cells. in the arthritis model, it's possible that ibmeca inhibits the synthesis of il-12, mip-1α, and no, which are all significant anti-inflammatory mechanisms [12-14]. it is widely known that il-12 plays a key role in the early events of the arthritis induction phase. the research best confirmed this, showing that il-12 may substitute mycobacteria and cause autoimmune arthritis in dba/1 mice inoculated with chicken cii in incomplete freund's adjuvant [15]. furthermore, these researchers discovered that il-12 could aggravate arthritis caused by chicken cii in cfa mice. the course of the disease was suppressed in il-12 deficient animals or mice treated with anti-mil-12 antibodies [13]. after il-12 initiates the inflammatory process, macrophages and other cell types become activated and release a range of mediators such as tnfa, il-, il-1, mip1α, and no, which keep the inflammatory state alive. the chemokine mip-1α, which is generated by fibroblasts or macrophages, appears to be a key pathogenic element in the development of arthritis, owing to its chemotactic action on inflammatory cells. the decrease of paw mip-1α levels may contribute to the reduction of neutrophil recruitment (as evidenced by the suppression of paw myeloperoxidase levels in the current investigation) [14-16]. the a3 has an inhibiting action. over production of this free radical, as well as its reactive reaction product, peroxynitrite, has been demonstrated to contribute to the pathogenesis of the inflammatory joint disease, therefore a receptor agonist on no release could be advantageous although recent research postulated alternate mechanisms of tyrosine nitration, connected to myeloperoxidase-dependent conversion of nitrite to no2cl and no2, nitrotyrosine production is largely acknowledged as a specific 'footprint' of peroxynitrite in the joints, nitrotyrosine could operate as a collective indication for the formation of reactive nitrogen species [17-20]. the current work shows that stimulating the a3 and a2 receptors reduces mip-1α production.by inhibiting the expression of its mrna furthermore, the current study shows that an a3 receptor agonist reduces inflammation in collagen-induced arthritis. downregulation of the proinflammatory mediators mip-1α, il-12, and no is one of the mechanisms underlying this anti-inflammatory action. the ability of drugs like methotrexate sulphasalazine and adenosine kinase inhibitors to release adenosine at the sites of inflammation is linked to their anti-inflammatory properties. these anti-inflammatory actions may be related to a3 and a2, at least in part, according to our hypothesis of activation of receptors. we believe that stimulating the adenosine receptor subtypes a3 and a2 could be a promising technique for the treatment of acute and chronic inflammatory diseases. references 1. standiford tj, kunkel sl, lukacs nw, et al. macrophage inflammatory protein-1 alpha mediates lung leukocyte recruitment, lung capillary leak, and early mortality in murine endotoxemia. j immunol [internet]. 1995 [cited 2023 apr 10];155(3):1515–24. 2. szabó c, lim lh, cuzzocrea s, et al. inhibition of poly (adpribose) synthetase attenuates neutrophil recruitment and exerts antiinflammatory effects. j exp med [internet]. 1997 [cited 2023 apr 10];186(7):1041–9. 3. tracey wr, magee w, masamune h, et al. selective adenosine a3 receptor stimulation reduces ischemic myocardial injury in the rabbit heart. cardiovasc res [internet]. 1997 [cited 2023 apr 10];33(2):410–5. 4. lubitz dkje, lin rc-s, popik p, et al. adenosine a3 receptor stimulation and cerebral ischemia. eur j pharmacol [internet]. 1994;263(1–2):59–67. 5. wolberg g, zimmerman tp. effects of adenosine deaminase inhibitors on lymphocyte-mediated cytolysis. ann n y acad sci [internet]. 1985;451(1):215–26. 6. al-mughales j, blyth th, hunter ja, et al. the chemoattractant activity of rheumatoid synovial fluid for human lymphocytes is due to multiple cytokines. clin exp immunol [internet]. 1996;106(2):230–6. 7. auchampach ja, rizvi a, qiu y, et al. selective activation of a 3 adenosine receptors with n 6 -(3-iodobenzyl)adenosine-5′ n -methyluronamide protects against myocardial stunning and infarction without hemodynamic changes in conscious rabbits. circ res [internet]. 1997;80(6):800–9. 8. beckman js, koppenol wh. nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. am j physiol cell physiol [internet]. 1996;271(5):c1424–37. 9. sheth s, brito r, mukherjea d, et al. adenosine receptors: expression, function and regulation. int j mol sci [internet]. 2014;15(2):2024–52. 10. connor jr, manning pt, settle sl, et al. suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase. eur j pharmacol [internet]. 1995;273(1– 2):15–24. 11. haskó g, cronstein b. regulation of inflammation by adenosine. front immunol [internet]. 2013;4:85. 12. cronstein bn, eberle ma, gruber he, et al. methotrexate inhibits neutrophil function by stimulating adenosine release from connective tissue cells. proc natl acad sci u s a [internet]. 1991;88(6):2441–5. 13. cronstein bn, naime d, ostad e. the antiinflammatory mechanism of methotrexate. increased adenosine release at sonwani et al. role of adenosine receptor agonists in inflammatory conditions vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 75 inflamed sites diminishes leukocyte accumulation in an in vivo model of inflammation. j clin invest [internet]. 1993;92(6):2675–82. 14. dubyak gr, el-moatassim c. signal transduction via p2purinergic receptors for extracellular atp and other nucleotides. am j physiol cell physiol [internet]. 1993;265(3):c577–606. 15. eiserich jp, hristova m, cross ce, et al. formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. nature [internet]. 1998;391(6665):393–7. 16. gadangi p, longaker m, naime d, et al. the anti-inflammatory mechanism of sulfasalazine is related to adenosine release at inflamed sites. j immunol [internet]. 1996 [cited 2023 apr 10];156(5):1937–41. 17. gallo-rodriguez c, ji x-d, melman n, et al. structure-activity relationships of n6-benzyladenosine-5’-uronamides as a3selective adenosine agonists. j med chem [internet]. 1994;37(5):636–46. 18. halliwell b. what nitrates tyrosine? is nitrotyrosine specific as a biomarker of peroxynitrite formation in vivo? febs lett [internet]. 1997;411(2–3):157–60. 19. haskó g, szabó c, németh zh, et al. adenosine receptor agonists differentially regulate il-10, tnf-alpha, and nitric oxide production in raw 264.7 macrophages and in endotoxemic mice. j immunol [internet]. 1996 [cited 2023 apr 10];157(10):4634–40. 20. haskó g, szabó c, németh zh, et al. suppression of il-12 production by phosphodiesterase inhibition in murine endotoxemia is il-10 independent. eur j immunol [internet]. 1998 [cited 2023 apr 10];28(2):468–72. how to cite this article: hari sonwani, virendra kumar sharma, steffi thomas, akhlesh kumar. adenosine receptor agonists suppress macrophage inflammatory protein -1α production and collagen induced arthritis. indian j pharm drug studies. 2023: 2(2) 69-75. funding: none conflict of interest: none stated mandumpala a caution for judicial antibiotic use vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 11 review article ceftriaxone resistance – a caution for judicial antibiotic use janice jacson mandumpala from, pharm d intern, nirmala college of pharmacy, ernakulam, kerala, india corresponding to: janice jacson mandumpala, pharm d intern, department of pharmacy practice, nirmala college of pharmacy, ernakulam, kerala, india. email: janice.jacson@gmail.com abstract ceftriaxone is a broad-spectrum antibiotic that is effective against both gram-negative and gram-positive isolates. ceftriaxone resistance research is useful in determining its current status. as a result, we aimed to understand the prevalence of ceftriaxone resistance in bacteria isolated from various clinical specimens. resistance to ceftriaxone has been steadily increasing and demands immediate attention. ceftriaxone resistance prevention mechanisms must be strictly enforced. the appropriate drugs should be chosen based on susceptibility patterns. in summary, ceftriaxone should be considered in the initial treatment of seriously ill patients with infections of unknown aetiology when gram-negative aerobes (other than pseudomonas species) are the suspected pathogens, and in patients with gram-negative organisms with suspected or demonstrated resistance to other antibiotics. this review summarizes the advent of ceftriaxone resistance among various organisms across the globe. keywords – ceftriaxone, resistance, gonorrhoea, salmonella, shigella, aeromonas, enterobacter he third-generation cephalosporin ceftriaxone has good effectiveness against numerous gram-negative pathogens and excellent activity against the majority of gram-positive infections. ceftriaxone has generally more antibacterial action against gram-negative bacteria than the "first" and "second generation" cephalosporins, but less antibacterial activity than the previous generations of cephalosporins against numerous gram-positive bacteria. ceftriaxone has a long elimination half-life, allowing for twice and once-daily treatment, the latter of which may result in significant cost savings. ceftriaxone may replace other thirdgeneration cephalosporins as the treatment of choice for a range of serious infections due to its proven efficacy, safety, and practical dose schedule [1]. in patients with bacterial meningitis, respiratory tract infections, urinary tract infections, soft tissue infections of the bone and joints, and gonorrhoea, clinical investigations have shown their effectiveness and safety. except for diarrhea, which has often not necessitated changing the course of treatment, ceftriaxone has been well tolerated. despite having modest action against pseudomonas aeruginosa, ceftriaxone is not currently advised as the only antibiotic therapy for pseudomonal infections based on the available data. infections caused by other "difficult" organisms, such as multidrugresistant enterobacteriaceae, have been successfully treated with ceftriaxone [2]. with the discovery of penicillin by sir alexander fleming in 1928, the modern era of antibiotics began [3]. since that time, antibiotics have revolutionized contemporary medicine and helped save countless lives [4]. in the 1940s, doctors began using antibiotics to treat severe infections. unfortunately, almost all antibiotics that have been produced have eventually been associated with resistance, and this is also true of cephalosporins, notably ceftriaxone [5]. mechanism of ceftriaxone resistance development there are multiple mechanisms that infectious agents use to resist the lethal action of antibiotics such as mutational resistance, acquisition of genetic material, increased drug efflux or, decreased drug intake [6]. other complex mechanisms involve are biofilm formation and quorum [7]. quorum sensing and biofilm formation is the common mechanism for antibiotic resistance among e. coli. in this, the cells adhere to each other on their surface using a self-produced matrix called the extra polymeric substance. this extra polymeric substance creates a barrier for the antibiotic to enter the bacteria, this promotes the emergence of antibiotic resistance and this is referred to as quorum sensing [8]. however, this mechanism may not be applicable to gonococci. gonococci develop resistance to beta-lactam antibiotics primarily by two mechanisms – one that is quickly acquired through the transfer of resistance plasmid that produces betalactamases and the second one is the gradual acquisition of multiple resistance genes that promotes beta-lactam resistance [9]. the production of extended-spectrum beta-lactamases is the most commonly attributed mechanism of ceftriaxone t mailto:janice.jacson@gmail.com mandumpala a caution for judicial antibiotic use vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 12 resistance in e.coli [10]. additionally, a molecular analysis of salmonella isolates with ceftriaxone resistance has demonstrated a higher production of the cmy-2 and the ctxm-3 beta-lactamases [11]. on the other hand, ceftriaxone resistance in neisseria gonorrhoeae is a result of chromosomally mediated mutations at the three loci of pena, mtrr, and penb [12]. furthermore, the production of cephalosporinases also degrades cephalosporins and this is one such mechanism that promoted ceftriaxone resistance among salmonella species. since extended-spectrum beta-lactamases are mobile genetic materials and bacteria can acquire them through horizontal gene transfer from resistant bacteria, sensitive bacteria can acquire resistance to cephalosporins [13]. also, several studies on the mechanism of resistance to third-generation cephalosporins, particularly ceftriaxone have suggested that clinical strains of enterobacter cloacae are linked to the excessive production of chromosomal beta-lactamases [10, 14]. neisseria gonorrhoeae antimicrobial resistance in neisseria gonorrhoeae is a major public health concern globally. the most frequently prescribed antibiotics for gonorrhoea are ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, and ofloxacin. neisseria gonorrhoeae was initially susceptible to the majority of antimicrobials since the mid-1930s [15, 16]. but recently the resistant patterns in neisseria gonorrhoeae have developed owing to its ability to develop or acquire antimicrobial resistance through most of the existing mechanisms. a few examples of the mechanisms neisseria gonorrhoeae undergo to cater to antimicrobial resistance are as follows – inactivation, degradation of antimicrobials, alteration of the target antimicrobial, decreased antimicrobial influx, and increased generation of efflux transporters. resistance of neisseria gonorrhoeae to fluoroquinolones and ceftriaxone came into prominence after its first appearance in japan which then further spread globally [17-20]. furthermore, the first global failure for treating pharyngeal gonorrhoea with dual antibiotics (both 500 mg ceftriaxone and 1g azithromycin) was first reported in the united kingdom in 2016 [21]. dual therapy has been instituted due to the emerging resistance patterns, but countries such as japan, china, azerbaijan, netherlands, belarus, and ukraine still use ceftriaxone 500mg-1g as empirical mono-therapy [22-24]. with the evolving resistance patterns, what are the options available to the medical community? repurposing the old antimicrobials, use of newer antimicrobials with mainly nonhuman or invitro data availability, and novel antibiotics in clinical trials. spectinomycin is a long-used antimicrobial for gonorrhoea with excellent susceptibility, but relatively lower success in treating pharyngeal gonorrhoea [25]. therefore, spectinomycin can be used as a part of the dual regimen along with azithromycin to prevent the emergence of antimicrobialresistant strains [26]. new antimicrobials with only non-human evidence have demonstrated relative potency against neisseria gonorrhoeae and include avarofloxacin, delafloxacin, sitafloxacin, tigecycline, 2-acyl carbapenems and many others which have been reviewed elsewhere [27-29]. however, many of them are in stages of early development thus posing a challenge for their use within the clinical setting. three antimicrobials solithromycin, zoliflodacin, and gepotidacin are novel orally administered drugs in clinical evaluation for uncomplicated gonorrhoea and particularly zoliflodacin appears very promising and requires attention [30]. until a promising new effective treatments, rational use of ceftriaxone and azithromycin will help treat gonorrhoea with its severe complications. aeromonas spp. aeromonas species can cause a wide spectrum of intestinal and extra-intestinal infections (gastrointestinal tract syndromes, wound and soft tissue infections, urinary tract infections, and rarely septicaemia) [31]. they are gram-negative rods, facultative anaerobes and oxidase-positive. aeromonas species that are pathogenic for humans include aeromonas hydrophila, aeromonas sobria, aeromonas trota, and aeromonas caviae and are most commonly found in the aquactic life forms particularly fishes [32, 33]. the sensitivity of aeromonas can vary based on geographical location and diversity. the most common antibiotics employed for aeromonas infections are fluoroquinolones and tetracyclines. furthermore, thirdgeneration cephalosporins have also been recommended but in recent years, there has been an upward trend for resistance to ceftriaxone attributed to the beta-lactamase activity of aeromonas [34]. aeromonas has been linked to the development of betalactamases (esbls, ampcbls, and carbapenemases), efflux pumps, and changes in the outer membrane that result in decreased permeability as the mechanisms of resistance to third-generation cephalosporins. aeromonas is known to carry various drug-resistance genes and has also demonstrated the ability to transfer such genes to various gram-negative bacteria. multiple drug resistance among aeromonas spp. have been reported from many parts of the world as well. also, a concerning aspect of drug resistance among the aeromonas spp. is the ability to bring about extra-intestinal infections that can be fatal with the existing multiple drugresistance genes [35]. a plausible cause for such increased spread of antibiotic resistance is rapid urbanization and the evolution of bacteria to survive such threats [36]. mandumpala a caution for judicial antibiotic use vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 13 salmonella spp. an alarming health issue that exists everywhere is salmonella infection. effective steps should be taken to stop the issue from getting worse [11]. the preferred treatment for typhoid fever is ceftriaxone, however, the introduction of salmonella typhi which is resistant to it creates serious treatment problems. there are more and more sporadic instances of ceftriaxone-resistant s. typhi, therefore reducing the chance that the patient won't respond to therapy and that outbreaks will spread throughout the neighbourhood must take precedence. since 1988, salmonella resistant to extended-spectrum cephalosporins has become an international problem. this public health issue had been reported in 43 countries by 2004. traditional extendedspectrum beta-lactamases, plasmid-mediated cephalosporinases, and most recently a class a carbapenemase were responsible for mediating resistance. the most extensively distributed of them is cmy-2. the two most prevalent serovars connected to extended-spectrum cephalosporins resistance in human infections are salmonella enterica serotype typhimurium and salmonella enterica serotype enteritidis [37]. the invasive infection caused by salmonella enterica serotype choleraesuis is usually typical. most often extendedspectrum cephalosporins or fluoroquinolones are utilized to treat resistant salmonella infections [38]. however, there are increasing reports of fluoroquinolone resistance in the invasive serotype of salmonella. in sc-b67, ceftriaxone resistance was linked to a plasmid-mediated blacmy-2 that is commonly present on a particular isecp1-blacmy-2-blc-suge structure [39]. this conserved dna segment, later given the designation tn6092, has been identified in numerous enterobacteriaceae and salmonella serotypes from across the globe [40, 41]. in addition, ceftriaxone-resistant serotype salmonella typhimurium infections, which were previously uncommon in western kenya, appeared quickly between 2009 and 2014 in countries in western kenya, restricting the range of available treatments for serious infections. in order to direct proper therapy in diverse asian and african nations, there is an urgent need for increased microbiologic diagnostic capability in clinical settings [42]. extended-spectrum –lactamases whose genetic elements can be found in plasmids or the chromosome, are typically responsible for the creation of resistance to ceftriaxone. organisms that produce ctx-m-type esbls constitute a particularly major public health danger globally among the variety of antibiotic-resistant gram-negative bacteria pathogens that are currently recognised [43]. an extremely invasive zoonotic bacterium called s. choleraesuis produces a devastating systemic infection in people. a significant treatment issue has arisen due to the emergence and growth of s. choleraesuis resistance to ceftriaxone and ciprofloxacin. sirirat et al have demonstrated high frequency of antimicrobial resistance in salmonella choleraesuis among 414 nontyphoidal salmonella isolates from bacteremic patients in thailand. s. choleraesuis isolates had high rates of ceftriaxone (58.3%) and ciprofloxacin (19.6%) resistance. the clonal spread of s. choleraesuis isolates harbouring blacmy-2 as well as the dissemination of the self-transferable blactx-m-14-carrying incfiis, incfii, and inci1 plasmids and the blacmy-2-carrying inca/c plasmid as well as the high frequency of resistance to extended-spectrum cephalosporins (3rd and 4th generation cephalosporins). the first occurrence of ceftazidime-hydrolyzing ctx-m-55 in s. choleraesuis isolates has been documented; between 2012 and 2016, this variant's abundance among esc-resistant s. choleraesuis isolates grew rapidly. the dispersion of inca/c plasmids carrying both blactx-m-55 and qnrs1 among ciprofloxacin-resistant s. choleraesuis isolates expressing d87g in gyra was the cause of the spread of clone pulsotype b3. the high frequencies of co-resistance to escs and ciprofloxacin (51.3%) from 2012 to 2016 were reportedly caused by these isolates. as a result of travel and commerce in animal food items, this study highlights the significance of having an action plan to prevent the spread of antibiotic resistance in s. choleraesuis [44-46]. shigella spp. in the underdeveloped world, shigellosis is one of the major causes of diarrheal illnesses. an estimated 165 million cases and 1.1 million fatalities worldwide (mainly in underdeveloped nations) occur each year. shigellosis patients have been advised to have antibiotic therapy because it can shorten the duration of the causative organism's faecal excretion and limit the clinical course of the illness, lowering the risk of complications and infection transmission [47, 48]. the growing resistance of shigella spp. to effective antimicrobial treatments, however, is a significant issue [49]. shigella isolates that are resistant to a variety of medications, including sulphonamides, tetracyclines, ampicillin, trimethoprim-sulphamethoxazole (sxt), and nalidixic acid, have been reported from many different nations over the years [50]. azithromycin, mecillinam, ciprofloxacin, ceftriaxone, and cefixime are examples of novel antimicrobials that have been proven to be successful in treating multidrug-resistant shigellaassociated illnesses. however, shigella strains resistant to ceftriaxone have emerged [51, 52]. fluoroquinolones are the sole available antibiotic therapy for such mdr shigellaassociated illnesses due to the dearth of other options. fluoroquinolones must be used carefully and wisely to prevent the rapid emergence and spread of resistance, as evidenced by the discovery of decreased susceptibility to fluoroquinolones in a significant fraction of shigella strains and completely fluoroquinolone-resistant s. dysenteriae type 1 [53, 54]. according to a study conducted by gu b et al, a lower average mandumpala a caution for judicial antibiotic use vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 14 prevalence of ceftriaxone resistance among shigella spp. was found in europe-america countries. in contrast, asia and africa saw a clear increasing trend in ceftriaxone resistance, especially after 2007. ceftriaxone resistance was present on average at a rate of 5% in asia and africa, which is 6 times higher than that of europe and north america. in addition, asia-africa had resistance rates to ceftriaxone, cefotaxime, and ceftazidime that were up to 142%, 226%, and 62%, respectively, between 2010 and 2012. this warrants rapid action [55]. this phenomenon could have a number of explanations. the major cause is regional variations, which have a direct impact on socioeconomic position. individuals in more developed regions (such as europe and america) typically receive better care after contracting an infection than people in less developed nations (asia-africa). second, flaws in medical infrastructure and facilities are also significant factors. pakistan and other asian-african nations lack the infrastructure necessary to track antimicrobial resistance on a national scale [56]. in addition, cheap antibiotics are widely accessible from both licensed and unlicensed sources in many asian and african nations, which contributes to drug addiction and a high rate of resistance [57]. third, the lack of access to safe drinking water and inadequate sanitation lead to the emergence of mdr strains of shigella. malnutrition is one of the underlying factors that raise the risk of diarrhoea and is widespread in asian and african nations [58]. enterobacter cloacae the environmental pathogen enterobacter cloacae complex (ecc), which is common, is a significant cause of nosocomial infections. the therapeutic importance of each species within ecc is less understood since standard techniques used in clinical laboratories cannot distinguish between species within ecc.gram-negative rods resistant to third-generation cephalosporins are included in the broad spectrum of antibacterial activity of carbapenems. the main cause of ceftriaxone resistance is the pre-existing presence of resistant clones (also known as "derepressed mutants") in the enterobacter populations. the majority of resistant clones generate substantial levels of beta-lactamases, and some of them exhibit decreased porin f expression together with enhanced porin c expression. in a hplc-based approach conducted by pechere et al the author used hplc to determine the outer membrane permeability in intact cells, and the results revealed that imipenem penetrated three times more quickly than ceftriaxone. furthermore, imipenem penetration was unaffected in a porin f defective mutant, in contrast to ceftriaxone. this implied that imipenem has a different route, possibly porin c, then ceftriaxone. the affinity for pbps, as determined by computation, and the rate of beta-lactamase hydrolysis under experimental settings that were believed to be physiologically relevant were comparable for the two antibiotics. thus, selective permeability of the outer membrane appears to be the main cause of imipenem's action against ceftriaxone-resistant e. cloacae [59]. because ecc isolates display intrinsic ampc -lactamases, such as cmh, act, and mir with numerous variations, they are innately resistant to ampicillin, amoxicillin-clavulanate, and first and secondgeneration cephalosporins [60]. antibiotic usage has led to the emergence and global spread of multidrug resistant (mdr) ecc strains [61]. third-generation cephalosporin resistance is also brought on by the acquisition of genes encoding extended spectrum -lactamase (esbl). additionally, treatment is becoming challenging due to the advent and rising prevalence of carbapenem-resistant ecc. mdr strain infections typically result in increased mortality, lengthier hospital stays, and higher expenses, having a significant influence on worldwide public health [62]. conclusion antibiotic resistance should be prevented by reducing the irrational misuse of antibiotics, but designing successful interventions to do so will require a greater knowledge of the practices and financial incentives associated with antibiotic administration. although increased cephalosporin resistance is frequently attributed to a single factor (pbp modification, betalactamase action, or impermeability), an organism's response to a drug frequently reflects the interaction of several factors. there is a need to develop methods, such as mathematical models, to aid in identifying the interplay of factors that lead to ceftriaxone resistance. references 1. nahata mc, barson wj. ceftriaxone: a third-generation cephalosporin. vol. 19, drug intelligence and clinical pharmacy. 1985;19(12) 900-906. 2. villegas mv, esparza g, reyes j. should ceftriaxone-resistant enterobacterales be tested for esbls? a pro/con debate. jacantimicrobial resist. 2021;3(2). 3. piddock ljv. the crisis of no new antibiotics-what is the way forward? the lancet infectious diseases. 2012, 12(3); 249-253. 4. spellberg b, gilbert dn. the future of antibiotics and resistance: a tribute to a career of leadership by john bartlett. clin infect dis. 2014;59; 71-75. 5. rossolini gm, arena f, 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pulsrikarn c, et al.. high prevalence of ceftriaxone resistance among invasive salmonella enterica serotype choleraesuis isolates in thailand: the emergence and increase of ctx-m-55 in ciprofloxacin-resistant s. choleraesuis isolates. int j med microbiol. 2018;308(4),447-453. 45. li wc, huang fy, liu cp, et al. ceftriaxone resistance of nontyphoidal salmonella enterica iolates in northern taiwan attributable to production of ctx-m-14 and cmy-2 β-lactamases. j clin microbiol. 2005;43(7),3237-3243. 46. müderris t, ürkmez fy, küçüker sa, et al. siprofloksasine dirençli salmonella serotip kentucky bakteriyemisi: olgu sunumu ve literatürün gözden geçirilmesi. mikrobiyol bul. 2016;50(4),598-605. 47. zalewska-piątek b, piątek r. bacteriophages as potential tools for use in antimicrobial therapy and vaccine development. pharmaceuticals. 2021,14(4);331. 48. keusch gt, bennish ml. shigellosis: recent progress, persisting problems and research issues. pediatr infect dis j. 1989;8(10),713719. 49. fontaine o. antibiotics in the management of shigellosis in children: what role for the quinolones? rev infect dis. 1989;11. 50. sack rb, rahman m, yunus m, et al. antimicrobial resistance in organisms causing diarrheal disease. clin infect dis. 1997;24(1 suppl.). 51. bhattacharya sk, sarkar k, balakrish nair g, et al. multidrugmandumpala a caution for judicial antibiotic use vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 16 resistant shigella dysenteriae type 1 in south asia. lancet infectious diseases. 2003,3(12);775. 52. ahamed j, gangopadhyay j, kundu m, et al. mechanisms of quinolone resistance in clinical isolates of shigella dysenteriae, antimicrobial agents and chemotherapy. 1999;43(4). 53. sur d, niyogi sk, sur s, et al. multidrug-resistant shigella dysenteriae type 1: forerunners of a new epidemic strain in eastern india. emerging infectious diseases. 2003;9(3),404-405. 54. talukder ka, khajanchi bk, islam ma, et al. genetic relatedness of 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[drug resistance and molecular epidemiology of shigella isolated from children with diarrhea]. zhonghua er ke za zhi. 2012;50(10). 57. bonkoungou ijo, haukka k, österblad m, et al. bacterial and viral etiology of childhood diarrhea in ouagadougou, burkina faso. bmc pediatr. 2013;13(1),36. 58. yang h, chen g, zhu y, et al. surveillance of antimicrobial susceptibility patterns among shigella species isolated in china during the 7-year period of 2005-2011. ann lab med. 2013;33(2),111-115. 59. pechere jc. why are carbapenems active against enterobacter cloacae resistant to third generation cephalosporins? in: scandinavian journal of infectious diseases, supplement. 1991. 60. fouhy f, guinane cm, hussey s, et al. high-throughput sequencing reveals the incomplete, short-term recovery of infant gut microbiota following parenteral antibiotic treatment with ampicillin and gentamicin. antimicrob agents chemother. 2012;56(11),5811-5820. 61. liu s, fang r, zhang y, et al. characterization of resistance mechanisms of enterobacter cloacae complex co-resistant to carbapenem and colistin. bmc microbiol. 2021;21(1),208. 62. kang ci, kim sh, wan bp, et al. bloodstream infections caused by enterobacter species: predictors of 30-day mortality rate and impact of broad-spectrum cephalosporin resistance on outcome. clin infect dis. 2004;39(6),812-820. how to cite this article: janice jacson mandumpala. ceftriaxone resistance – a caution for judicial antibiotic use. indian j pharm drug studies. 2023: 2(1); 11-16. funding: none conflict of interest: none stated evanjelene & velu jatropha curcasl vol 1 | issue 1 | apr jun 2022 indian j of pharm drug studies | 20 original article preliminary phytochemical, proximate and nutrient analysis indifferent leaf extracts of jatropha curcasl vasthi kennedy evanjelene1, ganesh velu1 from, 1proprietor, general manager, alpha omega research foundation, salem correspondence to: vasthi kennedy evanjelene, proprietor, alpha omega research foundation, 16, anbu nagar, gorimedu, salem, india – 636 008. email alphaomegabiovision@gmail.com abstract the current study looks at the phytochemical analysis, proximate composition, and nutrient content of different jatropha curcas l. leaf extracts in-vitro. preliminary phytochemical studies were also carried out to identify the classes of compounds found in different j. curcas leaf extracts. proximate analysis (moisture, ash, total protein, fats, fibre, carbohydrate, and energy content) was done using the association of official analytical chemists' methods. atomic absorption spectrometry was used to examine macronutrients (ca, mg, na, k) and micronutrients (fe, cu, pb, zn, ni, cr, cd).the results of the study revealed that j. curcas has a higher proximate and nutrient value, with a strong association between the two. j. curcas has also been discovered to be useful in medicine and food. in conclusion, the thesis demonstrated the biological potency of the plant and provided scientific support for its use in indian medicine. key word: phytochemical analysis, proximate analysis, micro and macro nutrients, j. curcas edicinal plants play an important role in providing primary health care to rural people, and they are used by around 80% of the world's marginal populations [1]. plants have been the foundation of many traditional medicine systems around the world since the beginning of time and have continued to provide mankind with new remedies.because of the unprecedented accessibility of complex chemical compounds, a wide range of medicinal plants, their distilled constituents, and natural products from these plants provide limitless prospects for new drug discovery [2] and have also been shown to have beneficial therapeutic potential. plants have medicinal value in the form of chemical compounds that have a beneficial physiological effect on the human body [3], and since plants synthesize such a wide variety of chemical compounds, they have a lot of potential for drug discovery and growth [4].synthetic products abound on the market, with high premiums, adverse side effects, and environmental consequences [5] medicinal plants and drugs extracted from them, on the other hand, are less expensive and have fewer side effects, making them common with the public [6]. while nonnutritive, most photochemical are considered to have diseasepreventive properties. as a result, they provide protection against pathogens [7], and samples are a source of sugars, minerals, organic acids, dietary fibres, and phenolics, which have a wide range of actions, including antioxidants, antimutagenic, cardio preventive, anti-bacterial, and antiviral activities [8].alkaloids, saponins, sugars, glycosides, flavonoids, gums, steroids, tannins, phenolic compounds, volatile oils, and other phytochemicals are synthesized from a variety of medicinal plants and are used to treat a variety of ailments [9]. the act of revealing below a certain limit results in a consistent manner in a physiologically essential function on the life system [10]. plant cell metabolic products are active elements in medicinal plants, and a trace element plays an important role in metabolism [11]. herbs provide us with nutritional and trace elements, as well as chemicals with medicinal value; each one plays an important role if a deficiency occurs, as it can contribute to irregular body growth [12]. some essential elements have been discovered in the aerial parts of various medicinal plants that are used in the treatment of various diseases. some doctors and locals used medicinal plants in high concentrations in various ways [13]. the excess of trace elements in soil, water, animals, and plants has been directly proportional to the pathological state of the human body. diets that are consistently high in a trace element may have an impact on the function of certain organs in the body. five medicinal plant species were examined to determine their elemental value for this purpose. protein and carbohydrates are essential nutrients for life. protein is essential for nutrient analysis [14]. carbohydrates and proteins are essential nutrients for plants. nutrients are essential for the body's physiological functions. these nutrients play an important role in satisfying human needs for m mailto:alphaomegabiovision@gmail.com evanjelene & velu jatropha curcasl vol 1 | issue 1 | apr jun 2022 indian j of pharm drug studies | 21 energy and life processes.moisture, ash, crude fibre, protein, and carbohydrate were measured in different species by [15]. the nutrient and proximate study of selected plant species' aerial parts is critical in determining their nutritional value. since a variety of medicinal plant species are also used as food in addition to their medicinal benefits, assessing their nutritional value may aid in recognising the importance of these plants’ species [16]. jatropha species are members of the euphorbiaceae family and are used in traditional folklore medicine in africa, asia, and latin america to treat a variety of ailments. physic nut, purging nut, or pig nut are both names for j. curcas. previous research has found that j. curcas leaf extract has substantial wound healing activity [17], antidiabetic activity [18], immunomodulatory effect [19] and antiparasitic/disinfectant activity.the plant's bark extract has been shown to have wound-healing properties[17] the root isolated fraction has been shown to have anti-diarrhoeal properties [20], curcin from seeds has been shown to have anti-tumour activity [21], and the latex contains alkaloids with anti-cancerous properties. the nutritional value of j. curcas medicinal plants species was assessed for this reason. materials and methods collection and extraction of plant material: the leaves of j. curcas were collected and preserved in the alpha omega research centre (aorc073). the plant materials were washed and dried in the shade using running tap water. the leaves have been crushed and ground into a coarse powder. these coarse powders (25 g) were extracted sequentially using soxhlet instruments in 250ml of each solvent (hexane, diethyl ether, ethanol, ethyl acetate, acetone, methanol, and aqueous). the extracts were processed before being taken up for further investigation. the dissolved solvents for these extracts are dmso (dimethyl sufloxide). table 1: preliminary phytochemical analysis of j. curcas class of compounds test performed extracts hexane diethyl ether ethanol ethyl acetate acetone methanol aqueous alkaloids mayer’s wagner’s + + ++ ++ ++ ++ flavonoids h2so4 lead acetate + + + + + + ++ ++ ++ ++ steroids liebermann burchard + + + + terpenoids salkowski + + + anthroquinone borntragers + ++ ++ phenols ferric chloride lead acetate + + + ++ ++ saponin foam + + + tannin braemer’s + + + + carbohydrates fehling + + + + + oils and resins filter paper + + + + note: “+” = present, “++” = more active, “-” = absent phytochemical screening: all extracts of j. curcas were subjected to preliminary phytochemical analysis using standard methods defined by brain and turner [22] evans [23]. proximate and chemical analysis: each collected plant sample was dried in the shade and finely ground to raw flour using an electric grinding machine (national model mx 491n). the researchers were then carried out using the association of official analytical chemists (aoac) standard techniques from [24].the moisture content of the sample was measured by drying it in the oven at 105°c until it reached a constant weight. the total organic nitrogen value of the sample was determined using micro-apparatus kjeldahl's to determine the crude protein value [25]. the crude lipids were extracted in petroleum ether at 40-60°c using the soxhlet apparatus, followed by rot-evaporation of the solvent to dryness.dry result of lipid estimation was ignited for fibre content estimation, and the ash contents were calculated and taken as equal to fibre contents [26]. the difference method was used to quantify the carbohydrate content of each sample as stated below: carbohydrate (%) = 100(moisture (%) + protein percentage (%) + lipid (%) + ash contents (%)). whereas, the energy value of each sample was determined by using the following formula: k calories/100 gm = 9 (crude fats (%)) + 4 (carbohydrates (%) + proteins (%)) elemental analysis:the plant was burned to ash, then the ash was dissolved in hcl to make it into a solution. the macro and micronutrients were thenmeasured using a perkin elmer single beam atomic absorption spectrometer. statistical analysis:each plant sample was subjected to proximate and elemental analysis three times, with the mean, standard deviation, and standard error determined for each parameter. statistical package for social sciences was used to conduct inter-element correlation (spss v.14) evanjelene & velu jatropha curcasl vol 1 | issue 1 | apr jun 2022 indian j of pharm drug studies | 22 results and discussion secondary metabolites found in medicinal plants, such as alkaloids, flavonoids, steroids, and their corresponding active metabolites, have a high medicinal value and are commonly used in the pharmaceutical and drug industries. (hexane, diethyl ether, ethanol, ethyl acetate, acetone, methanol, and aqueous) extracts of j. curcas demonstrate higher yield percentages. when compared to other extracts, methanol extracts (81.36 percent) and aqueous extracts (80.23 percent) have higher yield percentages. this suggests that the essence of the solvent accounts for not only the yield of the extracts, but also their biological activities. phytochemical screening the phytochemical analysis of various extracts of j. curcasis shown in table 1. the existence of alkaloids, flavonoids, steroids, terpenoids, anthraquinone, phenols, tannins, and carbohydrates were reported in the j. curcas methanol and aqueous extracts, according to the qualitative findings presented in table 1. other extracts with mild activity include hexane, diethyl ether, ethanol, ethyl acetate, and acetone. when methanol and aqueous extracts were compared to other extracts, the researchers discovered that methanol and aqueous extracts had more constituents. plant extracts were subjected to phytochemical examination, which indicated the existence of constituents with medicinal and physiological properties [27]. phytochemicals such as phenols, tannins, flavonoids, saponins, glycosides, hormones, terpenoids, and alkaloids were discovered in the plant extracts. total alkaloids, total flavonoids, total phenols, and total tannins were among the phytoconstituents investigated quantitatively. acetone extracts have a total alkaloids content of 8.24 ± 0.03 mg/g, methanol extracts have a total alkaloids content of 10.22 ± 0.02 mg/g, and aqueous extracts have a total alkaloids content of 9.55 ± 0.04 mg/g.ethanol extracts have a total flavonoid content of 4.23 ± 0.03 mg/g, ethyl acetate extracts have a total flavonoid content of 4.03 ± 0.03 mg/g, acetone extracts have a total flavonoid content of 4.38 ± 0.01 mg/g, methanol extracts have a total flavonoid content of 4.75 ± 0.03 mg/g, and aqueous extracts have a total flavonoid total phenols are 7.34 ± 0.04 mg/g in ethanol extract, 7.57 ± 0.01 mg/g in acetone extract, 7.95 ± 0.03 mg/g in methanol extract, and 7.87 0.01 mg/g in aqueous extract. total tannins are 2.56 ± 0.03 mg/g in ethanol extract, 2.84 ± 0.04 mg/g in acetone extract, 3.24 ± 0.03 mg/g in methanol extract, and 2.96 ± 0.02 mg/g in aqueous extract (figure1). one of the largest and most common classes of plant metabolites is phenolic compounds [28].they have biological properties such as anti-apoptosis, anti-aging, anti-carcinogen, anti-inflammation, anti-atherosclerosis, cardiovascular defense, and endothelial function enhancement, as well as angiogenesis and cell proliferation inhibition [29]. medicinal plants high in phenolic compounds have been studied for their antioxidant properties in several studies. plants produce natural antioxidants in the form of phenolic compounds like flavonoid, phenolic acids, and tocopherols [30]. tannins bind to proline-rich proteins, preventing them from being synthesised. flavonoids are hydroxylated phenolic compounds that plants produce in response to microbial infection and have been shown to have antimicrobial properties in-vitro against a wide range of microor-ganisms. their ability to complex with extracellular and soluble proteins, as well as the bacterial cell wall, is most likely the reason for their activity. they are also strong antioxidants with anticancer properties [31]. figure 1: quantitative phytochemical analysis of j. curcas nutritional analysis natural plant harvests have long contributed to the isolation of modern medicine and continue to plays an important role in the discovery of newer medications. humans consume a wide range of plant derivatives as food, drugs, and dietary supplements [32].plants offering rudimentary healthcare to the biosphere's outcasts. plants contained basic nutrients such as fats, protein, carbohydrates, and phytochemical inhabitants, which are essential medicinally and are responsible for the growth and change of living beings. plants are used to cure diseases locally, both raw and in liquid mixtures, without regard for their nutritional quality, which is essential for the proper physiological functioning of the human body [33]. nutritional and proximate evaluation plays an important role in the activities that plants perform in-vivo or in-vitro, whether they are used as vegetables or medicines [34].the chemical makeup of j. curcas offers useful knowledge regarding its medicinal and nutritional properties. the moisture, ash, protein, fats, fibre, carbohydrates, and energy values of a variety of medicinal plants are shown in table below (figure 2). the moisture content of ethanol extract (9.47%), ethyl acetate extracts (9.14%), acetone extract (10.66%), methanol extract (12.45%), and aqueous extract (12.45%) (13.74 %). evanjelene & velu jatropha curcasl vol 1 | issue 1 | apr jun 2022 indian j of pharm drug studies | 23 figure 2: nutritional value of j. curcas with the aid of a furnace, the percentage of ash was calculated at temperatures up to 600 degrees celsius. acetone extract (12.46 % ash) had the largest proportion of ash, followed by ethanol extract (10.13 % ash), ethyl acetate extract (9.23 % ash), methanol extract (11.22 % ash), and aqueous extract (11.22 percent ash) (10.22 %). methanol extract has a high protein content (16.24%) and a low protein content in other extracts, whereas acetone extract has a highfat content (7.23 %). the aqueous extract has a high fibre content (10.24%), while the methanolextract has a high carbohydrate content (88.43%) and a high energy content (490.36 kcal/100g) (figure 3).the samples were found to be a good source of carbohydrates and to some extent of protein. figure 3: energy content of j. curcas plants' nutritional value is determined by quanity of proteins, fibre, fats and oils, minerals, vitamins, and water they contain, all of which are essential forhuman and animal growth and development [35,36] carbohydrates, fats, and protein are the three most important nutrients for survival. the quantity and quality of proteins in seeds are critical factors in plant selection for nutritional value, systematic classification, and plant improvement programmers [37] table 2: correlation matrix of proximate parameters parameters correlation coefficient p value moisture vs ash 0.25 0.68 moisture vs protein 0.93 0.21 moisture vs fat 0.50 0.39 moisture vs fiber 0.98 0.003* moisture vscarbohydrate 0.64 0.24 moisture vs energy 0.93 0.02* ash vs protein 0.44 0.45 ash vs fat 0.72 0.06 ash vs fiber 0.35 0.56 ash vs carbohydrates 0.34 0.57 ash vs energy 0.15 0.80 protein vs fat 0.50 0.39 protein vs fiber 0.94 0.01* protein vs carbohydrates 0.84 0.06 protein vs energy 0.94 0.01* fat vs fiber 0.57 0.31 fat vs carbohydrates 0.13 0.83 fat vs energy 0.26 0.67 fiber vs carbohydrates 0.65 0.23 fiber vs energy 0.90 0.03* carbohydrate vs energy 0.81 0.09 note: * p< 0.05 (significant) elemental analysis k, mg, ca, na, fe, mn, zn, p, s, and al were included in all samples of medicinal plants, indicating that they are responsible for curing a variety of diseases as well as providing animal fodder. the elemental contents of the analysed plant j. curcas in various extracts such as acetone, methanol, and aqueous extracts show a wide range of diversity. plant species vary in the composition of elements and their balanced concentration [23]. the macronutrients and micronutrients included in table 2 were macronutrients and micronutrients, respectively.ca, mg, k, na, fe, cu, zn, cr, cd, pb, and ni are some of the nutrients. methanol extracts contain a lot of nutrients (table 2). table 3: concentration of macro and micro-nutrients of j. curcas in ppm (1 ppm = 1mg) nutrients acetone methanol aqueous ca 3620 5631 4892 mg 28243 30125 29426 k 3105 3486 3251 na 253 524 489 fe 64.3 98.2 86.6 cu 6.5 7.4 7.1 zn 25.3 31.4 28.2 cr 0.5 1.8 1.2 cd 0.3 1.3 0.9 pb 0.1 1.1 0.5 ni 1.7 2.8 2.1 evanjelene & velu jatropha curcasl vol 1 | issue 1 | apr jun 2022 indian j of pharm drug studies | 24 plants naturally produce primary and secondary metabolites that plays an important role in controlling some of the most important functions for plant growth and development. green leafy plants, for example, contain essential minerals such as ca, fe, zn, mg, cu, mn, and nutrients such as carbohydrates, proteins, fats, and crude fibre when eaten as fruit. plants also contain a large number of vitamins and hormone precursors [1] the role of medicinal plants in the treatment of various diseases can be seen in their widespread use from antiquity to the present day.the physicochemical studies are mainly concerned with the identification of adulterants as well as the efficacy and purity of the drug. maintaining ionic equilibrium necessitates the use of sodium and potassium. this relationship between sodium and potassium in foods helps to prevent hypertension and atherosclerosis[38]. calcium is a necessary mineral for strong bones and teeth, as well as providing structural rigidity to the body and aiding in blood clotting and cellular permeability. manganese is necessary for the structure of enzymes and is also essential for the formation of hemoglobin [39] phosphorus, an enzymatic component required for proper immune function, energy metabolism, and body acid-base balance, is in short supply. table 4: descriptive statistics of nutritional analysis parameters mean content std. deviation moisture 11.09 1.96 ash 10.65 1.23 protein 15.51 0.63 fat 6.60 0.46 fibre 9.78 0.36 carbohydrates 71.02 10.21 energy value 419.00 62.59 copper is a mineral that is used in many enzymes as a structural component. ceruloplasmin, a critical protein involved in iron oxidation, is made up of it. magnesium is essential for bone formation, energy metabolism, and enzymatic activity catalysis. as compared to other minerals, chromium was found to be moderately poor. it plays a unique role in a variety of important functions, including the development of muscle mass and the maintenance of blood glucose levels .zinc serves as a stimulator in the pancreas, causing beta cells to release insulin and allowing natural glucose tolerance to be maintained. it is also essential for tissue growth and repair. iron is needed for the production of haemoglobin, the transport of oxygen, and the body's immunity [37]. minerals such as mn, zn, mg, cu, fe, na, k, ca, and traces of chromium, all of which are present in prescribed quantities, can be helpful to diabetic patients [40]. furthermore, due to the plant's low lipid content, it may be used as a dietary supplement for obese people. furthermore, the total element concentration becomes a significant parameter for assessing plant stress, nutritive value, and toxicity, as well as a trademark on species-related data [34]. a better understanding of the importance of minerals and valuable biomolecules in plants paves the way for medicinal plants to be used as both food and medicine.minerals are also used by plants as structural components in carbohydrate and protein metabolism [35] conclusion to summarize, medicinal plants are rich in macro and micro secondary metabolites, as well as the potential elements needed by living organisms. the current study concludes that medicinal plants can plays an important role in the maintenance of a healthy life and normal body functioning by providing energy-rich nutrients for growth and development in medicinal plants' secret form. the plant j. curcas shows best activity in methanol and aqueous extract when compared to other extracts. in quantitative analysis methanol shows more quantity when compared to ethanol, ethyl acetate, acetone and aqueous extracts. nutritional parameters were carried out for moisture, ash, protein, fat, fiber, carbohydrates and energy in that methanol and aqueous extracts shows similar activity. the medicinal plants have the ability to provide vital nutrients to humans and domestic animals, according to the findings. nutrients of sufficient value are needed to live a healthy life and achieve maximum productivity, as in the case of animals producing milk. popular plant species, which provide a good quantity of these basic micro and macro elements, are included in their diet for this reason. it's worth noting that many of these plants are used in high concentrations for medicinal purposes by local physicians and ordinary citizens. it is therefore recommended that further research be done on proximate and elemental analyses of widely used plants by local people in order to assess their nutritional value and toxicity under a wider range of environmental conditions. references 1. uzoekwe nm, mohammed jj. phytochemical, proximate and mineral contents of leaves and back of ficus capensis. j appl sci environ manage.2015; 19(4):633-7. 2. vinotha sanmugarajah, ira thrabrew and sri ranjanisivabalan, comparative phyto–physicochemical studies on selected medicinal plants, 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some wild edible plants from meghalaya state, india. asian j. appl. sci. 2011;4(3):238 – 246. 40. indrayan ak, sharma s, durgapal d, et al. determination of nutritive value and analysis of mineral elements for medicinally valued plants from uttaranchal. curr sci.2005;89:1252-55. how to cite this article: vasthi kennedy evanjelene, ganesh velu. preliminary phytochemical, proximate and nutrient analysis indifferent leaf extracts of jatropha curcasl. indian j pharm drug studies. 2022; 1(1): 20-25. funding: none conflict of interest: none stated http://www.techno-preneur.net/timeis/technology.2002 maryam and ahmad safety comparison of hydroxychloroquine and belimumab vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 5 review article comprehensive analysis and critical review of randomized clinical trials on safety profiling of hydroxychloroquine and belimumab zahra maryam1, ausaf ahmad2 from, 1m s scholar, department of pharmaceutical chemistry, faculty of pharmacy, anadolou university, turkey, 2ms scholar, school of life and medical sciences. university of hertfordshire, uk. correspondence to: zahra maryam, m s scholar, department of pharmaceutical chemistry, faculty of pharmacy, anadolu university, eskişehir 26470, turkey. email: zahra.maryam489@gmail.com abstract systemic lupus erythematosus (sle), an autoimmune disorder is characterized by multiorgan damage, flares, and heterogeneity in its clinical symptoms. the treatment strategy for this notorious disease focuses on long-term antisle treatment to improve the quality of life, prevent flares, and suppress the immune system. thus, the chronic use of sle medicines makes it inevitable to study the safety and tolerability of drugs in large populations. in this comprehensive analytical study, hydroxychloroquine (hcq), which is known for its safe use in sle, and belimumab, which is comparatively a novel monoclonal antibody were critically analyzed for their adverse events. we performed the absolute risk, relative risk ratio, and odds ratio analysis after critically scru tinizing the clinical trials for significant adverse effects. hcq showed major effects on the eye and heart. however, based on the results, hcq was found to be safer than belimumab, as a few serious complications were found associated with belimumab. our results were found to be as per the previous metaanalysis. this comprehensive review is aimed at analyzing the safety profiles of two widely used drugs for sle ie hcq and belimumab. we have conducted this review, critical analysis was done to critically a nalyze the clinical trials, and to gather the information of interest from these trials. both anti-sle drugs; hcq and belimumab were found to be safe for use against sle. but comparatively, hcq showed a safer profile than belimumab which can be attributed to hcq’s longterm history of use since 1955. the observation emphasized the researchers for further clinical trials with a more standardized approach, specifically for belimumab to determine its safety and tolerability in the post-marketing stage. key words: systemic lupus erythematosus, belimumab, hydroxychloroquine, safety, risk analysis ystemic lupus erythematosus is a systemic disease, involving a multiorgan system and is chronic. it results in severe damage to the organ system and its dysfunction. sle is the result of a multistep cascade of immune system abnormalities starting with the inability of the cells to clear apoptotic bodies, which results in the activation of both arms of the immune system (adaptive and innate immunity), formation of immune complexes, and tissues’ inflammation that results in an autoimmune process [1]. apoptosis has a crucial role in the development of lupus. increased non-cleared apoptotic debris is linked to inflammation and the generation of autoantibodies [2]. autoantigens are released by apoptotic cells [3]. these can produce noticeable levels of type 1 interferon by activating tlr7 and tlr9 in dendritic cells [4, 5]. the aetiology of sle includes aberrant clearance of immunological complexes (ics) and apoptotic cells and low thresholds of b and t lymphocyte activation that result in loss of self-tolerance and autoantibody synthesis. interaction of genetic predisposition, immunological, and hormonal variables, and environmental triggers is necessary for the clinical commencement of sle. sle shows itself in a range of clinical manifestations. the symptoms can range from hair loss (that can be thought of as mineral deficiency) and light sensitivity to lethal conditions like inflammation of the myocardium, brain, and nephrons. thus, symptoms of sle are heterogeneous and may differ in different individuals or among the same individual for different periods [1]. the complex nature of sle makes it more difficult to treat because of its unpredictable symptoms [6]. sle can affect both genders, but it affects women more than men. the ratio of sle in males to females is 1 man against 13 women making it a feminine disease [7]. the disease is common in african lineage and african americans inhabiting european countries and the united states. interestingly and controversially, the disease is rare in africa itself [8]. according to the centre for disease control and prevention, there are around 322,000 probable or confirmed cases of sle, with the prevalence being higher in african americans, american indians, and alaska natives [9, 10]. for sle s mailto:zahra.maryam489@gmail.com maryam and ahmad safety comparison of hydroxychloroquine and belimumab vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 6 “standard of care” therapy consists of immunomodulators, antimalarials, corticosteroids, immunosuppressive, and cytotoxic agents. the purpose of this treatment includes 1) minimizing the activity level of the immune activation by using immunosuppressants and by avoiding the triggers 2) protecting the organs from damage 3) minimizing the risk of comorbid conditions associated with lupus 4) reduction of pain and lethargy. the treatment is necessary for the early stages of sle to avoid known triggers and to maximize the effects of immunomodulators. this critical review aims to analyse the safety profiles of the two drugs used for the treatment of sle i-e hcq and belimumab. sle is a challenging disease with current therapy aimed to restore the imbalance of the dysregulated immune system. b cells are important in the development of sle's pathophysiology. therefore, attempts to interfere with disease activity by targeting b lymphocytes for selective depletion using monoclonal antibodies is a promising strategy to increase therapeutic effectiveness. thus, recent therapies focus on the development of monoclonal antibodies. belimumab is an immunoglobulin monoclonal antibody that was approved by food and drug administration in 2011 for treating adults with sle [11]. it is a fully human antibody that inhibits soluble b lymphocyte stimulator (blys), which in turn inhibits the production of a crucial cytokine that is essential for b cell survival, proliferation, and differentiation [12 – 14]. therapy with belimumab is a helpful treatment option for patients with active sle despite conventional therapy due to the adaptability of the route of administration and the ease of the once-weekly regimen. the belimumab is metabolized by proteolytic enzymes and the proteolytic enzymes are not only restricted to hepatic tissue. moreover, the level of hepatic enzymes e.g., aspartate aminotransferase (ast) and alanine aminotransferase (alt) had no discernible effect on the pharmacokinetics of belimumab in trials. thus, no dose adjustment of belimumab is necessary for patients with hepatic impairment. the previous studies reported belimumab to cause bronchitis, diarrhoea, viral upper respiratory tract infections (urti), multifocal leukoencephalopathy, fatal hypersensitivity, and infusion reactions [15, 16]. hcq is an alkalinizing substance. it is a lysosomotropic medication having high cell permeability that is accumulated in lysosomes where it raises the ph [17]. figure 1 – chemical structure of hcq hcq was originally used as an antimalarial drug, but also found beneficial for treating autoimmune conditions like rheumatoid arthritis and sle. it has been demonstrated to slow the onset of disease, maintain remission, lessen the likelihood of complications, and decrease the frequency of disease flare-ups [18,19]. hcq has a broad spectrum of activity and safety profiles; thus, it can be given to the majority of sle patients and can be continued throughout pregnancy. hcq-associated toxicity is uncommon, minor, and typically reversible and it can reduce lupus activity in pregnant women without endangering the unborn child [20]. the adverse drug reactions (adr) requiring clinical aid include ocular adrs and effects on the heart [21]. the present review is aimed at analyzing the safety profiles of hcq and belimumab by the critical review as hcq was recently used in the global pandemic of covid-19 and recent data is available on hcq safety profiling. the review will focus on statistically analyzing the adverse drug effects of the two drugs: hcq and belimumab and theoretically choosing the best drug choice for sle. this can be done by signal detection from scientific databases; clinicaltrial.gov, pubmed, and other research articles. methodology this critical review is aimed at analyzing the pharmacovigilance studies and comparing the adverse effects of the two drugs used for treating sle. database selection: we searched authentic electronic databases pubmed, google scholar, ncbi (national center for biotechnology information), and sciencedirect to collect data. these scientific databases help find citations for sources, which can broaden the scope of the articles retrieved. for unpublished data extraction and collection of data directly from clinical trials, the website clinicaltrials.gov was used. for the clinical trials search, the keywords hcq, belimumab, and sle were employed. all clinical trials that focussed on the safety and efficacy of hcq and belimumab were considered acceptable for the study. because the number of clinical trials was small, thus, the area of research was broadened to the published articles as well. the trials were then scrutinized for inclusion and exclusion criteria. inclusion and exclusion criteria: a pico (patient, intervention, comparison, and outcomes) approach was applied to include the studies in this comprehensive review. all the participants in the clinical trials were included in this study. patients of both sexes (males and females) of age more than 18 were enrolled. the outcomes considered for this critical analysis were serious adverse effects, all-cause mortality, serious adverse events, and non-serious adverse events. the adverse effects of the drugs after treating the patients with hcq and belimumab were compared with the adverse effects observed in the placebo maryam and ahmad safety comparison of hydroxychloroquine and belimumab vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 7 group. the data relating to adverse events were considered for extraction. the data was extracted in the form of the number of persons having adverse events. figure 2 – flow diagram of search strategy statistical analysis: all the statistical analyses were performed using microsoft excel software. we extracted the number of persons from trials and then the percentage of persons having adverse events was determined. the formula employed for the percentage determination is given in equation 1. the absolute risk, relative risk, risk difference, and odds ratio were calculated. the absolute risk was calculated by dividing the number of participants having adverse effects (ae) by the total number of participants for both drugs (hcq and belimumab) separately. the formula for absolute risk is given in equation 2. the risk difference was calculated by equation 3. the odds ratio shows the likelihood that an outcome will occur given a specific exposure in comparison to the chances of the event occurring without that exposure [22]. the odds ratio ranges from 0 to 1 and is always positive. if the or is 1, it means that there is no difference in the outcome of interest between the treatment and the control. when the or is greater than 1, the treatment is more likely than the control to cause the desired outcome [23]. the odd ratio was calculated using the following formula mentioned in equation 4. relative risk compares the likelihood of a health event (in the present study, the adverse event) among two groups. relative risk was determined using equation 5. results we identified 90 records for belimumab and 509 studies on hcq. the clinical trials not focussing on safety and efficacy were excluded. the articles on safety and efficacy were then screened for inclusion in studies. among the selected trials, the trials having no results posted were also excluded and finally, four articles were included in this critical analysis. to compare the data on adverse effects between belimumab and hcq, the risk difference and relative risk between the two were calculated. table 1 shows the risk difference and relative risk between the two drugs. table 1 – risk difference and relative risk calculation ar bel ar hcq rd p1/p2 (rr) gastrointestinal disorders 31.88 15.66 16.22 2.04 general disorders 10.43 6.56 3.87 1.59 nervous system disorder 20.80 3.27 17.53 6.36 psychiatric disorders 9.61 2.18 7.43 4.41 skin disorders 3.60 4.61 -1.01 0.78 cardiac disorders 1.27 4.36 -3.09 0.29 eye disorders 0.33 0.49 -0.16 0.67 immune disorders 0.35 0.37 -0.02 0.95 *ar – absolute risk, rd – risk difference, rr – relative risk, hcq – hydroxychloroquine, bel – belimumab the results showed the major effects of belimumab on the gastrointestinal tract, nervous system, and heart. in some of the participants, belimumab was found associated with depression, suicidal thoughts, and changes in sleep patterns (insomnia). the positive value of risk difference in table 1 indicates that maryam and ahmad safety comparison of hydroxychloroquine and belimumab vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 8 belimumab has an increased risk of causing gastrointestinal disorders, general disorders, nervous systems, and psychiatric disorders compared to hcq in patients. while the negative value of risk difference for belimumab indicates the increased risk of skin disorder, eye disorders, immune disorders, and cardiac disorders by hcq. the results show the relative ratio of belimumab /hcq of 2 for git problems which indicates that the persons taking belimumab are twice as likely to suffer from gastrointestinal issues as the people taking hcq. similarly, the relative risk ratio for the general disorder is 2 (2x risk to suffer from general problems if the person is taking belimumab), for nervous disorders, the rr ratio came out to be 6.4, and for psychiatric disorders, it is 4.41. skin disorders show a relative risk ratio of 0.78 indicating that belimumab is less likely to cause skin problems as compared to hcq, in other words, hcq is 1.3 times more likely to cause skin problems than belimumab. likewise, the rr ratio for the cardiac disorder is 0.29 showing 3.5 times increased risk of having cardiac abnormality for people taking hcq with dyspnoea and chest pain as major effects. for eye disorder rr ratio is 0.66 showing a 1.5x risk for patients taking hcq, and the ratio for immune disorders is surprisingly 0.92 which somewhat shows the equal risk of getting an immune disorder with the use of hcq and belimumab. moreover, 2 cases of mortality were observed in patients taking belimumab, while no allcause mortality was observed when the patients were administered a placebo or hcq. then we compared the adverse effects of the drugs belimumab and hcq with respect to the placebo and calculated the odds ratio. table 2 shows the odds-ratio calculation for belimumab. table 2 odd ratio calculation for belimumab bel pl or gastrointestinal disorders 31.88 33.00 0.95 general disorders 10.43 10.45 1.00 nervous system disorder 20.80 23.84 0.84 psychiatric disorders 9.61 9.68 0.99 skin disorders 3.59 3.62 0.99 cardiac disorders 1.27 1.91 0.66 eye disorders 0.33 0.00 0.33 immune disorders 0.34 0.17 2.00 *bel – belimumab, pl – placebo, or – odds ratio the odds ratio for belimumab showed the value of 1 for a gastrointestinal disorder, general disorder, psychiatric disorders, and skin problems. this predicts an equal probability of belimumab and placebo showing these disorders. in the case of immune disorders, the odds ratio is 2, indicating two times increased risk of belimumab to induce immune abnormality compared to placebo. 0.33 odds-ratio for eye disorder, and 0.66 for cardiac disorder show rather a protective role of belimumab for these organs compared to placebo. for hcq, in one of the trials we selected, the adverse effects of hcq have been compared with ascorbic acid so, for this trial, the odds ratio was compared with ascorbic acid. the results of this are illustrated in table 3. in the present trial, ascorbic acid acted as a placebo equivalent. while the second trial of hcq as chemoprevention for covid-19 for high-risk healthcare workers had no placebo as the trial was during an ongoing pandemic situation. the participants enrolled in the study were on-duty high-risk healthcare workers receiving hcq and the placebo group might increase the chance of covid19. in the trial, hcq did not show any risk to the nervous system and psychiatric related disorders. however, oddsratio for eye disorder was found to be 98.96, indicating 99 times increased risk of having an eye disorder for a person taking hcq as compared to ascorbic acid. the odds ratios are 2.5, 277.5, and 3 for gastrointestinal disorders, skin disorders, and immune disorders respectively showing a multifold increase in the risk of these disorders if a patient is taking hcq compared to ascorbic acid. table 3 – odd ratio calculation for hcq hcq aa or gastrointestinal disorders 9.58 4.03 2.52 general disorders 4.42 7.58 0.56 nervous system disorder 0.01 0.01 1.00 psychiatric disorders 0.01 0.01 1.00 skin disorders 2.7 0.01 277.46 cardiac disorders 0.01 0.01 1.00 eye disorders 0.98 0.00 98.96 immune disorders 0.74 0.24 3.10 *hcq – hydroxychloroquine, aa – ascorbic acid, or-odds ratio discussion the recent advancements in the molecular mechanism of sle and sle’s association with the immune arm led to the development of novel monoclonal antibodies against sle. sle is an autoimmune disorder thus main clinical strategy behind antibody design is to target immune cells to inhibit the overactivation of the immune system and the overproduction of cytokines [24]. this critical analysis was focused on comparing the two sle drugs belimumab and hcq. there is no doubt that b cells are crucial to the development of sle and belimumab has a recognized function in sle treatment [25]. additionally, clinical trials have demonstrated the effectiveness of belimumab by demonstrating a beneficial improvement in disease activity. based on its efficacy, food and drug administration approved it in 2011 [26]. whereas, hcq has a long history of use in sle since it was approved in 1955 for sle treatment [27]. given its broad spectrum of activity, maryam and ahmad safety comparison of hydroxychloroquine and belimumab vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 9 recently, hcq was frequently used as a protective treatment for covid-19. the use of this drug globally provided a golden chance for clinicians and researchers to look widely at the adverse events associated with hcq. thus, we performed a critical review of the recent clinical trials conducted on diverse populations for hcq during the covid-19 pandemic. we compared the adverse events of hcq with a relatively novel monoclonal antibody belimumab by critically analyzing the clinical trials, and utilizing that data to calculate absolute risk, relative risk, and odds ratio for drugs. the trials of belimumab reported a range of adverse events from gastrointestinal discomforts to serious psychiatric disorders. however, the results of the trials observed under the scope of the study were not in harmonization. the results observed in one of the trials reported a 41.7% risk of gastrointestinal, and 28.61% risk of nervous system disorders. these results observed were almost double the aes observed in the other trial of the same drug. similarly, the risk for general disorders was almost 16 times greater than in another trial for belimumab. however, the trials presented an equal percentage risk for cardiac and eye disorders. the significant heterogeneity in results reported by the trials can be presented as the difference in the number of the study group and the ethnicity of the population selected for the trial as one trial employed americans and the other trial was conducted on americans, europeans, and asians. trials for hcq showed a safe profile in comparison to belimumab. however, we also observed discrepancies in the results of the hcq trials. the absolute risk for gastrointestinal, general, and skin disorders was found to be 21.74%, 8.7%, and 6.52% for one trial against the minute values of 9.58%, 4.42%, and 2.7% for another. interestingly, the trial also failed to provide any adverse effect on cardiac disorders, the more pronounced effects for which hcq is famous. again, this can be attributed to uneven sample size, differences in entry requirements, and the environment in which the trials are being conducted. belimumab caused 2 mortalities while hcq showed no mortality. the odds ratio calculated for hcq and belimumab trials presented an abrupt trend with a range of values from 0.3 to 277. the odds ratio for belimumab/placebo showed a value of 0.3 while 98.96 for hcq/placebo for eye disorders. 0.3 indicates three times more effect of placebo on eyes and rather a protective role of belimumab for eyes. on the other hand, 98.96 for hcq/placebo showed 99 times increased tendency of hcq to cause eye ailments. the most common cardiac effects reported in our study by hcq include chest pain, dyspnoea, qt prolongation, and qt syndrome. these results for hcq are in concordance with previous studies as the previous studies conducted for hcq showed the increased probability of the drug for cardiomyopathy, eye disorders, and skin hyperpigmentation [28]. in addition to corneal deposits, dysfunction in the ciliary body, posterior subcapsular lens opacity, uneven macular pigmentation, and hydroxychloroquine can result in side effects including a ring of macular pigment dropout. additionally, the hcq manufacturers claim that hcq is contraindicated in already existing maculopathy conditions [29]. in some studies, for example, hcq-induced retinopathy was found to be more common in those who used the drug for longer than five years, with a prevalence of 7.5%, and between 20 and 50% in people who used it for longer than twenty years [28]. the results of our clinical trials’ analysis revealed that belimumab has the highest drug-related adverse effects that range from gastrointestinal disorders, and general disorders, to more serious events in nervous systems, and psychiatric disorders. belimumab treatment presented one case of a successful suicidal attempt and 2 cases of mortality. the results of our study resonate with the belimumab assessment of safety study (base) trial and medicines and healthcare products regulatory agency uk drug update who suggested the increase in psychiatric disorders and mortality rate. after the critical analysis of the results, we concluded that except for the effects of hcq on heart and retinopathy, hcq was found to be safer than belimumab. previous studies also confirm the safety of hcq [17]. the safe safety profile of hcq can be due to the tolerability of the drug attributed to the well-established and long-term use of hcq in the general population while belimumab in turn is a novel agent. however, a critical point here is that the trials conducted for belimumab also showed adverse effects with placebo. thus, the odds-ratio calculated for belimumab showed neglected adverse effects with belimumab except for the immune system disorder that is again the known ae of belimumab. however, contrary to hcq, belimumab was found associated with suicidal attempts and depression cases which makes hcq a safer drug than belimumab. conclusion the critical analysis of the adverse effects of the drugs, belimumab and hcq showed the safe profile of both drugs. like the previous studies, hcq showed major adverse effects on the heart and eyes, while belimumab was found associated with immune system disorders. though both drugs were found to be safe for anti-sle treatment because of their long-term use, hcq was found to be safer, having less serious adverse effects than belimumab. thus, based on the results and observed heterogeneity in the results, it can be concluded that for result homogeneity and to further evaluate the safety and efficacy of belimumab, high-quality randomized controlled clinical trials on larger population sizes are needed. references 1. fava a, petri m. sle. diagnosis and clinical management. physiol behav. 2020;176(3). maryam and ahmad safety comparison of hydroxychloroquine and belimumab vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 10 2. thompson ra, haeney m, reid kbm, et al, white rhr, cameron ah. a genetic defect of the c1q subcomponent of complement associated with childhood (immune complex) nephritis. n engl j med. 1980; 303(1). 3. casciola-rosen l, rosen a, petri m, et al. surface blebs on apoptotic cells are sites of enhanced procoagulant activity: implications for coagulation events and antigenic spread in systemic lupus erythematosus. proc natl acad sci u s a. 1996; 93(4). 4. guiducci c, gong m, xu zg, et al. tlr recognition of self-nucleic acids hampers glucocorticoid activity in lupus. nature. 2010; 465(7300). 5. lehmann p, homey b. clinic and pathophysiology of photosensitivity in lupus erythematosus. autoimmunity reviews. 2009; 8. 6. xiong w, lahita rg. pragmatic approaches to therapy for systemic lupus erythematosus. nature rev rheumatol. 2014; 10. 7. danchenko n, satia ja, anthony ms. epidemiology of systemic lupus erythematosus: a comparison of worldwide disease burden. lupus. 2006; 15. 8. pons-estel gj, alarcón gs, scofield l, et al. understanding the epidemiology and progression of systemic lupus erythematosus. sem arthritis rheumatism. 2010; 39. 9. ferucci ed, johnston jm, gaddy jr, et al. prevalence and incidence of systemic lupus erythematosus in a populationbased registry of american indian and alaska native people, 2007-2009. arthritis rheumatol. 2014; 66(9). 10. helmick cg, felson dt, lawrence rc, et al. estimates of the prevalence of arthritis and other rheumatic conditions in the united states. part i. arthritis rheum. 2008; 58(1). 11. parodis i, axelsson m, gunnarsson i. belimumab for systemic lupus erythematosus: a practice-based view. lupus. 2013; 22(4). 12. ss k, ka k, d e belimumab in systemic lupus erythematosus: an update for clinicians. tacd. 2012; 3(1). 13. petri m, stohl w, chatham w, et al. association of plasma b lymphocyte stimulator levels and disease activity in systemic lupus erythematosus. arthritis rheum. 2008;58(8). 14. vana dr. belimumab: therapeutic mechanism and current status of clinical trials. biomed res. 2018;29(16). 15. fda. highlights of prescribing information these highlights do not include all the information needed to use benlysta safely and effectively. see full prescribing information for benlysta. benlysta (belimumab) for injection, for intravenous use benlysta (belimumab) injection, for subcutaneous use. 2022;15(12); available from: www.fda.gov/medwatch 16. benlysta | ema. 2022; 15(12). available from: https://www. ema.europa.eu/en/medicines/human/epar/benlysta 17. ponticelli c, moroni g. hydroxychloroquine in systemic lupus erythematosus (sle). expert opinion on drug safety. 2017; 16. 18. james ja, kim-howard xr, bruner bf, et al. hydroxychloroquine sulfate treatment is associated with later onset of systemic lupus erythematosus. lupus. 2007;16(6). 19. kaiser r, cleveland cm, criswell la. risk and protective factors for thrombosis in systemic lupus erythematosus: results from a large, multi-ethnic cohort. ann rheum dis. 2009;68(2). 20. ruiz-irastorza g, ramos-casals m, brito-zeron p, et al. clinical efficacy and side effects of antimalarials in systemic lupus erythematosus: a systematic review. 69, annals of the rheumatic diseases. 2010. 21. akarsu s. hydroxychloroquine: from pharmacological profile to neglected adverse reactions. j basic clin heal sci j basic clin heal sci. 2020; 4: 205–11. 22. szumilas m. explaining odds ratios. j can acad child adolesc psychiatry. 2010; 19(3): 227. 23. wang h, chow sc, li g. on sample size calculation based on odds ratio in clinical trials. j biopharm stat. 2002; 12(4): 471– 83. 24. davis ls, hutcheson j, mohan c. the role of cytokines in the pathogenesis and treatment of systemic lupus erythematosus. j interf cytokine res. 2011; 31(10): 781. 25. wise lm, stohl w. belimumab and rituximab in systemic lupus erythematosus: a tale of two b cell-targeting agents. front med. 2020; 7. 26. dubey ak, handu ss, dubey s, et al. belimumab: first targeted biological treatment for systemic lupus erythematosus. j pharmacol pharmacother. 2011; 2(4): 317. 27. dima a, jurcut c, chasset f, et al. hydroxychloroquine in systemic lupus erythematosus: overview of current knowledge. ther adv musculoskelet dis. 2022; 14. 28. frederick w, marmor mf. rates and predictors of hydroxychloroquine retinal toxicity in patients with rheumatoid arthritis and systemic lupus erythematosus. arthritis care res (hoboken). 2010; 62(6): 775–84. 29. yusuf ih, sharma s, luqmani r, et al. hydroxychloroquine retinopathy. eye (lond). 2017; 31(6): 828–45. how to cite this article: maryam z, ahmad a. comprehensive analysis and critical review of randomized clinical trials on safety profiling of hcq and belimumab. indian j pharm drug studies. 2023; 2(1) 5-10. funding: none conflict of interest: none stated http://www.fda.gov/medwatch padmakar neurofibromatosis associated with infective mass tumor vol 1 | issue 2 | jul – sep 2022 indian j pharm drug studies | 69 case report a case report on neurofibromatosis associated with infective mass tumor somanaboina padmakar from, research scholar, department of pharmacology, lovely professional university. correspondence to: research scholar, department of pharmacology, lovely professional university, phagwara –144001, punjab, india. email: spadmakar717@gmail.com abstract a rare genetic disorder with many non-cancerous (benign) tumors of the skin (neurofibrom as) and nerves is known as neurofibromatosis. this disorder includes type 1-neurofibromatosis 1 (nf1), type 2-neurofibromatosis 2 (nf2), and schwannomatosis as complex genetic abnormalities that may affect many different organ systems in the body. when alterations in the gene nf1, located in the 17th chromosome occur, it causes nf1 type disorder. nf1 gene controls the synthesis of a specific protein called neurofibromin that functions as a tumor suppressor. the predisposition to form tumors, often on the nerves, spine, brain, and skin, is a common characteristic of these disorders. but the types and severity of symptoms vary widely among individuals. here, we present a case of nf-1 with a severe infective tumor lesion on the left hand. a 56-year-old male was admitted to the dermatology department with a chief complaint of a mass lesion on the hand that was painful, severely itchily, and progressively increased in size. multiple neurofibromas were found on his body that are soft, fleshy tumors arising from a peripheral nerve sheath near the surface of the skin, or on the skin, and he had already been diagnosed in his childhood as neurofibromatosis. keywords: neurofibromatosis type 1, neurofibromas, schwannomatosis eurofibromatosis type 1 (nf1) is the most frequent single-gene disorder affecting the human nervous system, an estimated prevalence of 2 to 3 cases per 10,000 people. he inherited this disorder autosomally dominant, with an equal sex incidence. the neurocutaneous abnormalities like axillary freckling, cafe-au-lait spots, iris hamartomas (lisch nodules), and cutaneous neurofibromas were diagnosed in individuals with nf-1 type disorder [1]. neurofibroma consists of a varied mixture of schwann's cells, perineurial-like cells, and fibroblasts, which is a benign tumor deriving from peripheral nerve sheathing [2]. nf1 causes mutations in the nf1 gene, the neurofibromin-encoded 60exon tumor suppressor locus of 17q11.2. many malignancies of nf1 were observed for heterozygotic loss (loh) in chromosome 17 (including locus nf1) [3]. nf1 can be evaluated by a clinical examination and by a family history of the patient. the diagnosis of nf1 is based on the diagnostic criteria of the national health institutes (nih). these criteria typically appear in the following predictable order: café-au-lait macules, axillary freckling, lisch nodules, and neurofibromas [4]. the distribution of the affected nerve is evident for cutaneous neurofibroma in the skin palpating, tenderness to touch, and tingling. malignant changes occur rarely, and if removal intended, should consult expert assistance either from a soft tissue tumor/peripheral nerve surgeon, and the removal results occasionally in a neurological deficit [5]. the present study describes a case of an infective mass tumor over the left hand in an nf1 patient. case report a 56-year-old male had been admitted to the dermatology department, government general hospital, kadapa, with a chief complaint of mild fever, swelling lesion on the hand which is progressively increasing in size, painful and severe itchy for three months. the patient had a history of multiple raised nonitchy neurofibromas over the hands, followed by similar lesions on the face, legs, and body folds at two years of age. there was no family history of nf. there were no neurological abnormalities, skeletal malformations noted. multiple cafe-aulait spots disseminated on his body, several soft, fleshy tumors arose from a peripheral nerve sheath near the skin surface diagnosed in clinical examination. he was diagnosed in his childhood with neurofibromatosis. on cutaneous clinical examination, it revealed a severe infective mass tumor (fig. 1) over the left forearm, the biopsy was not performed. associated pruritus that was generalized moderate to severe in intensity with no diurnal variation and relieved by treatment with oral anti-histamines and topical calamine lotion. on physical examination, the patient have a high blood pressure of 170/90 mmhg that was newly diagnosed. on laboratory investigation, c-reactive protein values found to be increased 18mg/l (0.310mg/l). the patient was admitted to the n mailto:spadmakar717@gmail.com padmakar neurofibromatosis associated with infective mass tumor vol 1 | issue 2 | jul – sep 2022 indian j pharm drug studies | 70 hospital for seven days and treated with medications shown in table 1. after seven days the patient was discharged with the following medications tab. amoxycillin and potassium clavulanate 625mg, oint. fusidic acid 2%, tab. chlorpheniramine maleate 2mg od and asked to review for surgery after 1 week fig. 1 neurofibromas associated with infective mass tumor over left forearm table 1: drugs prescribed to the patient drug name dose route freque ncy dura tion inj. amoxycillin and potassium clavulanate 1.2g iv bid 6 d inj. diclofenac sodium 75mg iv bid 4 d inj. pantoprazole 40mg iv bid 6 d oint. fusidic acid 2% w/w topical tid 7 d tab. chlorpheniramine ma 4 mg oral od 6 d note: iv intravenous bid-twice a day; tid-three times a day; od-once a day discussion nf1 is an inherited neurocutaneous condition and characterized by a multi-system tumor with a risk of malignant transformation across the skin in the central nervous system. neurofibromas are a distinctive feature of the nf1, which are benign tissue-based tumors that occur at the periphery of the nerves of schwann cells. they comprise fibroblasts, macrophages, and mast cells in addition to neoplastic schwann cells [6]. 52.5 % of individuals with pruritus had localized in one or more cutaneous neurofibromas. pruritus pathogenesis is not well known in nf1. it hypothesized that mast cells and components produced from their degranulation were considered the main source of pruritus, cnf microenvironment was known to have mast cells, which may contribute to tumor initiation, progression, and angiogenesis [7]. type 1 neurofibromatosis is described as a benign tumor disorder nevertheless, in 2% or 4.2% of patients older than 21 years malignant transformation was reported [8]. there is currently no effective treatment method for people with type 1 neurofibromatosis. one approach is surgical excision, although it is usually impossible to remove all lesions due to excess of nf-1 and the progression of the disease. surgery is required when suspected of malignancy. operation indicated if tumors pressure on other organs [9]. in preoperative, perioperative, and postoperative conditions, radiation treatment is used. adjuvant radiation treatment produced a statistically significant decrease in rates of recurrence of local diseases [10]. conclusion benign (harmless) tumors of neurofibromatosis do not need treatment. but subcutaneous tissues are often affected by superficial tumors and may evoke significant infection or cellulitis. the dermatologist, neurologist, and general surgeon should follow these patients routinely to ensure no mass lesions were developing. note: written informed consent was taken from the patient who participated in this study. references 1. north k. neurofibromatosis type 1. am j med genet. 2000; 97(2):119-127. 2. nallanchakrava s, mallela mk, jeenepalli vs, et al. a rare case report of neurofibromatosis type 1 in a 12-year-old child: a 15month follow-up. j oral maxillofacial pathol. 2020; 24:106-109. 3. rasmussen sa, overman j, thomson sa, et al. chromosome 17 loss‐of‐heterozygosity studies in benign and malignant tumors in neurofibromatosis type 1. genes chromosomes cancer. 2000; 28(4):425-431. 4. bergqvist c, servy a, valeyrie-allanore l, et al. neurofibromatosis 1 french national guidelines based on an extensive literature review since 1966. orphanet j rare dis. 2020; 15(1):37. 5. ferner re, huson sm, thomas n, et al. guidelines for the diagnosis and management of individuals with neurofibromatosis 1. j med genet. 2007; 44(2):81-88. 6. karaconji t, whist e, jamieson rv, et al. neurofibromatosis type 1: review and update on emerging therapies. asia pac jophthalmol. 2019; 8(1):62-72. 7. ortonne n, wolkenstein p, blakeley jo, et al. cutaneous neurofibromas: current clinical and pathologic issues. neurology. 2018; 91:5-13. 8. rallis e, ragiadakou d. giant plexiformneurofibroma in a patient with neurofibromatosis type i. dermatol online j. 2009; 15(5):7. 9. fijałkowska m, antoszewski b. clinical picture and treatment of cutaneous lesions in patients with neurofibromatosis type 1. postepy dermatol alergol. 2020; 37(5):781-784. 10. ferner re, gutmann dh. international consensus statement on malignant peripheral nerve sheath tumors in neurofibromatosis. cancer res. 2002; 62(5):1573-1577. how to cite this article: padmakar s. a case report on neurofibromatosis associated with infective mass tumor. indian j pharm drug studies. 2022; 1(2) 69-70. funding: none conflict of interest: none stated thaker et al. formulation and evaluation of levocetrizine dihydrochloride vol 2 / issue 1 / jan mar 2023 indian j pharm drug studies | 23 original article formulation and evaluation of rapidly dissolving film containing antihistaminic drugs (levocetirizine dihydrochloride) vamakshi thaker1, jyoti verma1, suchita waghmare1, navinraj mourya1 from, 1assistant professor, school of pharmacy, rai university, ahmedabad, gujarat, india. correspondence to: vamakshi thaker, assistant professor, school of pharmacy, rai university, ahmedabad, gujarat india. email: vidhivamakshi@gmail.com abstract the oral dispersible tablets of levocetirizine dihydrochloride were prepared by direct compression method. various combinations of sodium starch glycolate (ssg), croscarmellose sodium (ccs) and crospovidone (cp) were used as superdisintegrants for formulating the oral dispersible. it was seen that increasing the concentration of the superdisintegrants decreased the wetting time and disintegration time of the formulations. the combination of ssg & cp was more effective in decreasing the disintegration time as compared to the combination of ssg & ccs and cp & ccs. the in-vitro dissolution study showed that the formulation containing ssg (6%) and cp (4.5%) was more effective in enhancing the rate of drug release from the oral dispersible tablets. the comparison of effect of individual superdisintegrant on wetting time, disintegration time and dissolution showed that ssg was more suitable for the formulation of oral dispersible tablets of levocetirizine dihydrochloride as compared to other superdisintegrants used in the current study. hence, from the present study, it can be concluded that the superdisintegrants ssg and cp in appropriate concentration can be used to develop oral dispersible tablets of levocetirizine dihydrochloride by dire ct compression method. key words: orodispersible tablet, superdisintegrants, levocetirizine dihydrochloride, surfactants, starch, co-surfactants istamine is an organic nitrogenous compound involved in local immune responses, as well as regulating physiological function in the gut and acting as a neurotransmitter for the brain, spinal cord, and uterus. it consists of an imidazole ring attached to an ethylamine chain; under physiological conditions, the amino group of the side-chain is protonated [1]. properties: histamine base, obtained as a mineral oil mull, melts at 83–84 °c. hydrochloride and phosphorus salts form white hygroscopic crystals and are easily dissolved in water or ethanol, but not in ether. in aqueous solution, the imidazole ring of histamine exists in two tautomeric forms, identified by which of the two nitrogen atoms is protonated. the nitrogen farther away from the side chain is the 'tele' nitrogen and is denoted by a lowercase tau (τ) sign and the nitrogen closer to the side chain is the 'pros' nitrogen and is denoted by the pi (π) sign. the tele tautomer, nτ-h-histamine, is preferred in solution as compared to the pro-tautomer, nπ-h-histamine (fig. 1) [2]. histamine has two basic centres, namely the aliphatic amino group and whichever nitrogen atom of the imidazole ring does not already have a proton. under physiological conditions, the aliphatic amino group (having pka around 9.4) will be protonated, whereas the second nitrogen of the imidazole ring (pka ≈ 5.8) will not be protonated. thus, histamine is normally protonated to a single charged cation. histamine is a monoamine neurotransmitter [3]. synthesis and metabolism: histamine is derived from the decarboxylation of the amino acid histidine, a reaction catalyzed by the enzyme l-histidine decarboxylase. it is a hydrophilic vasoactive amine (figure 2). once formed, histamine is either stored or rapidly inactivated by its primary degradative enzymes, histamine-n-methyltransferase or diamine oxidase. in the central nervous system, histamine released into the synapses is primarily broken down by histaminen-methyltransferase, while in other tissues both enzymes may play a role. several other enzymes, including maob and aldh2, further process the immediate metabolites of histamine for excretion or recycling [4]. fig. 1 the tele tautomer (nτ-h-histamine), on the left is more stable than the pros tautomer (nπ-hhistamine) on the right. h thaker et al. formulation and evaluation of levocetrizine dihydrochloride vol 2 / issue 1 / jan mar 2023 indian j pharm drug studies | 24 fig. 2 -conversion of histidine to histamine by histidine decarboxylase bacteria also are capable of producing histamine using histidine decarboxylase enzymes unrelated to those found in animals. a non-infectious form of foodborne disease, scombroid poisoning, is due to histamine production by bacteria in spoiled food, particularly fish. fermented foods and beverages naturally contain small quantities of histamine due to a similar conversion performed by fermenting bacteria or yeasts. sake contains histamine in the 20–40 mg/l range; wines contain it in the 2–10 mg/l range [5]. mechanism of action: in humans, histamine exerts its effects primarily by binding to g proteincoupled histamine receptors, designated h1 through h4. as of 2015, histamine is believed to activate ligandgated chloride channels in the brain and intestinal epithelium [6]. anti-histaminic drugs: antihistamines are drugs which treat allergic rhinitis and other allergies. typically, people take antihistamines as an inexpensive, generic, over-thecounter substitutes that can provide relief from nasal congestion, sneezing, or hives caused by pollen, dust mites, or animal allergy with few side effects. antihistamines are usually for short-term treatment. chronic allergies increase the risk of health problems which antihistamines might not treat, including asthma, sinusitis, and lower respiratory tract infection. consultation by a medical professional is recommended for those who intend to take antihistamines for longer-term use [7]. although people typically use the word “antihistamine” to describe drugs for treating allergies, doctors and scientists use the term to describe a class of drugs that opposes the activity of histamine receptors in the body. in this sense of the word, antihistamines are subclassified according to the histamine receptor that they act upon. the two largest classes of antihistamines are h1antihistamines and h2antihistamines. classification of anti-histaminic drugs [8] h1-antihistamines: meclizine, clemastine, hydroxyzine, brompheniramine, dimetindene, doxylamine, etc. h2antihistamines: loratadine, cetirizine, levocetirizine, azelastine, fexofenadine, etc. h3-antihistamines: ranitidine, cimetidine, famotidine, etc. medical uses: histamine produces increased vascular permeability, causing fluid to escape from capillaries into tissues, which leads to the classic symptoms of an allergic reactiona runny nose and watery eyes. histamine also promotes angiogenesis. antihistamines suppress the histamine-induced wheal response (swelling) and flare response (vasodilation) by blocking the binding of histamine to its receptors or reducing histamine receptor activity on nerves, vascular smooth muscle, glandular cells, endothelium, and mast cells. itching, sneezing, and inflammatory responses are suppressed by antihistamines that act on h1-receptors [9]. hence, for an antihistamine drug like levocetirizine dihydrochloride, a quick-disintegrating dosage form is suitable, since the disintegration and dissolution of the dosage form occur rapidly, thus providing a rapid onset of action. it was thought worth formulating oro-dispersible a formulations of the drug, so that the patient can ingest the dosage form anywhere and at any time, without the aid of water, which would be helpful, especially in cases of unavailability of water, motion sickness, sudden episodes of allergic attacks, and deglutition problems. mouth-dissolving tablets of levocetirizine dihydrochloride were prepared by a direct compression method using different concentrations of spraydried mannitol (perlitol sd 200), menthol, and camphor [10]. pharmacology: levocetirizine is an antihistamine. it acts as an inverse agonist that decreases activity at histamine h1 receptors. this in turn prevents the release of other allergy chemicals and increases the blood supply to the area, and provides relief from the typical symptoms of hay fever [11]. clinical data • trade name :levazyl • other name :levocetirizine dihydrochloride • route of administration : by mouth pharmacokinetics data • bioavailability :high • protein binding :90% • metabolism :liver 14% • elimination half-life :6 to 10 hour • excretion :kidney and fecal identification • iupac:-2-(2-{4[(r)-(4chlorophenyl) (phenyl)methyl] piperazine-1yl}ethoxy) • formula :c21h26cln2o3 • molar mass :388.89 g.mol-1 plan of work formulation 1. materials: levocetirizine dihydrochloride was used as the active ingredient. croscarmellose sodium, sodium starch glycolate and crosspovidone were used as the thaker et al. formulation and evaluation of levocetrizine dihydrochloride vol 2 / issue 1 / jan mar 2023 indian j pharm drug studies | 25 superdisintegrants. the other ingredients used were mannitol, aerosol, magnesium sterate, aspartame, mint flavor and microcrystalline cellulose ph 102. the active drug was obtained as a gift sample from sr drug laboratories pvt. ltd, kathamandu. crospovidone was received as a gift sample from lomus pharmaceuticals pvt. ltd, kathmandu. the other excipients and chemicals used in experimental works were obtained from nova genetica pharmaceuticals pvt. ltd, dhading, nepal. all reagents used were of analytical grade [11]. 2. methods: preparation of orodispersible tablets of levocetirizine dihydrochloride the composition of different formulations of levocetirizine dihydrochloride orodispersible tablets. levoretirizine dihydrochloride and all other excipients were weighed separately and passed through sieve number 60. the active drug was mixed with mcc ph102. then the remaining excipients except the lubricants were blended with the active drugmcc blend. the lubricants were then blended to the mix to form the final blend. the final blend was then compressed on a 10 stations rotary compression machine using an 8 mm punch. 3. in-vitro wetting time studies: circular tissue papers of 10cm diameter were placed in a petri-dish containing 10 ml of buffer solution simulating saliva, ph 6.8, and amaranth. a tablet was placed on the paper and the time taken to complete it wetting it was noted. three tablets from each formulation were randomly selected and the average wetting time was recorded [12]. 4. invitro disintegration studies: the disintegration time for all formulations was carried out using tablet disintegration test apparatus. six tablets were placed individually in each tube of disintegration test apparatus and discs were placed. water was used at the media for the study. the water was maintained at a temperature of 37°±0.5°c and time taken for the entire tablet to disintegrate completely was noted. 5. invitro dissolution studies : in vitro dissolution studies for all the fabricated tablets was carried out by using usp type ii apparatus (usp xxiii dissolution test apparatus) at 50 rpm in 900 ml of phosphate buffer ph 6.8, maintained at 37±0.5ºc. 5 ml aliquot was withdrawn at the specified time intervals, filtered through whatmann filter paper and assayed spectrophotometrically at 231nm using dissolution medium as blank. an equal volume of fresh medium, which was prewarmed at 370c was replaced into the dissolution medium after each sampling to maintain the constant volume throughout the test. dissolution studies were performed in triplicate. then the cumulative percentage of drug release was calculated using the following formula [12]. results & evaluation 1. evaluation of films thickness: all the batches were evaluated for thickness by using a calibrated digital micrometer. three readings from all the batches were taken and the mean thickness was evaluated at 10. 2. folding endurance: the folding endurance was measured manually on the prepared films. a strip of film was cut and repeatedly folded in the same place till it broke. the number of times the film could be folded in the same place without breaking gave the value of folding endurance to 11. 3. drug content: the drug content of all nine batches was determined by the uv-spectrophotometric method. for this, a 1.4x1.2 cm2 strip from each batch was cut and dissolved in 50ml of methanol. then 5 ml of this solution was diluted to 50 ml. so a 4ppm solution was made, filtered and absorbance was recorded at 231 nm in comparison with the 4ppm standard solution. drug content was calculated by comparison method 10. 4. uniformity of drug content: for determining the uniformity of drug content in the film at least 10 strips (1.4x1.2 cm2) ware taken and assayed. same procedure was repeated for all the 6 batches 10. 5. in vitro disintegration time: the disintegration test was performed on the usp disintegration time testing apparatus. simulated salivary fluid (ph 6.8) was used as a medium. the films were placed in the tubes of the container and the disks were placed over it. 6. bursting strength: it is also known as tensile strength. it is the maximum tolerance power of an oral fast dissolving film. how many grams of weight when applied through a pointed subject in the film will be the bursting strength of that film? 7. moisture permeation: this test is to check weather a film is moisture permeable or not. for this in a vial desiccated silica was kept, and on the mouth of vial oral film was bounded and placed in 90% rh for 2 days. 8. in-vitro dissolution studies: dissolution study was carried out in usp basket type apparatus using the stimulated salivary fluid (ph 6.8) as a dissolution medium at 50 rotations per minute. 10 ml aliquots were withdrawn at one-minute time intervals and same amount of fresh dissolution medium was added. the aliquots were assayed for drug content at 231nm wavelength using uvspectrophotometer. the cumulative percentage drug release was calculated. discussion the comparison of the effect of individual superdisintegrants on the wetting time, disintegration time and dissolution showed that ssg was more suitable for the formulation of oral dispersible tablets of levocetirizine dihydrochloride as compared to other superdisintegrants used in the current study. hence, from the present study, it can be concluded that the thaker et al. formulation and evaluation of levocetrizine dihydrochloride vol 2 / issue 1 / jan mar 2023 indian j pharm drug studies | 26 superdisintegrants ssg and cp in appropriate concentration can be used to develop oral dispersible tablets of levocetirizine dihydrochloride by direct compression method. conclusion the oral dispersible tablets of levocetirizine dihydrochloride were prepared by direct compression method. various combinations of sodium starch glycolate, croscarmellose sodium and crospovidone were used as the superdisintegrants for formulating the oral dispersible. it was seen that increasing the concentration of the superdisintegrants decreased the wetting time and disintegration time of the formulations. the combination of ssg & cp was more effective in decreasing the disintegration time as compared to the combination of ssg & ccs and cp & ccs. the in-vitro dissolution study showed that the formulation containing ssg (6%) and cp (4.5%) was more effective in enhancing the rate of drug release from the oral dispersible tablets. references 1. m. aruna, samreen, shaik harun rasheed. formulation and evaluation of fast disintegrating tablets of metoprolol succinate using various superdisintegrants. int j res pharm sci technol. 2019, 1: 10.33974/ijrpst.v1i2.150 2. joshi p, patel h, patel v, et al. formulation development and evaluation of mouth dissolving film of domperidone. in: journal of pharmacy and bioallied sciences. 2012. 10.4103/09757406.94159 3. yurtdaş kirimlioğlu g, öztürk aa. levocetirizine dihydrochloride-loaded chitosan nanoparticles: formulation and in vitro evaluation. turkish j pharm sci. 2020, 17: 10.4274/tjps.galenos.2018.34392 4. khan s, kataria p, nakhat p, et al. taste masking of ondansetron hydrochloride by polymer carrier system and formulation of rapid-disintegrating tablets. aaps pharm sci tech. 2007, 8: 10.1208/pt0802046 5. labib gs. novel levocetirizine hcl tablets with enhanced palatability: synergistic effect of combining taste modifiers and effervescence technique. drug des devel ther. 2015, 9: 10.2147/dddt.s92245 6. deluca pp: aaps pharmscitech volume 8, issue 4 editorial. aaps pharmscitech. 2007, 8: 10.1208/pt0804080 7. warren f. handbook of pharmaceutical excipients. am j heal pharm. 1987, 44: 10.1093/ajhp/44.8.1946 8. dey p, maiti s. orodispersible tablets: a new trend in drug delivery. j nat sci biol med. 2010, 1: 10.4103/0976-9668.71663 9. jacob s, nair ab, boddu shs, et al. an updated overview of the emerging role of patch and film-based buccal delivery systems. pharmaceutics. 2021, 13: 10.3390/pharmaceutics13081206 10. gupta mm, gupta n, chauhan bs, et al. fast disintegrating combination tablet of taste masked levocetrizine dihydrochloride and montelukast sodium: formulation design, development, and characterization. j pharm. 2014, 2014: 10.1155/2014/568320 11. chaturvedi a, srivastava p, yadav s, et al. fast dissolving films: a review. curr drug deliv. 2011, 8: 10.2174/156720111795768022 12. dinge a, nagarsenker m. formulation and evaluation of fast dissolving films for delivery of triclosan to the oral cavity. aaps pharmscitech. 2008, 9: 12249-008-9047-7. how to cite this article: vamakshi thaker, jyoti verma, suchita waghmare, navinraj mourya. formulation and evaluation of rapidly dissolving film containing antihistaminic drugs (levocetirizine dihydrochloride). indian j pharm drug studies 2023; 2(1) 23-26. funding: none conflict of interest: none stated ahmad et al rp-hplc method validation of empagliflozin vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 76 research article development and validation of novel stability indicating rp hplc method for quantitative estimation of empagliflozin in tablets ausuf ahmad1, zahra maryam2 from, 1department of clinical and pharmaceutical sciences, school of life and medical sciences, university of hertfordshire, hatfield, uk, 2department of pharmaceutical chemistry, faculty of pharmacy, anadolu university, eskişehir 26470, turkey. correspondence to: zahra maryam, department of pharmaceutical chemistry, faculty of pharmacy, anadolu university, eskişehir 26470, turkey. tel: +90-5411666124, e-mail: zahra.maryam489@gmail.com abstract the current study was conducted to develop and validate a novel method for the qualitative and quantitative determination of empagliflozin in bulk granules and tablets. this method was successively applied to the analysis of the approved dosage form of empagliflozin available in the market with the brand name of jardiance tablets. the developed method was found to be precise, economic, and rapid with a run time of 20 minutes. the empagliflozin peak was eluted at 5.0 minutes. this elution was done on the c18 column (150 x 4.6mm) having a 5μm particle size using an isocratic mobile phase, a mixture of phosphate buffer ph 3.0 and methanol in the ratio of 70:30 at a flow rate of 1.0ml/min with pda detector (maximum wavelength of 224nm) and column temperature 30°c. the method was found to be linear with lod and loq, 0.068 μg and 0.35 μg, respectively. the correlation coefficient r2 calculated was 0.996. precision and accuracy of the method evaluated by interday, intraday, and recovery study was found to be within limits. the forced degradation study proved the stability of the method. objectives: this research article is aimed at developing a new method for developing and validating a novel method for the qualitative and quantitative determination of empagliflozin in bulk granules and tablets. methods: to conduct this research, all the validation parameters were employed according to international council for harmonization (ich) guidelines and all the validation parameters e.g., accuracy, specificity, linearity, lod, loq, robustness, and degradation studies were performed. results: results of the proposed technique were found to be trustworthy, accurate, suitable, and robust for everyday application. conclusion: conclusively the results of the study indicated that the current method is simple, precise, accurate, stable, and economic, and thus can be used for routine quality control testing of empagliflozin. keywords: empagliflozin, rp-hplc, method validation, qualitative determination hemically, empagliflozin is (1-chloro-4[b dglucopyranos-1-y1]-2-[4-([1]-tetrahydrofuran-3-yloxy) benzyl]-benzene [1-2]. empagliflozin controls the excess glucose level in the blood. it is an inhibitor of sodiumglucose cotransporter-2(sglt-2) and promotes the excretion of excess glucose in urine by reabsorbing it in the kidney, thus is used in the treatment of type 2 diabetes. [1,3-4]. the empirical formula of empagliflozin is c23h27clo7 and its molecular mass is 450.91 a.m.u [5-6]. empagliflozin is available in 10 mg and 25mg oral tablet dosage forms alone or in combination with other drugs. jardiance, glyxambi, synjardy, and synjardy xr are examples of empagliflozin tablets [7-10]. figure 1 – structure of empagliflozin the development of a quantitative method and its validation are fundamental and essential phases in pharmaceutical analysis to ensure that the results of the proposed technique are trustworthy, accurate, suitable, and robust for everyday application. the literature review revealed that empagliflozin can be detected both qualitatively and quantitatively by using liquid chromatography (lc), ultraviolet spectrophotometer (uv), high-performance liquid chromatography (hplc), and ultra performance liquid chromatography (uplc) techniques [11-15]. however, few reports are available for the rp-hplc measurement of empagliflozin in pharmaceutical preparations. in this article, we discussed the validation of a novel technique for precise empagliflozin quantification in pharmaceutical dosage forms and bulk pharmaceuticals. the validation is important as drug regulatory governing bodies play a vital role and have placed emphasis on drug manufacturing companies to offer comprehensive information regarding the process for the validation of analytical methods c mailto:zahra.maryam489@gmail.com ahmad et al rp-hplc method validation of empagliflozin vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 77 [15]. we performed the validation in accordance with ich [1618]. the current strategy, which is brand-new to the market, uses several mobile phase compositions to estimate empagliflozin in a quality control laboratory. in accordance with the recommendations of the international council for harmonization, the method's performance was validated. this study was conducted to develop and validate the novel, accurate, precise, and stability-indicating rp-hplc method for the analysis of empagliflozin in bulk granules and tablets. in the development stage, the wavelength detection study was conducted by scanning the sample between 200nm and 400nm. a major peak was found at 224 nm (λ max). then the method was validated for validation parameters i-e accuracy, precision (repeatability, intermediate precision), specificity, the limit of detection, the limit of quantification, linearity, range, and robustness as per ich and united states pharmacopeia (usp) guidelines using hplc [11, 19]. stress study was performed in acid, base, peroxide solutions, thermal, and photolytic stress conditions to check the stability of dosage form as well as the robustness of the method [20-21]. materials and methods all the chemicals, methanol, acetonitrile, hydrochloric acid, hydrogen peroxide, sodium hydroxide, and monobasic hydrogen phosphate were purchased from science center merk distributor islamabad. all the chemicals were used without any further purification. instrumentation: gradient hplc model lc20 (shimadzu japan) (lc20at) equipped with pda detector (spd m-20a), autosampler (sil-20aht), column oven (cto-20a) and degassing unit (dgu-20asr) was used for the study. computer-supported software-based spectrophotometer model 1800 (shimadzu japan) for wavelength detection and ft-ir model alpha (germany) with atr diamond atr module for identification of empagliflozin were employed. calibrated analytical balance metler tolendo was used for weighing purposes. ph meter was served for the adjustment of the ph of the buffer for the mobile phase. a neutronic photostability chamber was used for the photostability study, and thermal degradation was checked using a memart oven. an ultrasonic bath was used to dissolve the samples. chromatographic conditions: in the isocratic mobile phase, the mixture of phosphate buffer ph 3.0 and methanol in the ratio of 70:30 with a flow rate of 1.0 ml/minute and column temperature of 30 °c was used. the flow rate was set at 1.0 ml/minute and the column temperature was kept at 30 °c. we kept the injection volume at 20 µl. the mobile phase was also used as diluent and chromatograms were recorded at 224nm. the summary of chromatographic conditions is mentioned in table 1. table 1 – chromatographic conditions preparation of solutions preparation of mobile phase: 6.8 grams of monobasic potassium phosphate was dissolved in 900 ml deionized water in a 1000ml volumetric flask, the ph was adjusted to 3.0 with orthophosphoric acid and the solution was filtered through a 0.5-micron filter and degassed. then phosphate buffer and methanol were mixed in a ratio of 70:30 to make the isocratic mobile phase. preparation of standard solution: after weighing carefully, the empagliflozin working standard equivalent to 25mg, was dissolved in a diluent in 50 ml volumetric flask. the solution was filtered and 2ml of the filtrate was taken to 50 ml volumetric flask, mixed well, and the volume was made with diluent to 50 ml and sonicated for 5.0 minutes. preparation of sample solution: we took 20 tablets, weighed, and powdered them. then the powder equivalent to 25 mg empagliflozin was dissolved in diluent and the volume was made up to 50 ml in the volumetric flask. after filtration, 2.0 ml of this solution was transferred to another 50 ml volumetric flask, mixed well with diluent, and sonicated for 5.0 minutes. method development: we performed some preliminary tests for the development of a stable and effective method for qualitative and quantitative estimation of empagliflozin. first, the wavelength was detected using a uv-visible spectrophotometer by scanning the sample and standard solution between 200nm to 400nm. a major peak of empagliflozin was observed at 224nm (figure 2). figure 2 – uv spectrum of empagliflozin mobile phase phosphate buffer: methanol (70:30%v/v) ph adjusted to 3.0 column ods c18, 250×4.6 mm, 5μ wavelength 224 nm flow rate 1.0 ml/min injection volume 20 μl run time 10 min diluent phosphate buffer: methanol (70:30%v/v) ph adjusted to 3.0 ahmad et al rp-hplc method validation of empagliflozin vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 78 then many injections of the sample were applied on hplc using water, methanol and acetonitrile, and phosphate buffer in different compositions (to determine the optimal mobile phase combination) as mobile phase. the mobile phases tested included methanol and acetonitrile in the ratios of 60:40 and 70:30, phosphate buffer, and methanol with 65:35, 60:40, and 70:30 ratios to observe the separation and characteristics of the peak. it was observed that the best symmetry of peak with less retention time and low pressure of hplc pump was achieved using phosphate buffer and methanol in the ratio of 70:30 as mobile phase. the optimum flow rate selected after hit and trial basis was 1.0 ml per minute. system suitability: a suitability study of the system was conducted to confirm its performance. figure 3 – suitability chromatogram all critical parameters including percentage relative standard deviation (rsd), tailing factor, usp plate count, and usp resolution were verified by injecting six injections of standard solution. the results of system suitability are depicted in table 2. table 2 – system suitability figure 4 – specificity chromatogram specificity: the analytical method should have the ability to resolve all its process impurities, known and unknown degradation impurities during stress study. a placebo should not interfere with the retention times of active drugs and impurities. the specificity of the method was confirmed by injecting the blank, placebo, and standard solution. linearity: the linearity of the method was checked by injecting 5 standard solutions of concentration ranging from 25 µg/ml to 125 µg/ml. then the graph was plotted between concentration and peak areas. a straight line was obtained which confirmed the linear response of the method (fig. 5). figure 5 – linearity curve the intercept, slope, correlation coefficient of regression, and sum of squares were calculated. the linearity results are presented in table 3. precision a) repeatability: for repeatability testing, we applied six replicate injections of the same concentration of sample solution on the same day. all the parameters including percentage rsd, tailing factor and usp plate were checked and are mentioned in table 4. accuracy: the accuracy of a method describes how close the results are to a true value. the accuracy study was conducted by calculating the percentage recovery of empagliflozin. it was processed by injecting three replicates of three different concentrations of the analyte. in the end, the percentage recovery of the analyte was determined to confirm the accuracy of the system and depicted in table 6. table 3 – linearity y = 845241x r² = 0.9976 0 1000000 2000000 3000000 4000000 5000000 6000000 0 1 2 3 4 5 6 7 ahmad et al rp-hplc method validation of empagliflozin vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 79 table 4 repeatability table 5 – intermediate precision figure 6 – accuracy chromatogram limit of detection (lod) & limit of quantitation (loq): qualitative analysis of an analyte in a tablet at the lowest possible concentration is called the limit of detection. it can be calculated by the formula lod = 3.3σ/s, where σ is the standard deviation of response and s is the slope of the calibration curve. whereas the lowest possible concentration of an active ingredient in a tablet that can be detected quantitatively is called the limit of quantitation. in the recent study, the limit of quantitation was calculated in the sample with acceptable precision and accuracy limits by the formula loq=10σ/s. the results of the limit of detection of empagliflozin calculated in xr tablets were found to be 0.068μg/ml while the limit of quantitation came out to be 0.35 μg/ml. figure 7 – limit of detection figure 8 – limit of quantitation robustness: the robustness of the analytical method is the capacity to remain unchanged by a small change in conditions. high system robustness ensures a positive indication in normal use. the robustness of the method was checked by making minor changes in the composition of the mobile phase, flow rate, temperature of the column, and the ph of the buffer. it was confirmed that no significant changes occur after deliberately changing the system conditions. the robustness results are depicted in table 7. table 6 – accuracy of the system ahmad et al rp-hplc method validation of empagliflozin vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 80 table 7 – robustness of the method table 8 – stress stability study stress stability study: this study was conducted to assess the stability of the method under stressed conditions. the forced degradation parameters like acid, alkali, oxidative, photolytic, thermal, and hydrolytic analyses were carried out under the scope of this study. acid degradation study: in the acid degradation study, 1ml of 2n hcl was added to 1ml of stock solution of empagliflozin and the mixture was refluxed for 30 minutes at 60°c. the resultant solution was diluted with diluent to 5 µg/ml. then three replicate injections of 10 μl solution were injected into the chromatograph and chromatograms were observed to check the stability of the solution. the percentage degradation was checked by comparing the results with the standard. alkali degradation study: this study was conducted by taking 1ml of stock solution and 1ml of 2n naoh solution and the mixture was refluxed for 30 minutes at 60°c. then the solution was diluted to 5 µg/ml. three replicate injections of 10 μl solution were then injected into the chromatograph and the behavior of the peak area was observed. this peak area was then compared with that of the standard. dry heat degradation study: thermal degradation was carried out by placing powder of empagliflozin in a petri dish at 105°c for 1 hour. then 5 µg/ml solution of empagliflozin was made and triplicate injections were applied to the chromatograph and the degradation was observed by comparing the peak areas with the standard solution. photo stability: for photolytic degradation, a standard stock solution of empagliflozin was placed in a photostability chamber for 5 days. then after 5 days, the powder was diluted to make the concentration of about 5 µg/ml and then triplicate injections were applied to the chromatograph and the results for possible degradation were observed. the standard solution kept in normal condition was studied versus the photolytic standard solution and then the percentage degradation was calculated. the results of degradation studies were depicted in table 8. results and discussion with the advancements in technology and instrumentation, there comes the requirement to design an efficient, dependable, and selective quantification approach for active pharmaceutical components. though numerous analytical techniques have been employed to estimate drugs, researchers continue to develop alternative and more efficient hplc methods to quantify a wide range of compounds. we also employed a novel method for the pharmaceutical assay of empagliflozin tablets. we developed an accurate, simple, and stable reverse phase hplc method for both qualitative and quantitative estimation of empagliflozin using different ratios of mobile phase, different chromatographic conditions, diluents, and flow rates for analysis. in optimal conditions of isocratic mobile phase with the combination of phosphate buffer ph 3.0 and methanol (70:30), c18 column (150x4.6mm) with particle size 5μm and flow rate of 1.0 ml per minute, accurate and precise results were obtained. the results of system suitability, specificity, linearity, and robustness were all within good acceptable limits. there was no interaction of solvents like mobile phase, diluents, and excipients with the peak of empagliflozin. additionally, statistical analysis revealed negligible differences between the peak areas during precision, suitability, and repeatability studies. a fit value of 0.9996 was obtained for the regression equation y = 845241x, indicating a strong correlation. it was discovered that the minimal detection and quantification limits were 0.068 µg/ml and 0.35 µg/ml, respectively. due to the proper selection and composition of the mobile phase, the reduced detection values in our investigation were noted. lower standard deviations and coefficients of variation were found in the drug assay results, indicating good procedure accuracy. the peak purity of a sample depends on the selectivity and specificity of an analyte. for confirmation of peak purity, the drug solution was also tested against a placebo mixture (with excipients used in tablet formulations).a lack of co-elution peaks during the drug sample's retention time indicated that the current approach was suggestive of both selectivity and specificity. since there was no peak of any additive during the analytical run of empagliflozin, the devised method can be effectively used to quantify the medicine in the marketed tablets. the underlying methodology was confirmed to be reliable because no concentration change was seen when the flow rate, column temperature, or mobile phase composition ahmad et al rp-hplc method validation of empagliflozin vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 81 was changed. previous studies also confirmed the reliability of analytical methods, frequently by altering the solvent composition, flow rate, and column temperature to a limited extent [22]. based on the results, it can be concluded that the present method can be employed in pharmaceutical labs for routine quality control testing and can serve as a basis for further modifications in the method in the future. conclusion validation parameters mentioned in the recent study were checked according to ich guidelines and satisfactory results were obtained. a stress stability study confirmed that the method is stability-indicating. therefore, based on the study conducted it can be ensured that the method can be applied to the pharmaceutical lab for routine analysis of tablet dosage form. references 1. frampton je. empagliflozin. a review in type 2 diabetes. drugs [internet]. 2018; 78 (10):1037–48. 2. lamos em, younk lm, davis sn. empagliflozin, a sodium glucose co-transporter 2 inhibitor, in the treatment of type 1 diabetes. expert opin investig drugs. 2014; 23(6): 875–82. 3. empagliflozin monograph for professionals". drugs.com. ahfs. retrieved dec 2018. 4. davies mj, d’alessio da, fradkin j, et al. management of hyperglycemia in type 2 diabetes, 2018. a consensus report by the american diabetes association (ada) and the european association for the study of diabetes (easd). diabetes care. 2018; 41(12):2669–701. 5. kinduryte schorling o, clark d, zwiener i, et al. pooled 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molnupiravir and favipiravir in sars-cov-2 vol 1 | issue 3 | oct dec 2022 indian j pharm drug studies | 73 review article molnupiravir and favipiravir in the therapeutics of sars-cov-2 a review ayush singh1, 2, riya sehgal1, karamjit kaur1, manju1, jatin isher1, 3, neeru kumari1, 4 from, 1department of pharmacy, global group of institutes, 2department of production, alkem laboratories limited, baddi, solan, himachal pradesh, 3department of quality assurance, kwality pharmaceuticals ltd., 4department of quality control, kwality pharmaceuticals ltd., amritsar, punjab, india correspondence to: ayush singh, department of production, alkem laboratories limited, baddi, solan, himachal pradesh 173205 and department of pharmacy, global group of institutes, amritsar, punjab, india-143501. email: ayushsingh28659@gmail.com abstract sars-cov-2 led to several unwanted deaths all across the globe since 2020. high mortality rates are seen by this virus. as per the various theories, numerous variant and deadly strains are come into existence in the world due to covid-19 pandemic. to treat deadly strains of this virus, various anti-viral are drugs that are utilized in the therapeutics of covid-19. few antivirals are molnupiravir, favipiravir, remdesivir, alisopirivir and many more. molnupiravir was originally developed to treat influenza at emory university but also reports abandoned for mutagenicity concerns. favipiravir is a prodrug has been approved to treat the influenza rather than the seasonal influenza and this medication selectively inhibition of rdrp. this review generally covers the two potent oral antiviral molnupiravir and favipiravir investigation. key words: molnupiravir, favipiravir, covid-19, sars-cov-2, clinical data, chemical. oronavirus disease 2019 (covid-19), the illness caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has had a devastating effect on the world’s population resulting in more than 5.4 million deaths worldwide and emerging as the most significant global health crisis since the influenza pandemic of 1918. since being declared a global pandemic by the world health organization (who) on march 11, 2020, the virus continues to cause devastation, with many countries continuing to endure multiple waves of outbreaks of this viral illness [1]. covid-19 outbreak in china, led to tremendous breakdown in the healthcare sector. with emerging therapies including vaccines and anti-viral therapies, it is necessary to research on their clinical effects on treatment. most of the drugs used in clinical practice have limited clinical experience [2]. with the vast expanding knowledge of the sars-cov-2 virology, newer potential targets are being identified. we have summarized the clinical picture of two anti-virals molnuoiravir and favipiravir. molunipiravir is the first oral antiviral medicine to show considerable and convincing antiviral activity in vitro and in animal models. shreds of evidence suggest that molnupiravir reduces hospitalization and mortality among unvaccinated individuals [3]. favipiravir is also one such drug with a crucial role in treating mild to moderate covid-19. with its oral form it can be easily administered and meant for treatment in mildly ill covid-19 patients [4]. variants of sars-cov-2 since the covid-19 pandemic first began in december 2019, the severe acute respiratory syndrome coronavirus 2 (sarscov-2) virus has continuously evolved with many variants emerging across the world. these variants are categorized as the variant of interest (voi), variant of concern (voc), and variant under monitoring (vum). as of september 15, 2021, there are four sars-cov-2 lineages designated as the voc (alpha, beta, gamma, and delta variants). vocs have increased transmissibility compared to the original virus, and have the potential for increasing disease severity [5]. all the variant of sarscov-2 are enlisted in the table no. 1. molnupiravir molnupiravir is the drug that came into existence worldwide for the treatment of covid-19. molnupiravir (emory institute of drug development-2801 [eidd-2801]/mk-4482) is one of the upcoming oral drugs which is promising. this oral agent was developed by drug innovation ventures at emory university, and later acquired by ridgeback therapeutics in partnershipwith merck & co, usa. in general, antiviral drugs tested so far usually terminate the elongation of rna-chain by targeting the viral polymerases but such anti-virals have not shown a very promising role in the treatment of sars-cov-2 c singh et al. molnupiravir and favipiravir in sars-cov-2 vol 1 | issue 3 | oct dec 2022 indian j pharm drug studies | 74 infections [14]. this drug affects the rna-dependent rnapolymerase enzyme used by the coronavirus for transcription and replication of its viral rna genome [15]. figure 1: chemical structure of molnupiravir molnupiravir is an isopropyl ester prodrug of the nucleoside analogue β-d-n4–hydroxycytidine (nhc or eidd-1931). molnupiravir interferes with the replication of various viruses, including sars-cov-2. it inhibits sars-cov-2 replication in human lung tissue, blocks sars-cov-2 transmission in ferrets and reduces sars-cov-2 rna in patients [15].it is β-d-n4hydroxycytidine (eidd-1931) is an orally bioavailable ribonucleoside analogue and has broad-spectrum activity against numerous rna viruses in animal models [16-18]. molnupiravir (eidd-2801), is a prodrug of β-d-n4hydroxycytidine (eidd-1931) and is rapidly converted into eidd-1931 in the plasma by the host’s esterase. after entering host cells, eidd-1931 is intracellularly transformed into its active form, β-d-n4-hydroxycytidine-triphosphate, which inhibits viral replication through its incorporation into the viral genome. consequently, the accumulation of mutations results in the viral error catastrophe. mechanism of action previously, it was developed to treat influenza and was recognized as another candidate for antiviral drugs. understanding the mechanism of molnupiravir at the molecular level is critical to the further development of antiviral drugs. the drug is activated through metabolism in the body. once inside the cell, it becomes an rna-like component. in the first step, rna polymerase incorporates these components into the rna genome of the virus. in the next step, rna-like components are paired with viral genetic material components. viral rna contains several mutations when it multiplies to produce new viruses, preventing the reproduction of the pathogen. this viral drug causes mutations in other rna viruses and prevents them from expanding. molnupiravir, a promising drug, is in the third phase of studies. when molnupiravir enters the cell, the active molnupiravir forms nhydroxycytidine hydrate (nhc triphosphate (mtp), which can be replaced by ctp or utp by rdrp of sars-cov-2. initially, when rdrp uses positive-strand genomic rna for the synthesis sub-genomic rna and negative-strand genomic rna as a template, it regularly substitutes m for u or c. in the next step, +grna or +sgmrna can be used from rna including m as a template. then mutations are formed in positive-stranded genomic rna products due to the presence of m in negative strand genomic rna, and these products prevent the formation of healthy new viruses. at the end of this two-step mechanism, the mechanism of molnupiravir and its activated type were shownto result in rna mutations through polymerases of other viruses. according to previous studies, molnupiravir-induced lethal mutagenesis was determined by a relatively high selectivity of mtp for incorporation as a ctp analogue and the indiscriminate incorporation of either atp or gtp when mnp is centralized in the template strand [19]. favipiravir favipiravir (6-fluoro-3-hydroxypyrazine-2-carboxamine) which is a prodrug, is an anti-influenza drug that has shown broad-spectrum antiviral activity against a variety of other rna viruses. this antiviral drug metabolized intracellularly into its active ribonucleoside 5′-triphosphate form that acts as a nucleotide analogue to selectively inhibit rdrp and induce lethal mutagenesis. recently, several studies reported in vitro inhibitory activity of favipiravir against sars-cov-2 with 50% ec50. based on these results, more than 20 clinical trials on the management of covid-19 by favipiravir are ongoing. table 1: variant of sars-cov-2 variant strain description references voc alpha detected in oct 2020 and correlated with a significant increase in the rate of covid19 infection. [11] voc beta detected in 18 december 2020 in south africa. [12] voc gamma detected in tokyo on 6 january 2021 by national institutes of infectious diseases [13] voc delta detected in 6 may 2021 and it is globally dominant variant [14] voc omicron detected in 26 november 2021 in south africa. [15,16] voi lambda detected in peru in august 2020 [17] voi mu detected in colombia in january 2021. [18] singh et al. molnupiravir and favipiravir in sars-cov-2 vol 1 | issue 3 | oct dec 2022 indian j pharm drug studies | 75 figure 4(b): mechanism of action of favipiravir (favipiravir-rmp phosphorylates and produce favipiravir-rtp in active form) table 2 miscellaneous anti-viral drugs used in the treatment of covid-19 and their respective deadly strains drug structure mechanism of action remdesivir the active metabolite of this drug interferes with the action of rdrp and evades profeeding by viral exoribonucleaases causing a reduction in the synthesis of rna alisopirivir/ debio 025/ unil-025 abolish cyclophilin a or rotamase a figure 2: mechanism of action of molnupiravir fig 3: chemical structure of favipiravir figure 4 (a): mechanism of favipiravir singh et al. molnupiravir and favipiravir in sars-cov-2 vol 1 | issue 3 | oct dec 2022 indian j pharm drug studies | 76 umifenovir it suppress the membrane fusion of influenza virus and orifice impinging betwixt the virus and target host cell. galidesivir/ bcx4430/ immucillina this is under phase 1 human trail in brazil for sars-cov-2 and possess broad spectrum antiviral potency against virus belongs to rna families involving bunyaviruses, marburg virus disease, zika virus, ebola virus, arenaviruses, flaviviruses, phleboviruses, paramyxoviruses nelfinavir this medication is a competitive inhibitor and protease inhibitor with activity against hiv-1. lopinavir this drug also inhibit the hiv protease enzyme by producing an enzyme inhibitor complex, thus preventing the segmentation of the gag-pol polyproteins ritonavir generally, this medication abolishes the hiv viral proteinase enzyme that normally breaks the structural and replicative proteins that arises from major hiv genes. table 3: clinical trial of several medications that are utilized in the therapeutics of sars-cov-2 identifier number drug sponsor sponsor status enrollment nct04323527 chloroquine diphosphate fundação de medicina tropical dr. heitor vieira dourado completed 278 nct04343729 methylprednisolone fundação de medicina tropical dr. heitor vieira dourado completed 416 nct04853199 quercetin hôpital universitaire sahloul recruiting 200 nct04334148 hydroxychloroquine adrian hernandez completed 1360 nct04510493 canakinumab university hospital, basel, switzerland completed 116 nct04842747 veru-111 veru inc. recruiting 300 nct04602000 ct-p59 celltrion completed 1642 nct04978025 colloidal silver hôpital universitaire sahloul recruiting 50 nct04646044 bempegaldesleukin nektar therapeutics completed 30 nct04560231 remdesivir lahore general hospital recruiting 30 singh et al. molnupiravir and favipiravir in sars-cov-2 vol 1 | issue 3 | oct dec 2022 indian j pharm drug studies | 77 nct04477993 ruxolitinib vanderson geraldo rocha terminated 5 nct04780581 dexamethasone fundación instituto de estudios de ciencias de la salud de castilla y león recruiting 290 nct04406246 nitazoxanide materno-perinatal hospital of the state of mexico completed 150 nct04473274 pioglitazone samaritan health services completed 10 nct04668209 cx4549 university of arizona recruiting 40 nct04414618 opaganib redhill biopharma limited completed 42 nct04409509 csl312 csl behring completed 124 nct04341116 tj003234 i-mab biopharma co. ltd. recruiting 384 nct04632381 zotatifin effector therapeutics recruiting 36 nct04672564 carrimycin shenyang tonglian group co., ltd recruiting 300 mechanism of action favipiravir-rtp binds to and inhibits rdrp, which ultimately prevents viral transcription and replication. favipiravir is a purine base analogue that is converted to active favipiravir-rtp by intracellular phosphoribosylation. it is a selective and potent inhibitor of rdrp of rna viruses. favipiravir is incorporated into the nascent viral rna by error-prone viral rdrp, which leads to chain termination and viral mutagenesis [20]. the rdrp existing in various types of rna viruses enables a broader spectrum of antiviral activities of favipiravir. after rna viral incorporation, favipiravir-rtp works as a mutagen, which is capable of fleeing coronavirus repair machinery. the favipiravir-rtp adds to the pressure on cov nucleotide content, which already has a low cytosine in the sarscov-2 genome. in total, along with the increased frequency of mutation, favipiravir-rtp has a positive effect on sarscov2 by a cytopathic effect, which is induced by the virus, reduction in the number of viral rna, and infectious particles. favipiravir has a strong binding affinity to rdrp with a docking score of 6.925. hence, targets the achilles heel (rdrp complex) of sars-cov-2. miscellaneous anti-viral agents in the therapeutics of sars-cov-2 we have also elaborated the list of other drugs that are being employed in the treatment of covid-19. there are several kinds of anti-viral drugs other than molnupiravir and favipiravir for the treatment of covid-19. clinical data major drugs that are under clinical trial of various drugs under several phases for the development of new pharmaceutical medicament for the treatment of sarscov-2 from different genera (or different classes of drugs) are entitled in the table 3. conclusion and future scope the review concludes that antivirals used in the treatment of coronavirus can serve excellent in treatment. anti-viral drugs prevent viral replication through several kinds of mechanisms or physiology. this review justifies that molnupiravir (mk4482) is a prodrug used to treat mild-to-moderate covid-19. mk-4482 is the first oral, direct-acting antiviral that shows highly effective at reducing nasopharyngeal sars-cov-2 infection. favipiravir act as a prodrug and undergoes ribosylations and phosphorylation intracellularly to become active favipiravir-rtp. this drug inhibits rdrp at long last prevent viral transcription and replication. both drugs are not preferred in pregnancy because several studies and evidence show that molnupiravir causes fetal harm. during clinical treatment, these newer generation drugs serve as a potential drug for the therapeutics of covid-19. intensive investigations related to antiviral drugs that are utilized in the therapeutics of sars-cov-2 for specific pharmacological actions, and their mechanism of action, safety, and efficacy could be the future research interest to explore the drugs exhaustively. there is still an ever-increasing need for high quality evidence to investigate newer and effective therapies for covid-19. abbrevatons +sgmrna: positive-strand subgenomic mrna; atp: adenosine triphosphate; ctp: cytidine triphosphate; ec: effective concentrations; eidd: emory institute for drug development; favipiravir-rtp: favipiravir ribofuranosyl5b-triphosphate; gtp: guanosine-5’-triphosphate; hiv: human immunodeficiency virus; rdrp: rna-dependent rna polymerase; sars-cov-2: severe acute respiratory syndrome corona virus 2; utp: uridine triphosphate; voc: variant of concern; voi: variant of interest. references 1. singh ak, singh a, singh r, et al. molnupiravir in covid-19: a systematic review of literature. diabetes metab syndr [internet]. 2021; 15(6): 102329. 2. zarenezhad e, marzi m. review on molnupiravir as a promising oral drug for the treatment of covid-19. med chem res [internet]. 2022; 31(2): 232-43. 3. kabinger f, stiller c, schmitzová j, et al. mechanism of molnupiravir-induced sars-cov-2 mutagenesis. nat struct mol biol [internet]. 2021; 28(9): 740-6. singh et al. molnupiravir and favipiravir in sars-cov-2 vol 1 | issue 3 | oct dec 2022 indian j pharm drug studies | 78 4. baranovich t, wong s-s, armstrong j, et al. t-705 (favipiravir) induces lethal mutagenesis in influenza a h1n1 virusesin vitro. j virol [internet]. 2013; 87(7): 3741-51. 5. reynard o, nguyen x-n, alazard-dany n, et al. identification of a new ribonucleoside inhibitor of ebola virus replication. viruses [internet]. 2015; 7(12): 6233-40. 6. toots m, yoon j-j, hart m, et al. quantitative efficacy paradigms of the influenza clinical drug candidate eidd-2801 in the ferret model. transl res [internet]. 2020; 218: 16-28. 7. urakova n, kuznetsova v, crossman dk, et al. β-d -n4hydroxycytidine is a potent anti-alphaviru compound that induces a high level of mutations in the viral genome. j virol [internet]. 2018; 92(3). 8. painter, george r, bluemling, et al. n4-hydroxycytidine and derivatives and anti-viral uses related there to published 2020-0903, assigned to emory university. 9. clinicaltrials.gov nct04405739. the safety of molnupiravir (eidd-2801) and its effect on viral shedding of sars-cov2 (end-covid) 10. clinicaltrails.gov nct04402203. study on safety and efficay of favipiravir (favipira) for covid-19 patient in selected hospitals of bangladesh. 11. new evidence on vui-2020/2021 and review of the public health risk assessment. 15 december 2020. 12. lowe d, “the new mutations” american association for the advancement of science 22 december 2020. 13. faria nr, claro im, candido d, et al, “genomic characterisation of an emergent sars-cov-2 lineage in manaus: prelimary findings.march 2021. 14. cutler s. “nepal variant”: what we’ve learned so far. the conversation [internet]. 2021 jun 16 [cited 2022 15. aug 1]; 15. torjesen, ingrid, 2021. covid-19; omicron may be more transmissible than other variants. 16. callaway, ewen. heavily mutated corona virus 2021. 17. covid-19 coronavirus: ultra-contagious lambda variant. 2021; 6(7). 18. o’neill l. mu: everything you need to know about the new coronavirus variant of interest. the conversation [internet]. how to cite this article: ayush singh, riya sehgal, karamjit kaur, manju, jatin isher, neeru kumari. molnupiravir and favipiravir in the therapeutics of sarscov-2 a review. indian j pharm dug studies. 2022: 1(3) 73-78. funding: none conflict of interest: none stated sakshi and singh cisplatin induced nephrotoxicity vol 1 | issue 1 | apr – jun 2022 indian j pharm drug studies | 5 review article role of membrane transporters in cisplatin induced nephrotoxicity sakshi1, gaaminepreet singh1 from, 1department of pharmacology, isf college of pharmacy, moga, punjab. correspondence to: gaaminepreet singh, isf college of pharmacy, moga, punjab, india. tel.: +91 7696579358; email: gpsinghcologist@hotmail.com. abstract transporters are important mediators of specific cellular uptake and thus, not only for effects, but also for side effects, metabolism, and excretion of many drugs such as cisplatin. cisplatin is a potent cytostatic drug, whose use is limited by its severe acute and chronic nephro-, oto-, and peripheral neurotoxicity. for this reason, other platinum derivatives, such as carboplatin and oxaliplatin, with less toxicity but still with antitumoral action have been developed. several transporters, which are expressed on the cell membranes, have been associated with cisplatin transport across the plasma membrane and across the cell: the copper transporter 1 (ctr1), the copper transporter 2 (ctr2), the ptype copper-transporting atpases atp7a and atp7b, the organic cation transporter 2 (oct2), and the multidrug extrusion transporter 1 (mate1). some of these transporters are also able to accept other platinum derivatives as substrate. since membrane transporters display a specific tissue distribution, they can be important molecules that mediate the entry of platinum derivatives in target and also non-target cells possibly mediating specific effects and side effects of the chemotherapeutic drug. this paper summarizes the literature on toxicities of cisplatin compared to that of carboplatin and oxaliplatin and the interaction of these platinum derivatives with membrane transporters. keywords: nephrotoxicity, ototoxicity, neurotoxicity, mate1, oct2, ctr2. n the last several decades, novel cancer drugs have been developed and used in clinical practice, being more specific against cancer cells and extremely effective against several previously untreatable malignancies, the so-called molecularly targeted agents, but also suffer from nephrotoxicity which limits the efficacy of the treatment and impact their quality of life and overall survival [1]. most of the chemotherapeutic agents developed so far exert their action in the cell and therefore have to cross the cell membrane to reach their targets [2]. however, they are often poorly lipophilic compounds, which cannot easily pass the cell membrane and thus need to be transported into the cell by specific systems of protein nature called transporters [3]. general concept of drug movement across biological membranes is that they can pass cell membranes via passive diffusion at a rate related to their lipophilicity. however, it is becoming evident that membrane transporters are also important determinants of in vivo drug disposition, therapeutic efficacy, and adverse drug reactions [3]. in epithelial tissues, which are constituted by polarized cells, transporters are even specifically expressed on the apical or baso-lateral cell membrane [4]. in this way, a specific drugtransporter interaction can be exploited to target drugs to selected cells and tissues, but of course can also explain specific undesired adverse effects [5]. membrane transporters such as the copper transporter-1 (ctr1), the copper transporter-2 (ctr2), the p-type copper transporting atpases atp7a and atp7b, the organic cation transporter-2 (oct2), and the multidrug extrusion transporter-1 (mate1) mediate cellular transport of cisplatin [6]. transporter mediated uptake has been shown to be an important process mediating cellular accumulation of cisplatin. cisplatin is one of the most widely utilized antitumor drugs in the world [7]. cisplatin was the first platinum-based drug that revolutionized the treatment of neoplastic diseases. for example, before the introduction of cisplatin as chemotherapeutic agent, testicular cancer was associated with a survival rate of only 5% [8]. today, treatment of this cancer with a combination of new surgical techniques and cisplatin chemotherapy allows to achieve a cure rate of over 90%. currently, cisplatin is widely used for the therapy of solid tumors [9]. however, its use is limited by severe side effects such as nephroand ototoxicity and peripheral neurotoxicity. therefore, there is a need to put an effort in developing less toxic platinum derivatives [10]. action of cisplatin on cell growth was unexpectedly discovered by rosenberg in 1965 by investigating the effects of an electric field on the growth of escherichia coli bacteria [11]. when placed in an electric field using platinum-conducting plates, bacteria ceased to divide. rosenberg hypothesized that if cisplatin could inhibit bacterial cell division it could also suppress tumor cell growth. cisplatin was approved by the fda i mailto:gpsinghcologist@hotmail.com sakshi and singh cisplatin induced nephrotoxicity vol 1 | issue 1 | apr – jun 2022 indian j pharm drug studies | 6 in 1978 for the treatment of metastatic testicular or ovarian cancer and is also administered for many other types of solid tumors [12]. a common event happening when platinating agents enter a cell is their aquation that is losing of chloride or oxalate ions and gaining two water molecules to form aquaions. the low intracellular concentration of chloride ions facilitates this process [13]. positively charged aquated form is more reactive to the cellular targets, such as nucleophilic molecules within the cell, including dna, rna, and proteins [14]. it is generally accepted that dna is the preferential cytotoxic target for cisplatin and other platinating agents: these substances bind preferentially the imidazole ring of the purines guanosine and adenosine forming monoadducts, intrastrand crosslinks, and interstrand crosslinks [15]. all crosslinks distort the structure of the dna duplex and begin the dna damage response signaling, resulting in cell cycle arrest and apoptosis [16]. cisplatin toxicity cisplatin treatment, even though effective against tumors, has severe side-effects such as nephrotoxicity, which is often doselimiting, ototoxicity, and peripheral neurotoxicity [5]. nephrotoxicity in patients cisplatin-induced nephrotoxicity manifests acutely and/or chronically. clinically, cisplatin nephrotoxicity develops after 10 days of cisplatin administration and is manifested as lower glomerular filtration rate, higher serum creatinine, and reduced serum magnesium and potassium levels interestingly, striking differences between patients in susceptibility to progressive nephrotoxicity are even though nephrotoxicity can be controlled by diuretics and prehydration of patients [17]. it is recognized that the prevalence of cisplatin nephrotoxicity is high, occurring in about one third of patients undergoing cisplatin treatment in animal studies it has been shown that the kidney accumulates more cisplatin than other organs and that the proximal tubules are principally damaged by cisplatin [18]. ototoxicity ototoxicity is an9 untypical side effect for a chemotherapeutic drug. cisplatin treatment causes a hearing loss, which can also lead to deafness [19]. ototoxicity remains an unresolved clinical problem especially in infants and younger children, where it leads to a considerable risk of delayed language development due to impaired perception of higher frequency consonant sounds that is of great importance in the presence of background noise [20]. incidence of ototoxicity is reported to be between 23 and 50% in adults and greater than 50% in children, clinical symptoms of toxicity consist of bilateral symmetrical high-frequency sensori-neural hearing loss, ear pain, or tinnitus [21]. damage induced by cisplatin begins at the cochlea base, where high-frequency sounds are processed, and proceeds towards the apex, affecting also hearing at lower frequencies as the cumulative dose increases [21]. in the cochlea, cisplatin seems to induce the generation of reactive oxygen species and/or the depletion of scavenging enzymes causing cell apoptosis [22]. neurotoxicity most patients treated with cisplatin develop a symptomatic and clinically detectable sensory neuropathy, caused by its preferential uptake in the dorsal root ganglia, which produces a dose-related large fibre sensory neuropathy [5]. symptoms include unpleasant distal paresthesias (tingling in the extremities) and numbness, associated with large fibre sensory -loss (reduced vibration and joint position sensations) and diminished or absent muscle stretch reflexes [23]. sensory ataxia (incoordination) may be disabling in those patients who have severe neuropathy. these symptoms may appear as soon as one month after initiating treatment [24]. the neuropathy may only partially recover or not recover at all. in rodents, cisplatin affects sensory nerve structure and function, showing preferential toxicity to large diameter neurons and proprioceptive sensory modalities, while motor nerves are spared from toxicity. the mechanism of platinum neurotoxicity remains in completely understood although it may involve platinum accumulation within the dorsal root ganglia (drg) leading to atrophy or loss of peripheral sensory neurons [25]. cellular transport of cisplatin several different transporters seem to be involved in the cellular transport of cisplatin: ctr1, ctr2the p-type coppertransporting atpases atp7a and atp7b, oct2 and mate1 [5]. copper transporter 1 (ctr1) copper transporter 1 (ctr1, solute carrier 31a1-slc31a1) is a membrane protein that plays a significant role in the cellular cisplatin uptake. down-regulation of ctr1 extensively reduced cisplatin uptake in yeast and inmouse embryonic fibroblasts [26]. the natural substrate of ctr1 is monovalent copper (cu+). cu+ uptake by ctr1 triggers transporter internalization [27]. however, whether this phenomenon also happens upon cisplatin transport is debated. as observed for cu+, cisplatin binds to methionine-rich motifs of the extracellular domain of ctr1. ctr1 carries out vital physiological function supplying the cell with copper, which is an essential cellular nutrient used in a broad range of enzymatic reactions. because of its important biological role, ctr1 is almost ubiquitously expressed and perhaps may not be the decisive transporter for specific cisplatin toxicities. since several cell lines from human tumor samples express ctr1mrna, this transporter could represent the uptake route of cisplatin in cancer cells. indeed, high sakshi and singh cisplatin induced nephrotoxicity vol 1 | issue 1 | apr – jun 2022 indian j pharm drug studies | 7 expression levels of ctr1 have been associated with cisplatin therapeutic success whereas ctr1 mutations are associated with cisplatin resistance. ctr1 has been also associated with the cellular transport of carboplatin and oxaliplatin [28]. copper transporter 2 (ctr2) copper transporter 2 (ctr2, slc31a2) is a copper transport protein with substantia structural homology to ctr1. ctr2 is mainly expressed in late endosomes and lysosomes , where it probably mediates the efflux of copper under conditions of low environmental copper concentration [12]. a similar function of ctr2 was proposed for cisplatin. studies in ctr2-deficientmice suggested that ctr2 functions as an indirect regulator of cu+uptake and intracellular flux by stabilizing the biosynthesis of cleaved ctr1. the cleaved ctr1 is a transporter form which lacks metal binding methionineand histidine-rich motifs and of consequence has decreased cu+ and also cisplatin uptake function [29]. therefore, high expression of ctr2 seems to be associated with resistance to the cytotoxic effect of cisplatin and knockdown of ctr2 was associated with an increased cisplatin accumulation and cytotoxicity [30]. copper-transporting (atp7a and atp7b) the p-type copper-transporting atpases atp7a and atp7b are also involved in cellular cisplatin handling [31]. these transporters play an important role in regulating the cellular copper levels, because too high intracellular copper concentrations are toxic for the cell [32]. inactivation of these transporters, as present for example in menkes’ disease (inactivation of atp7a) and in wilson’s disease (inactivation of atp7b), is associated with copper deficiency because of impaired copper efflux from enterocytes into the blood or massive cellular copper overload, respectively [26]. while atp7a is mainly expressed in intestine, choroid plexus, vascular smooth muscle and endothelial cells, as well as in cerebrovascular endothelial cells, atp7b is principally expressed in the liver and the brain [33]. regarding the transport of cisplatin, atp7a and atp7b mediate its efflux from the cell or its distribution to specific sub-cellular compartments [34]. for this reason, the expression of these transporters is correlated with cisplatin cellular sensitivity and resistance atp7b is stronger associated with the acquisition of resistance than ctr1 or atp7a. besides cisplatin, atp7a and b transporters also interact with carboplatin and oxaliplatin [35]. even though the effects of atp7a and b transporters on cisplatin cellular distribution are very similar to those observed for copper, platinum drugs are not readily exported after vesicular sequestration [35]. interestingly, copper transport systems are expressed and active in drg, which are sensitive to toxicity from platinum derivatives. here, ctr1 is expressed in large-sized neurons and atp7a in small drg neurons, suggesting that large neurons are especially sensitive and small neurons are protected from toxic effects of platinum derivatives [36]. organic cation transporters (oct1-3, slc22a1-3) a specific interaction of cisplatin with octs has also been demonstrated. since octs have a specific organ distribution, with high renal expression, the cisplatin-oct interaction is of pecial interest to explain selective organ toxicity of cisplatin [26]. octs are highly expressed in excretory organs such as the liver and the kidneys, where they mediate the electrogenic uptake of heir substrates in hepatocytes and proximal tubule cells. octs are defined as polyspecific transporters, because they can transport several unrelated substances. the driving force for the cellular transport by octs is the electrochemical gradient of the substrate [37]. in excretory organs, octs mediate the first step of secretion process, consisting of substrate uptake through the baso-lateral plasma membrane (the blood-faced part of plasma membrane). the subsequent substrate efflux through the luminal membrane (the bileor urine-faced part of plasma membrane) is the final secretion step, resulting in a vectorial substrate movement from the blood to the bile or urine in the liver or kidneys, respectively. in humans the paralogs hoct1 and hoct2 are specifically expressed in the basolateral membrane of hepatocytes and renal proximal tubule cells, respectively [38]. cisplatin seems to interact preferentially with hoct2, suggesting that hoct2 is the critical transporter for renal cisplatin uptake in humans. also the secondand third generation platinum derivatives oxaliplatin are substrates of octs [38]. for the interpretation of translational studies, it is important to underline that the rodent oct orthologs have a different organ distribution and kinetic properties compared with human octs: for example, in mice oct1 is expressed in renal proximal tubules at higher level than oct2. competition of oct-mediated cisplatin transport is able to reduce cisplatin uptake and toxicity in vitro and in vivo. oct2 has been demonstrated to be expressed in the mouse cochlea in hair cells of organ of corti and in the cells of the stria vascularis and in mouse and human drg), structures that are specially sensitive to toxicity by platinum-derivatives. in animal models it has been demonstrated that octs are critical mediators of cisplatin ototoxicity and oxaliplatin peripheral neurotoxicity [5]. multidrug and toxin extrusion protein 1 (mate1, slc47a1) several evidences indicate that mate1 mediates secretion of cisplatin into the urine. mice with genetic deletion of mate1 are more sensitive to cisplatin nephrotoxicity [39]. furthermore, cell transfected with mate1 displayed a higher cisplatin uptake than control cells. interestingly, mate1 and sakshi and singh cisplatin induced nephrotoxicity vol 1 | issue 1 | apr – jun 2022 indian j pharm drug studies | 8 mate2-k, another member of mate family which is solely expressed in human kidneys, seem to transport oxaliplatin with higher affinity than cisplatin, offering a possible explanation of the low oxaliplatin nephrotoxicity. as outlined above, inhibition of oct2 may be a protective strategy against cisplatin nephrotoxicity [40]. however, some inhibitors of oct2 such as cimetidine and ondansetron interact with higher potency with mate1, blocking cisplatin efflux from the cells and potentially increasing cisplatin renal toxicity [41]. indeed, co-treatment of mice with cisplatin and cimetidine was effective in protecting the animals from ototoxicity but not from nephrotoxicity [42]. there are some investigations suggesting a role of novel organic cation transporters (octns) for oxaliplatin transport. these transporters are expressed on the apical membrane of renal proximal tubule cells and in rat drgs. when transfected in human embryonic kidney cells, rat and human octn1 and octn2 mediate significant oxaliplatin uptake, suggesting that octns are involved in oxaliplatin neurotoxicity [26]. apart from these not directly atp-dependent transporters, multidrug resistance-associated protein 2 (mrp2) transporter seems to be involved in the efflux of cisplatin and its conjugates from kidney cells, and for this reason to play an important role for control of cisplatin renal toxicity [43]. conclusion cellular transport of platinum derivatives is mediated by several transport systems. some transporters, such as octs, are specifically expressed in organs, which are 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transport mechanisms of cytotoxic metallodrugs: an overview beyond cisplatin. molecules. 2014, 19:15584–610. 43. leslie em, deeley rg, cole spc: multidrug resistance proteins: role of p-glycoprotein, mrp1, mrp2, and bcrp (abcg2) in tissue defense. toxicol appl pharmacol. 2005, 204:216–37. how to cite this article: sakshi, gaaminepreet singh. role of membrane proteins in cisplatin induced nephrotoxicity. indian j pharm drug studies. 2022: 1(1); 5-9. funding: none conflict of interest: none stated sujeetha & anand cholesterol and alzheimer’s vol 1 | issue 1 | apr – jun 2022 indian j pharm drug studies | 10 review article the link between cholesterol and a brain disease sujeetha purushothaman1, arumugan vijaya anand1 from, 1medical genetics and epigenetics laboratory, department of human genetics and molecular biology, bharathiar university, coimbatore, tamil nadu, india. correspondence to: sujeetha purushothaman, medical genetics and epigenetics laboratory, department of human genetics and molecular biology, bharathiar university, coimbatore 641046. tamil nadu, india. email: suji648@yahoo.co.in abstract lipids comprise the immensity dry mass of the brain and these have been allied with healthy function as well as the most common pathological circumstances of the brain. genetics and lifestyles are the most important factor that influences the lipid metabolism and the key components of lipid disruption in alzheimer’s disease (ad). additionally, the most common genetic risk factor of ad, apoe e4 genotype, is involved in lipid transport and metabolism. under healthy conditions, lipid homeostasis bestows a balanced cellular environment that enables the proper functioning of brain cells. however, under pathological conditions, dyshomeostasis of brain lipid can result in disturbed bbb, abnormal processing of app, dysfunction in endocytosis/exocytose/autophagocytos is, altered myelination, disturbed signaling, unbalanced energy metabolism, and enhanced inflammation. this lipid instability may contribute to abnormalities in brain function that are the hallmark of ad. in this review, we focus on the lipid and cholesterol metabolism, with an overview of the various lipid and cholesterol metabolic pathways and changes that have been linked to ad. key words: alzheimer’s disease, lipid homeostasis, inflammation, apolipoprotein e eregulated lipid and cholesterol homeostasis in the body and particularly in the brain has been demonstrated in neurodegenerative diseases such as alzheimer’s disease (ad), as well as parkinson’s disease (pd) and huntington’s disease (hd) [1]. the changes in lipid metabolism may affect the disease progression or pathogenic mechanism. amyloidogenic peptide is believed to play a key role in the ad pathogenesis mounting the evidence that dyslipidemia provoke the production or reduce of amyloid beta (aβ) clearance [2]. moreover, other dyslipidemic-related conditions have also been linked in the ad pathogenesis, including obesity, hypertension, inflammation, insulin resistance, and type 2 diabetes [3]. in this review, we focus on the lipid and cholesterol metabolism, with an overview of the various lipid and cholesterol metabolic pathways and changes that have been linked to ad. cholesterol and lipid metabolism rising evidence suggest the changes in the metabolism of lipids, predominantly cholesterol, is implicated directly in the pathogenesis of many neurodegenerative disease including ad [2]. this part describes the cholesterol synthesis and metabolism followed by some vital information about the lipid transport and metabolism, in order to explain the normal roles of these lipids in the body and the brain. some changes in lipid metabolisms which have been detected in ad [4]. cholesterol synthesis and metabolism cholesterol plays a vital function in the structure and function of cell membranes. it influences the inflexibility of lipid bilayers, there in affects the transfer and process through the membrane. cholesterol in the brain accounts for 25% of the total body cholesterol [5]. about 70% of its cholesterol is there in the myelin, 20% is present in the glial cells, while the remaining 10% is present in the neurons [6]. the cholesterol requirements in the body are met by dietary intake of animal fats, or by synthesis. cholesterol productiongets activated when acetyl-coa is transformed into 3-hydroxy-3methylglutarylcoa (hmg-coa). cholesterol is formed when its precursor lanosterol is channeled into either the bloch pathway to generate desmosterol or lathosterol respectively; these are then converted to cholesterol [7]. cholesterol is used widely in the body, particularly by the cell membrane structures. other important physiological functions include the production of bile by the liver and hormone synthesis. brain cholesterol metabolism is different and independent from that of peripheral tissues, due to the blood–brain barrier (bbb) preventing rapid transport into and out of the brain [8]. in the adult brain, cholesteroloriginate in the form of non-esterifies form, rest being in the form of desmosterol and cholesteryl esters [9]. major part of cholesterol (about 70–80%) is seen in the myelin sheaths formed by d mailto:suji648@yahoo.co.in sujeetha & anand cholesterol and alzheimer’s vol 1 | issue 1 | apr – jun 2022 indian j pharm drug studies | 11 oligodendrocyte that insulates the axons and in the plasma membranes of astrocytes and neurons, where it maintains cellular morphology, plays important roles in lipid rafts, and helps in the synaptic transmission [10]. during adolescence, the cholesterol turnover reduces because of myelin sheath formation with the half-life between six months and five years. this is a contrast turnover that occurs during the general circulation [6]. the role of lipid rafts in neurodegenerative diseases lipid rafts are buoyant membrane glycolipoprotein micro domains rich in cholesterol and sphingolipids [11]. these provide the frame for signaling molecules and protein transduction, trafficking and immunoglobulin functions and neurotransmitters [12]. lipid rafts have been linked to various diseases conditions including cardiovascular disease and certain cancers. they are also important for the entry, replication, assembly, and budding of various types of viruses [13]. some proteins are linked with lipid rafts include gpi-anchored proteins, src family kinases, components of the heterotrimeric g-proteins and many of the proteins linked to ad linked with the metabolism of app [14]. some of the later include the γsecretase complex (processes over 20 proteins, including γ-site cleavage of app), bace-1 (β-site app cleaving enzyme, the enzyme responsible for βsite app cleavage), adam10 (a disintegrin and metalloprotease-10, enzyme which cleaves app at the α-site, also cleaves tnf-α and e-cadherin), and neprilysin, an aβ-degrading enzyme [15]. studies have demonstrated that by increasing the dietary cholesterol content, aβ production can be elevated [16]. in animal studies where rabbits were placed on a highcholesterol diet, a greater level of brain aβ accumulation was found [17]. lim et al., [18] concluded that a high-fat and highcholesterol (hfhc) diet results in increase in brain cholesterol esters, these effects were seen in older apo e e4 knock-in mice compared to apo e e3 mice. conversely, the reduction of cell membrane cholesterol has been shown to reduce γ-secretase activity and also to increase the non-amyloidogenic α-secretase cleavage of app [19, 20]. studies have shown that the membranes of the lipid content raft, from early-stage ad brain frontal and entorhinal cortex tissue, has greater microviscosity which correlated with bace-1/app interaction levels [21]. this was found not to be due to increased cholesterol or sphingomyelin levels, but due to a lower content of unsaturated fats [14]. this adds to the evidence that dyslipidaemia is central in ad neurodegeneration and that a diet high in polyunsaturated fatty acids may provide benefit in slowing or preventing ad pathogenesis. transgenic ad mouse models and mathematical modeling studies support this theory, with evidence suggesting that increasing the cholesterol and long-chain polyunsaturated fatty acid (mainly dha) content of membranes may delay the onset and/or progression of ad [22] alzheimer’s disease ad is the most common type of dementia, characterized by the progressive loss of memory and other cognitive functions of the brain. an affected individual gradually becomes totally dependent upon others, culminating in their death approximately 3– 10 years after diagnosis [23]. the neurodegenerative changes that are characteristic of an ad brain include widespread synaptic and neuronal loss, the accumulation of extracellular aβ fibrils and plaques, intracellular neurofibrillary tangles (nft) composed of hyperphosphorylated tau filaments, microglial infiltration, and brain atrophy, particularly in the regions important for memory, such as the hippocampus, amygdala, and frontal cortex [24]. genetic mutations in genes whose proteins are involved in the processing of the app to aβ peptides, such ps1 and ps2, as well as ad-related app mutations, are predisposing risk factors for ad. however, these mutations are involved only in earlyonset familial alzheimer’s disease (eoad) which occurs before age 65, but sometimes as young as 30. these cases are relatively rare and account for less than 5% of all ad cases [25]. the main risk factor linked to the much more common late-onset form of alzheimer’s disease (load) is apoe e4 allele status. other significant risk factors include dyslipidemia, hypertension, obesity, chronic inflammation, insulin resistance, and type 2 diabetes. all of these are also risk factors for cardiovascular disease. when these conditions occur together, this is termed ‘metabolic syndrome’ [28, 29]. it is believed that metabolic changes caused by these conditions lead to a greater production or reduced clearance (or both) of aβ peptides. many of these risk factors can be prevented by altering diet and physical exercise, which are known to reduce the risk of cardiovascular disease. there is a growing body of evidence that reducing these same risk factors would considerably slow or possibly prevent ad pathogenesis [30, 31]. effect of amyloid-β on lipid metabolism it has been suggested that app processing affects cellular lipid metabolism [32]. in cultured neurons and in transgenic mice, aβ with 42 amino acids (aβ42) can activate neutral sphingomyelinases and down regulate sphingomyelin levels, whereas aβ40 reduces de novo cholesterol synthesis by inhibiting the activity of hmg-coa reductase. therefore, maintaining lipid homeostasis could be a biological function of app processing grimm et al., [33] and the pathological accumulation of aβ could lead to abnormal lipid metabolism. furthermore, both studies in vitro and in ad patients suggest that aβ causes oxidative stress, leading to lipid oxidation that might contribute directly to neurodegeneration [32] studies also suggest that aβ induces ozonolysis of cholesterol, leading to the formation of peroxiderivatives that accelerate aggregation of aβ monomers [35] and that aβ oxidizes cholesterol at positions of 7-β and 3-β, thus leading to h2o2 production [36]. therefore, sujeetha & anand cholesterol and alzheimer’s vol 1 | issue 1 | apr – jun 2022 indian j pharm drug studies | 12 a deleterious feedback loop between aβ accumulation and altered lipid metabolism could be one of the molecular mechanisms underlying the link between lipid disorders and ad. cholesterol metabolism-related genes and alzheimer’s disease apo e protein plays a central role in lipid transport while possession of the apoe e 4 allele influences ad risk. the human apoe gene is located on chromosome 19, and its three most common alleles are designated e2, e3 and e4(williams et al., 2020). of these, e3 is the most common allele in humans (78%) followed by e4 (14%), then e2 (8%). however, it is usually apo e e4 which is associated with increased levels of circulating ldl cholesterol, higher triglyceride levels, and a greater risk of coronary artery disease (linton et al., 2019). this is despite apo e e4 having higher affinity for the ldl receptors than apo e e3 and e2, and increased levels of lipoprotein internalization [37, 38].the increased affinity of apo e e4 for the ldl receptor in the periphery may be a limiting factor for cellular processing of lipoproteins, for it has been observed that apo e e4 is poorly recycled by cells, yet is readily internalized which results in raised intracellular cholesterol levels [39]. apo e in the brain whilst apo e is clearly a molecule of great importance in the cns, it is not imported from the periphery [40] where the liver produces it in the largest amounts. rather it is synthesized locally by astrocytes in significant quantities [41]. lrp-1 is primarily expressed in neurons, whereas glial cells mostly express the ldl-receptor [42]. these receptors (particularly lrp-1) are not only used for lipoprotein metabolism, but they also bind some proteins involved in brain development (e.g. sonic hedgehog, wnt, and reelin), as well as proteases, protease inhibitors, vitamin transporters, and proinflammatory molecules [43]. receptor-mediated endocytosis transit lipid particles to late endosomes. immediately after endocytosis, apo e is detached from the lipid components and is not sent to lysosomes but recycled back to the plasma membrane [44]. apoe 4 and alzheimer’s disease one of the strongest genetic risk factors for ad risk is apoe e4 allele, the mechanisms is still implicit. a study that examined the physical distribution of the resultant apoe e4 protein shows that it gravitates towards the larger, less dense particles of cns lipoprotein whereas apoe e2 and apoe e3 tend to associate with smaller, denser lipoprotein fractions [45]. like their peripheral counterparts, the cns apoe proteins have differential effects, where apoe e2 and apoe e3 appear to be more effective than apoe e4 [46]. early studies showed that plasma levels of apoe may be an important factor in ad, as higher levels were observed in the plasma of ad individuals relative to non-ad individuals [47, 48]. other early studies suggested that polymorphisms in the apoe promoter region may influence the probability of developing ad [49, 50]. this region, which belongs to the tata box family, regulates the production of apoe protein where a genotype of -491tt produces much less apoe than -491at and -491aa. the initial studies demonstrated that the apoe -491at and aa genotypes lead to increased risk for ad independent of apoe allele [51]. it was soon realized that the risk for ad may be compounded when apoe e4 alleles and the 491 aa genotype are combined [42]. these results suggested that the improved production of the apoe protein may boost disease progression but did not account for any mechanism of action in ad by the apoe e4 protein itself. however, more recent studies now suggest that low plasma apoe levels increase the risk of ad [53]. furthermore, baker-nigh et al., 2016 concluded that the levels of aapo e and cns are not correlated but it is well correlates csf aβ levels which are more relevant to ad risk. conclusion reducing the risk of ad will involve dietary changes and healthy lifestyles that can reduce the risk of dyslipidaemia, insulin resistance, type 2 diabetes, cardiovascular disease, and chronic inflammation, which are all known risk factors for ad. despite the many studies on the influence of the apoe e4 allele on aβ aggregation, binding, and clearance, the overall pathological effect of this allele is still not known. it is hoped this will pave the way towards effective treatments, whilst highlighting the importance of preventing dyslipidaemia. references 1. martín mg, pfrieger f, dotti cg. cholesterol in brain disease: sometimes 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disease? cortex. 2005:1; 41(4):452-63. sujeetha & anand cholesterol and alzheimer’s vol 1 | issue 1 | apr – jun 2022 indian j pharm drug studies | 14 49. lambert mp, barlow ak, chromy ba, et al. diffusible, nonfibrillar ligands derived from aβ1– 42 are potent central nervous system neurotoxins. proceedings of the national academy of sciences. 1998:26;95(11):6448-53. 50. rasmussen kl, tybjærg-hansen a, nordestgaard bg, et al. absolute 10-year risk of dementia by age, sex and apoe genotype: a population-based cohort study. cmaj. 2018:4; 190(35):e1033-41. how to cite this article: sujeetha purushothaman, arumugan vijaya anand. the link between cholesterol and a brain disease. indian j pharm drug studies. 2022; 1(1): 10-14. funding: none; conflict of interest: none stated shabaraya a & chandur k spectrophotometric characterization of lycopene vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 118 original article spectrophotometric characterization of lycopene phytosomes from fruit peels of lycopersicum esculentum ramakrishna shabaraya a1, viresh k chandur2 from, 1prof. and principal, 2asso. prof.department of pharmaceutics, srinivas college of pharmacy mangalore. abstract among all colored fruits tomatoes are one of the rich source of lycopene, which is essentially known for its anti-oxidant activity and effective in treating major diseases like cancer, heart diseases. it is insoluble in water and sensitive to light and temperature but, it is soluble with various proportions in aprotic solvents like petroleum ether, diethyl ether, and acetone. best combination of these solvents was made used to extract lycopene and characterized for its standardization by means of phytochemical testing, melting point, λ max, ftir and dsc studies showing the effective way of extraction with good yield up to 8%, phytochemical investigation showed presence of flavonoids, turpinoids and steroids. uv spectrophotometric analysis shows λ max at 471nm. ftir studies show the presence of conjugated carbon double bonds essential to get red color to the product and open ring structure on both the ends. dsc endotherms, the melting peak obtained for the phytosomal complex was less than the melting point obtained for the lycopene and is due to the less cohesive force energy of crystal lattice of the complex. key words: lycopene, ftir, uv spectrophotometer, dsc, endotherms. ecent intriguing possibilities for the treatment of chronic diseases stand out as phytopharmaceuticals. new therapy options for a variety of diseases are made possible by phytochemicals that have fewer side effects and cost less. natural pigments called carotenoids are produced by microbes and plants to protect cells from photosensitization and act as light-blocking substances during photosynthesis [1]. about 60 of the more than 700 carotenoids that have been classified can be present in the human diet [2]. carotenes and xanthophylls, including lutein and beta-cryptoxanthin, are important dietary groups [3]. up to 95% of the total plasma carotenoid concentration is made up of six different compounds: -carotene, -carotene, -cryptoxanthin, lutein, zeaxanthin, and lycopene. food is one factor that affects plasma concentrations of carotenoids [4]. in human plasma and tissue, lycopene is a very significant carotenoid and accounts for up to 50% of the body's total carotenoids. access this article online received – 24th march 2023 initial review – 04th april 2023 accepted – 14th april 2023 quick response code lycopene is a carotenoid pigment that is primarily present in foods that are red in hue. due to its conjugated double bonds, lycopene is a potent antioxidant. lycopene prevents oxidation of vascular cells and lipoproteins through its antioxidant action. in vitro tests on human lymphoid cells, low-density lipoprotein, and plasma have shown that lycopene is an efficient singlet oxygen quencher [5]. lycopene-rich meals have been demonstrated to reduce the risk of cancer and cardiovascular disease. the primary red colour in fruits, lycopene is a linear, unsaturated hydrocarbon carotenoid. lycopene, which is present in tomatoes, belongs to a class of carotenoids with a broad polyene chain that contains 35–40 carbon atoms. some of these polyene chains are ended by two 6-carbon rings.antioxidant capabilities of carotenoids have the potential to slow down the ageing process and many degenerative diseasbes. lycopene is a necessary nutrient that must be consumed daily. lycopene's potential health _______________________________________ correspondence to: viresh k chandur, asso. prof. department of pharmaceutics, srinivas college of pharmacy, mangalore. karnataka, india. tel: +91 9742565141, email: viresh.chandur2009@gmail.com r mailto:viresh.chandur2009@gmail.com shabaraya a & chandur k spectrophotometric characterization of lycopene vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 119 consequences have also recently been researched. lycopene is believed to have antioxidant characteristics that are primarily responsible for these positive effects [6]. materials & methods collection of tomato fruits: tomatoes were collected from local market at mangalore of karnataka in the month of january 2018. the fruit was authenticated by botanical department regional science center pilikula mangalore. morphological study of tomato fruit tomato fruit: the tomato is the edible, often red, fruit/berry of the plant solanumlycopersicum, syn: lycopersicumesculentum, commonly known as a tomato plant. the plant belongs to the nightshade family, solanaceae. tomato is consumed in diverse ways, including raw, as an ingredient in many dishes, sauces, salads, and drinks. while tomatoes are botanically berry-type fruits, they are considered culinary vegetables as an ingredient or side dish for savory meals. numerous varieties of tomato are widely grown in temperate climates across the world, with greenhouses allowing its production throughout the year. the plants typically grow to 1–3 meters (3–10 ft.) in height and have a weak stem that sprawls. it is a perennial in its native habitat, and cultivated as an annual. fruit size varies according to cultivator, with a width range of 0.5–4 inches (1.3–10.2 cm) preliminary preparation for the peel of tomato: the ripe tomatoes stored at 4ºc were used within 48 h for isolation of lycopene. the peels of tomato were separated using blanching method. the removed peels were dried at room temperature (24-25°c) for 2-3 h and then packed in zip-lock polyethylene bags. method 1: extraction of lycopene from tomato peel: the dried peels were stored at 4°c before grinding with a small amount of solvent system (n-hexane: acetone: ethanol in 2:1:1 v/v) in the presence of carbon dioxide. it was then sonicated using ultrasonic crusher followed by magnetic stirring for 4 h in an inert environment (carbon dioxide) under dark conditions. the resulting extract was collected in an amber color glass container with the presence of carbon dioxide environment and stored at 4ºc for further processing. the extract was then dried using rotary flash evaporator by heating at 60°c at 50 rpm in dark condition [1-2]. method 2: raw tomato slices were blended with water (1:1) and boiled for 10 minutes. the aqueous suspension was vacuum filtered to reduce water to 15% and treated with acetone (1:1) under high speedy blender and filtered, filtrate containing βcarotenes and other carotenoids was separated. residue was treated with diethyl ether (1:1) under high speedy blender and filtered. filtrate containing lycopene was separated and lyophilized. pre-formulation investigation of lycopene extract 1. shinoda test: a little quantity of extract was dissolved in alcohol + few fragments of mg turnings + conc. hcl drop wise. 2. lead acetate test: lead acetate solution was added to small amount of extract. 3. alkaline reagent test: increasing amount of sodium hydroxide was added to the sample of extract. 4. ferric chloride test: extract + ferric chloride solution detection of steroids 1. lieberman burchard’s test: 2mg of dry extract was dissolved in acetic anhydride, heated to boiling, cooled and then 1 ml of conc. h2so4 added. 2. salkowski reaction: 2mg of dry extract was shaken with chcl3. to the chcl3 layer, h2so4 was added slowly along the sides of test tube [4]. uv-vis spectrophotometric determination of lycopene in tomato peel extract. determination of λ max: solution of lycopene in diethyl ether was prepared and scanned using uv–vis spectrophotometer to read λ max in the wavelength of 200–600 nm. determination of standard graph: 100 mg of tomato extract (lycopene) was dissolved in diethyl ether and the obtained solution is serially diluted to obtain descending concentrations and the final volume was made up to 10 ml using diethyl ether. finally the absorbance values were plotted on a graph with variable concentration on x-axis and absorbance at 471 nm on y-axis. the regression coefficient value and the equation for the graph were also derived so as to ease the calculations of concentration of lycopene in tomato samples [8]. ftir spectrum of lycopene was taken by ftir spectrophotometer: lycopene was scanned between wave number ranges of 4000 cm-1 to 650 cm-1. major peaks of the spectra were interpreted to determine the respective functional groups present. to authenticate the spectrum, ftir spectrum of the extracted lycopene was compared with spectrum of imported lycopene, spectrum given by hisarphytoextracts [9]. differential scanning calorimetric (dsc) studies of lycopene phytosomes: lycopene phytosomeswere shabaraya a & chandur k spectrophotometric characterization of lycopene vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 120 subjected to differential scanning calorimetric using sdt q600 v20.9 build 20 calorimeter. the instrument comprised of calorimeter, flow controller, thermal analyser and operating software. the instrument was designed to supply heat to the sample, so that its temperature was raised precisely. the sample was heated in sealed aluminium pans under nitrogen flow (30 ml/min) at a scanning range of 30 to 800oc. empty aluminium pan was used as a reference. the heat flow as afunction of temperature was measured for the lycopene phytosomes. the dsc of lycopene phytosomeswas obtained from which melting point was recorded [10]. results phytoconstiuents investigation found to possess flavonoids and terpenoids in tomato peel extract (table 1 – 3). qualitative determination of λmax and ftir studies. uv-vis spectrophotometric detection of lycopene in tomato peel extract. identification of lycopene: i) determination of λmax figure 1: determination of λmax of lycopene form tomato peel extract. as reported λ max for solution of lycopene in diethyl ether is 472 nm and during investigation it was found to be 471 and 502 nm (figure 1 & figure 2). quantitative determination –the percentage yield from marketed powder and tomato peels was found to be 8.5% and 8.0 % respectively. from fruit authentication and from the qualitative & quantitative determination it was concluded that selected component from tomato was lycopene. the phytochemical studies confirms the presence of the above said constituents and further the spectrophotometric determination was compared with that of the marketed tomato extract containing lycopene, confirming the presence of lycopene which showed same λ max as that of the standard figure 2. ftir spectral studies confirms about the presence of the same functional groups as that of the standard (figure 3, 4, 5). drug excipients compatability study ftir studies of tomato extract figure 2: standard graph of lycopene. y = 0.4419x r² = 0.9961 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 0.5 1 1.5 2 2.5 3 a b s a t 4 7 1 n m conc in mg/ml standard graph of lycopene series1 linear (series1) shabaraya a & chandur k spectrophotometric characterization of lycopene vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 121 figure 3: ft-ir spectrum of lycopene figure 4: ft-ir spectrum of phosphatidylcholine table 1: peak picking of lycopene frequency amide lipid c-h bending c-c and c-c-h stretching water reported 1650 and 1540 cm-1 1730-1765 cm-1 and 3000-2800 cm-1 1100-1400 cm-1 3700-3000&1600-1700 observed 1610 1735 1049 2922 & 1610 table 2: peak picking of phosphatidylcholine frequency amide lipid c-h bending c-c and c-c-h stretching water reported 1650 and 1540 cm-1 1730-1765 cm-1 and 3000-2800 cm-1 1100-1400 cm-1 3700-3000&1600-1700 observed 1610 1735 1049 2922 & 1610 table 3: peak picking of formulation c1 origin frequency (cm−1) observed given c−c−h stretch, 3338.78 3072−3055, 3033−3007 m methylene/methyl h stretch, alkene 2924.09 2852.72 2981−2962, 2926−2894 2894 m to s c=c stretch 1602.85 1636, 1558 m 500750100012501500175020002500300035004000 1/cm 75 80 85 90 95 100 105 %t 34 44 .8 7 29 22 .1 6 28 52 .7 2 23 62 .8 0 17 35 .9 3 16 08 .6 3 14 58 .1 8 13 77 .1 7 12 20 .9 4 11 51 .5 0 10 47 .3 5 92 5. 83 83 3. 25 71 9. 45 f 500750100012501500175020002500300035004000 1/cm 45 60 75 90 105 %t 29 22 .1 6 28 52 .7 2 17 35 .9 3 16 10 .5 6 14 63 .9 7 12 17 .0 8 10 49 .2 8 82 5. 53 71 9. 45 51 3. 07 pc shabaraya a & chandur k spectrophotometric characterization of lycopene vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 122 c=o stretch 1734.01 1735 c−h deformation, methyl 1373.32 1399−1375, 1364−1350 m c−h out-of-plane, (e) disubstituted double bond 1004.91 976−946 s figure 5: ft-ir spectrum of formulation c1 differential scanning calorimetric studies figure 6: dcs of lycopene phytosomes. discussion the maxima are decidedly red-shifted from the maxima at 505, 475, and 445 that are observed for lycopene in organic solvent such as hexane as observed by davis ar et al. sanchit s and oishee c observed the absorbance maxima of lycopene at 502 nm using hexane. the ftir spectra showed typical bands same as by aghel n et al., observed arising from amide (1650 and 1540 [cm-1]) and lipid (1730-1765 [cm-1] and 3000-2800 [cm-1]) groups. other bands occur at 1477-1400 [cm-1] (c-h bending), 1100-1400 [cm-1] (c-c and c-c-h stretching), and 11701115 [cm-1] (c-o stretching). strong and broad absorption bands of water are shown in the 3700-3000 [cm-1] and 1600-1700 [cm-1] range. the frequency region between 1200 and 900 [cm-1] shows intense bands attributed to (co-c) 1352 vibrational modes of various carbohydrates and acids, which are abundant groups in tomatoes. frequencies of all types of deformations are found below 1000 [cm-1]. the spectral signal obtained at a frequency of 957 [cm-1] can be attributed to the presence of trans ch out-of-plane deformation vibration of lycopene. the dsc thermogram obtained for the phytosome is shown in figures 6. the thermogram of the lycopene phytosomes showed endothermic peaks at 66.43oc. these melting peaks obtained were less than the melting point obtained for the lycopene (173.28°c). the decrease in the melting point of the lycopene might be due to the less cohesive force energy of crystal lattice of the complex formed as showed by haixiang w et al one peak around 44 ◦c, probably because of loss of water, another peak at about 162.5 ◦c, likely due to its melting point. however, the dsc curve of the inclusion complexes showed different features of free molecules and the physical mixtures, indicating that there was probable interaction between the lycopene and β-cd. these results evidenced that the lycopene was embedded into the cavity of the βcd. phytochemical screening showed the presence of flavonoids, terpenoids & steroids as seen by sravanthi j et al. better yield was seen as compared with marketed spray dried tomato powder of 8%. conclusion hence, tomato can be a good natural source of lycopene. methods have been developed for extraction and analytical process for quantification and qualification. an absorption 500750100012501500175020002500300035004000 1/cm 60 67.5 75 82.5 90 97.5 105 %t 33 38 .7 8 29 24 .0 9 28 52 .7 2 23 62 .8 0 23 37 .7 2 17 34 .0 1 16 02 .8 5 13 73 .3 2 12 32 .5 1 11 49 .5 7 10 04 .9 1 92 7. 76 83 7. 11 60 9. 51 f-1 shabaraya a & chandur k spectrophotometric characterization of lycopene vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 123 maximum was found to be at 471 nm. ftir, a better tool for understand drug excipient interactions and identifying the functional groups which complies with screening tests. in the dsc endotherms, the melting peak obtained for the phytosomal complex was less than the melting point obtained for the lycopene and is due to the less cohesive force energy of crystal lattice of the complex. references 1. sandra k and iris ep. the tomato (solanumlycopersicum l., solanaceae) and its botanical relatives, the tomato genome, compendium of plant genomes; springer: 2016, 721. 2. aghel n, ramezani z, amirfakhrian s. isolation and quantification of lycopene from tomato cultivated in dezfoul, iran, journal of natural pharmaceutical products 2011; 6(1): 9-15. 3. bunghez ir, raduly m, doncea s, et al. lycopene determination by different spectral techniques (uv-vis, ftir, and hplc), digest journal of nanomaterials and biostructures, 2011; 6(3): 134956. 4. barku vy, boye a, adinortey c, et al. preliminary phytochemical screening, antimicrobial examination and wound healing potential of the root extract of amaranthusspinosus. journal of basic & applied sciences, 2015; 1 (2): 84-95. 5. butnariu m. methods of analysis (extraction, separation, identification and quantification) of carotenoids from natural products, j eco sys eco graph, 2016; 6 (2): 1-19. 6. sravanthi j, gangadhar s., phytochemical and antioxidant composition in lycopersicumesculentum, journal of medicinal plants studies, 2015; 3(4): 107-10. 7. alain cl, ement, martine d, et al. non-destructive measurement of fresh tomato lycopene content and other physicochemical characteristics using visible-nir spectroscopy, j. agric. food chem, 2008; 56: 9813–18. 8. olives barba ai, ca´mara hurtado m, sa´nchez mata mc, et al. application of a uv–vis detection-hplc method for a rapid determination of lycopene and b-carotene in vegetables, food chemistry, 2006: 95; 328–36. 9. davis ar, fish ww, perkins-veazie p. a rapid spectrophotometric method for analyzing lycopene content in tomato and tomato products. postharvest biology and technology, 2003; 28: 425-30. 10. haixiang w, shaofeng w , hua z , suilou et al. inclusion complexes of lycopene and β-cyclodextrin: preparation, characterization, stability and antioxidant activity. antioxidants, 2019; 8 (314): 1-13. 11. sanchit s, oishee c. quantitative analysis of lycopene extract using pretreated tomato samples, international journal of biotech trends and technology, 2016; 6(1): 32-5. how to cite this article: ramakrishna shabaraya a, viresh k chandur.spectrophotometric characterization of lycopene phytosomes from fruit peels of lycopersicum esculentum. indian j pharm drug studies. 2023: 2(3) 118-123. funding: none conflict of interest: none stated kareenmulla et al efficacy, safety of vildagliptin-metformin versus glimepiride-metformin vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 63 original article a comparitive study on efficacy and safety of vildagliptin-metformin versus glimepiride metformin in type 2 diabetes mellitus patients prathap bingi1, shaik kareemulla2, b. himabindu3, d. sheklane khan3, g. ramu3, s. mahammed rafi3 from, 1assistant professor (ggh-rims), kadapa, 2associate professor, 3pharmd intern, department of pharmacy practice, p. rami reddy pharmacy college, kadapa andhra pradesh. correspondence to: shaik kareemulla, associate professor, department of pharmacy practice, p. rami reddy memorial college of pharmacy (prrmcp), kadapa, andhra pradesh, email: tanveerkareems@gmail.com abstract background: inadequate control of blood glucose in patients leads to micro or macrovascular complications. metformin is an initial drug used to treat diabetes. in case of inefficient glucose control with metformin, add-on therapy is recommended. the motto of our study is to compare the efficacy and safety of vildagliptin-metformin with glimepiride – metformin in type 2 diabetes. aim & objectives: this study was aimed to compare the efficacy and safety of vildagliptin-metformin with glimepiride metformin for patients with type 2 diabetes mellitus at government general hospital (ggh-rims), kadapa. objective: to assess the prognosis report, and compare the effectiveness and safety profiles of group i (vildagliptinmetformin) patients with group ii (glimepiridemetformin) patients and counsel them about a multi-disciplinary treatment approach by prov iding patient information leaflets to exhibit a better life. methodology: a single-center prospective observational study was conducted for 6 months in the general medicine department at a government general hospital, in india. a total of 100 patients were recruited for this study and divided into 2 groups of 50 patients each. vildagliptin-metformin group 1 and glimepiride-metformin group 2. results: glycated hemoglobin (hba1c), fasting blood sugar levels (fbs), and random blood sugar levels (rbs) parameters are taken as primary and secondary endpoints to compare groups as per efficacy. in terms of safety weight gain, hypoglycemia, and other gastrointestinal adverse effects are considered. the difference between values of baseline and final followup of hba1c, fbs, rbs is 0.50%, 20mg/dl, 85mg/dl in group 1 (v-m); whereas 0.35%, 15mg/dl, 55mg/dl in group 2 (gm) respectively. 24 adrs with respect to hypoglycemia, weight gain, abdominal pain, muscle tenderness, and diarrhea are observed in group 1 (vm) group and 46 adrs are observed in group 2 (g-m). conclusion: vildagliptinmetformin treatment provided favorable glucose control comparable to that of glimepiride-metformin treatment. it also resulted in better adverse event profiles with lower ris ks of hypoglycemia and weight gain. key words: type 2 diabetes, metformin, vildagliptin, glimepiride, hba1c, adr’s ccording to an idf report published in 2013, the global prevalence of diabetes in adults (20-79 years old) was 8.3% (382 million people), with 14 million more males than females (198 million males vs 184 million females). this disease majorly occurs between 40 to 59 years of age and the number will rise beyond 592 million by 2035 with a 10.1% global prevalence [1]. the ada has developed and provided diabetes care guidelines and related documents since 1989. its clinical practice guidelines are integral resources for all health care professionals. according to ada, metformin is the preferred initial pharmacological agent for the treatment of t2dm. if metformin is not tolerated, other options for first-line therapy include sulfonylureas, dipeptidyl peptidase 4 inhibitor (dpp-4i), and sodium-glucose linked transporter 2 inhibitor (sglt2i), alpha-glucosidase inhibitors [2]. some studies documented that long-term vildagliptin combination therapy is safe and effective in japanese t2dm patients [3]. sanjay et al. concluded that saxagliptin in combination with metformin was generally well-tolerated in indian t2dm patients [4]. studies by zang et al. concluded that compared with dual oad non-vildagliptin combination therapies, vildagliptin add-on to metformin is effective and safe to achieve glycaemic control in chinese patients with t2dm [5]. harika et al. concluded that vildagliptin and metformin combination provided better efficacy comparable to that of glimepiride and metformin combination and resulted in a better adverse effect profile with lower risks of hypoglycemia and weight gain [6]. yavropoulou et al. concluded that vildagliptin is well tolerated either as monotherapy or in combination but the majority of patients require add-on therapy shortly after the beginning of treatment [7]. our study attempted to compare the safety and efficacy of vildagliptin –metformin with glimepiride –metformin in type 2 diabetes mellitus patients. materials and methods a kareenmulla et al efficacy, safety of vildagliptin-metformin versus glimepiride-metformin vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 64 study design: this study was a prospective observational study and was carried out at a tertiary care hospital in southern india over a period of 6 years (from december 2020 to may 2021) after due clearance from the institutional ethical committee. inclusion criteria: male and female patients aged from 2070 years who gave consent for participation, who are consulting both ip and op general medicine departments suffering from type 2 diabetes mellitus with or without comorbid conditions with respect to the cardiovascular system and pulmonary system and receiving vildagliptin metformin and glimepiride metformin as a treatment for type 2 diabetes mellitus, are included in the study. exclusion criteria: pregnant women, breastfeeding women, neonates, children, and geriatric (>70 years), those who were not willing to participate, and those who were suffering from type 1 diabetes mellitus, chronic renal or hepatic disorders, and receiving insulin treatment are excluded from the study. study method our study was a prospective observational study conducted on 100 patients with type 2 diabetes mellitus who were on treatment with either vildagliptin metformin or glimiperide – metformin at a government general tertiary care hospital. this study was conducted over a period of 12 weeks –follow-up was conducted every 4 weeks and the obtained values were evaluated. the present study aimed to compare the efficacy and safety of the combinations of two different drug regimens i.e., vildagliptin – metformin and glimiperide – metformin in type 2 diabetic patients. patients diagnosed with type 2 diabetes mellitus and prescribed metformin-vildagliptin (group i), and metformin-glimepiride (group ii) were screened. screened patients were recruited after taking informed consent and then, data was copied in our data collection form for every follow-up that was conducted once in 4 weeks. group i and group ii patients continued the treatment for a duration of 6 months. after the end of 6 months, the efficacy and safety profiles of both the groups were estimated and compared simultaneously. data were processed and analyzed, and framing of results, preparation of dissertation book, and communication of the research work through publications were performed. results were represented as frequencies, percentages, mean, and medians. software graph pad prism was applied to evaluate and analyze the data. in some cases, inferential statistics like analysis of variance (anova) followed by student– tests, using spss software version 21.0 were also implemented. permission for collecting patient data was approved by the institutional ethical committee of rims hospital and the clinical guide of the general medicine department. in addition, hospital management also allowed us to utilize the other facilities for the project. results a total of 100 patients were taken as the sample size in this study. among them, 65(65%) were male patients and 35(35%) were female patients. based on their treatment, 50 patients were grouped in the v-m group and 50 patients were grouped in the g-m group. the in v-m group, 35 (70%) patients were male and 15(30%) were female patients. in the g-m group, 30(60%) were male patients and 20(40%) were female patients. the details were shown in figure 1. figure 1: graphical representation of male & female patients (v-m; g-m) a total of 50 patients were in the v-m group, the majority of them i.e., 22 (44%) were in the 50-59 years age group, 2(4%) patients belonged to the 20-29 years age group, 6(12%) patients belonged to 30-39 years age group, 9(18%) patients belonged to 40-49 years age group, and 11(22%) patients belonged to 60-69 years age groups. a total of 50 patients were in the g-m group, the majority of them i.e., 20(40%) were in the 40-49 years age group, 4(8%) patients belonged to the 20-29 years age group, 2(4%) patients belonged to 30-39 years age group, 16(32%) patients belonged to 50-59 years age group and 8(16%) patients belonged to 60-69 years age group. of a total of 100 patients, the majority of them i.e., 38 were in the geriatric age group i.e., 50-59 years, 6 patients belonged to the 20-29 years age group, 8 patients belonged to the 30-39 years age group, 29 patients belonged to 40-49 years age group, and 19 patients belonged to 60-69 years age groups. the details have been represented in figure 2. figure 2: graphical representation of patients-based age groups (v-m; g-m) v m 35 v m 15 g m 30 g m 20 0 5 10 15 20 25 30 35 40 males females gender 2 6 9 22 11 4 2 20 16 8 0 5 10 15 20 25 20-29 30-39 40-49 50-59 60-69 age groups kareenmulla et al efficacy, safety of vildagliptin-metformin versus glimepiride-metformin vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 65 in the total of 50 patients in the v-m group, 33 comorbid conditions were present. among all co-morbidities, majorly i.e., 13(40%) co-morbidities were found to be hypertension, 4(12%) co-morbidities were found to be cardiac arrhythmia, 3(9%) comorbidities were found to be coronary artery disease, 9(27%) co-morbidities were found to be copd, and 2(6%) comorbidities were found to be pneumonia and asthma. in the total of 50 patients in the gm group, 35 co-morbid conditions were present. among all co-morbidities, majorly i.e., 14(40%) comorbidities were found to be hypertension, 5(14%) comorbidities were found to be coronary artery disease, 10(28%) co-morbidities were found to be copd, and 2(6%) comorbidities were found to be cardiac arrhythmia, pneumonia, and asthma. in the total of 100 patients, 68 co-morbid conditions were present. among all co-morbidities, majorly i.e., 27(40%) comorbidities were found to be hypertension, 6(9%) comorbidities were found to be cardiac arrhythmia, 8(12%) comorbidities were found to be coronary artery disease, 19(27%) co-morbidities were found to be copd, and 4(6%) comorbidities were found to be pneumonia and asthma. details with regards to co-morbidities were represented in figure 3. figure 3: graphical representation of patients based on comorbidities (v-m; g-m) discussion on evaluation, it was found that a significant reduction of hba1c, fbs, and rbs levels was noted in vm group patients when compared to gm group patients. in terms of the safety profile, more adrs were noted in gm group patients than in vm group patients. we observed that the vildagliptinmetformin combination is a more effective safety treatment for type 2 diabetes mellitus than glimepiridemetformin combination. metformin is the first-line drug used to treat diabetes mellitus. it suppresses hepatic gluconeogenesis and glucose output from the liver and enhances insulin-mediated glucose uptake and disposal in skeletal muscle and fat. thus, insulin resistance exhibited by type-2 diabetes is overcome. episodes of hypoglycemia are rare while using metformin [8]. if blood sugar levels are not controlled by metformin, then dual therapy will be recommended. sulfonylureas (glimepiride) is the first add-on drug to metformin in glucose intolerance. glimepiride will increase insulin secretion. they are widely used for type 2 diabetes because they improve glycemic control, and lack stomatic side effects other than hypoglycemia, and are very inexpensive [9]. insulin production is not provoked even at low-glucose concentrations risking the production of severe and unpredictable hypoglycemia, which may increase the mortality rates. to overcome this effect, some other add-on therapies are recommended. dpp-4 inhibitors are one of the therapies among them, however, they are considered adjuvant therapy agents. vildagliptin is an active and potent dpp-4 inhibitor that binds to enzymes covalently. this complex dissociates very slowly resulting in the persistent dpp-4 inhibition even after the clearance of the free drug from circulation. it decreases the degradation of the incretin hormones glucagon-likepeptide1(glp-1) and glucose-dependent insulinotropic peptide (gip). this activity increases levels of active incretins and enhances pancreatic islet cell responsiveness to glucose, thus improving insulin secretion and reducing inappropriate glucagon production, improving insulin sensitivity, reducing fasting, postprandial glucose, and hba1c. vildagliptin treatment is charcharacterizedweightneutral and lipid-neutral effects, a very low risk of edema [10]. the episodes of hypoglycemia are also rare in dpp-4 inhibitors treatment. nasopharyngitis and cough are common side effects of dpp-4 inhibitors. angioedema, acute pancreatitis, and skin lesion are rare side effects of dpp-4 inhibitors. hba1c levels have been decreased rapidly within the initial three months of vildagliptin [11]. biswanath et al., cheli et al., conducted studies that concluded that vildagliptin and metformin combination provided better efficacy comparable to that of glimepiride and metformin combination and resulted in better adverse effect profile with lower risks of hypoglycemia and weight gain [12, 13]. a study conducted by li-nong ji et al. concluded that the use of combination therapy with vildagliptin and metformin will provide good glycemic control and will be better tolerated than up-titration of metformin monotherapy [14]. a study conducted by hye-soon kim et al., concluded that glimepiride metformin combination therapy was more effective in glycemic control than metformin up-titration and was well tolerated in type 2 diabetes mellitus patients inadequately controlled by lowdose metformin monotherapy [15]. a study conducted by manuel gonzalez et al. showed that glimepiridemetformin demonstrated to be more efficacious than glibenclamide metformin at reaching the glycemic control goals with less hypoglycemic events in patients with uncontrolled type 2 diabetes mellitus [16]. in the present study, the average hba1c levels of v-m group patients were 7.25% at the start of the study and 6.75% at the end of the study. the average hba1c levels of g-m patients were 7.50% at the start of the study and 7.15% at the end of the study. the difference between initial i.e., at the start of the study kareenmulla et al efficacy, safety of vildagliptin-metformin versus glimepiride-metformin vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 66 (week 0) and final i.e., at the end of the study (week 12) of both groups were 0.50% and 0.35% respectively. we observed that there was a significant reduction of hba1c levels in v-m group patients than in gm group patients, represented in table 1. table 1: comparison of average hba1c levels (v-m; gm) hba1c levels monitoring group at week 0 at week 12 difference v-m 7.25% 6.75% 0.50% g-m 7.50% 7.15% 0.35% while fbs monitoring, the average value at the initial reading (week 0) was 145 mg/dl and 138mg/dl, 133mg/dl, 125mg/dl on subsequent i.e., 1 st, 2nd, 3rd follow-ups respectively in v-m group patients. the average value at the initial reading (week 0) was 142mg/dl and 137mg/dl, 135mg/dl, 130mg/dl on subsequent follow-ups i.e., 1 st, 2nd, 3 rd follow-ups respectively in g-m group patients. the difference between the initial reading and final follow-up was 20mg/dl in the v-m group and 12mg/dl in g-m group patients. it was evaluated that fbs reduction was significantly higher in v-m group patients than in g-m group patients, tabulated in table 2. table 2: comparison of average fbs levels (v-m; g-m) fbs (mg/dl) monitoring group initial reading 1st follow up 2nd follow up 3rd follow up difference between initial reading and 3rd follow up v-m 145 138 133 125 20 g-m 142 137 135 130 12 the average value of rbs levels at the initial reading (week 0) was 270 mg/dl and 245mg/dl, 210mg/dl, 185mg/dl on subsequent i.e., 1st, 2nd, 3rd follow-ups respectively in the v-m group patients. the average value of rbs levels at the initial reading (week 0) was 250mg/dl and 235mg/dl, 205mg/dl, 195mg/dl on subsequent follow-ups i.e., 1st, 2nd, 3rd follow-ups respectively in g-m group patients. the difference between the initial reading and final follow-up was 85mg/dl in the v-m group and 55mg/dl in the g-m group patients. it was noticed that rbs was notably depleted in v-m group patients than in gm group patients. the details were summarized in table 3. in terms of safety profile, adrs were considered. in the total of 50 patients in the v-m group, 24 adverse drug reactions were noted in 18 patients. the number of adrs with respect to abdominal pain was 8, adrs of muscle tenderness were 6, adrs of weight gain, and diarrhea were 4, and adr of hypoglycemia was 2. table 3: comparison of average rbs levels (v-m; g-m) rbs (mg/dl) monitoring group initial reading 1st follow up 2nd follow up 3rd follow up difference between initial reading and 3rd follow up v-m 270 (mg/dl) 245 (mg/dl) 210 (mg/dl) 185 (mg/dl) 85 (mg/dl) g-m 250 (mg/dl) 235 (mg/dl) 205 (mg/dl) 195 (mg/dl) 55 (mg/dl) in the total of 50 patients in the g-m group, 46 adverse drug reactions were noted in 32 patients. the number of adrs with respect to abdominal pain was 12, adrs of muscle tenderness were 6, adrs of weight gain, and diarrhea were 10, and adrs of hypoglycemia was 8, which were represented in table 4. table 4: comparison of adverse drug reactions (v-m; g-m) adverserug reactions v-m (n=24) g-m (n=46) number of adr’s(n=70) hypoglycemia 2 8 10 weight gain 4 10 14 muscle tenderness 6 6 12 abdominal pain 8 12 20 diarrhea 4 10 14 in a total of 100 diabetic patients who were on the treatment of either vildagliptinmetformin or glimepiridemetformin; 70 adverse drug reactions were experienced by 50 patients. the number of adrs with respect to abdominal pain was 20, adrs of muscle tenderness were 12, adrs of weight gain, diarrhea were 14, and adrs of hypoglycemia wwas10. in comparison, it was found that 24 adverse drug reactions were experienced in the v-m group, while 46 adverse drug reactions were experienced in the g-m group. details were outlined in table 5. on evaluation, it was observed that there was a significant variation between the average hba1c levels at the initial reading and at the end of the study i.e., at the end of 12 weeks in both groups and the greatest reduction was observed in v-m group patients than in g-m group patients. table 5: comparison of overall adverse drug reactions (vm; g-m) regimen number of adrs percentage of adrs v-m 24 36% g-m 46 65% there was a significant reduction in fbs and rbs levels on subsequent follow-ups in each group and a slightly highest reduction was observed in the v-m group than in gm group patients. in safety profile, there was a significant difference between both groups, and most of the adrs were observed in kareenmulla et al efficacy, safety of vildagliptin-metformin versus glimepiride-metformin vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 67 g-m group patients than in v-m group patients. for years, the american diabetes association (ada) has recommended that all people with diabetes aim for a target hemoglobin hba1c level below 7 percent. even more stringent, the american association of clinical endocrinologists (aace) recommends hba1c targets below 6.5 percent. although there's no cure for type 2 diabetes. studies show that it is possible to reverse diabetes through diet changes and weight loss can hold normal blood sugar levels without medication. comparing the efficacy and safety profiles of the metformin-vildagliptin regimen with metforminglimepiride regimen is the main objective of the research study. this research study may contribute to clinical oriental sails in the future. yet, some more studies are needed to make statistically significant conclusions. conclusion clearly, it is proved that a combination of vildagliptin and metformin provided better blood glucose control, compared to that of glimepiride-metformin treatment. on evaluating the adverse drug reactions, treatment with vildagliptinmetformin does not appear to be associated with many adverse events as treatment with glimepiride-metformin. also, the risk of weightgain is lower in v-m group when compared to g-m group. the investigators concluded vildagliptin-metformin regimen is not only safer but also more effective than glimepiride-metformin regimen. although there is no permanent cure for type 2 diabetic patients it is recommended that, people with diabetes should choose a variety of fiber-foods such as legumes, fiber-rich cereals (≥ 8 gm fiber/serving), fruits, vegetables, and whole grain products as they provide vitamins, minerals and other substances important for good health. studies shows that it is possible to reverse diabetes and maintain normal blood sugar levels without medication through diet changes and weight loss. this research study may contribute clinical oriented trails in future. yet, some more studies are needed to make statistically significant conclusions. abbrevatons ada: american diabetes association; adr: adverse drug reaction; anova: analysis of variance; bmd: bone mineral density; bmi: body mass index; cdc: centres for disease control and prevention; chd: coronary heart disease; ci: class interval; cvd: cardio vascular diseases; dm: diabetes mellitus; dme: diabetic macular edema; dpp4: di peptidyl peptidase inhibitor; fda: food and drug administration; fpg: fasting plasma glucose; gcp: good clinical practice; gfr: glomerular filtration rate; ggh: government general hospital; gm: glimepiride – metformin; hba1c: haemoglobin a, c glucohemoglobin; icd: informed consent document; ich: international council of harmonisation; iddm: insulin dependent diabetes mellitus; idf: international diabetes federation; idh: intra dialytic hypotension; iec: institutional ethical committee; igt: impaired glucose tolerance. references 1. nam han cho (chair), david whiting (deputy chair). international diabetes federation. sixth edition, 2013. online version of idf diabetes atlas: www.idf.org/diabetesatlas. 2. chiang jl, kirkman ms, laffel lmb et al. type 1 diabetes through the life span: a position statement of the american diabetes association. diabetes care. 2014; 37: 2034 54. 3. hayashi t, murayama h, shinfuku y, et al. safety and efficacy of vildagliptin: 52-week post marketing surveillance of japanese patients with type 2 diabetes in combination with other oral anti diabetics and insulin. expert opinpharmacother. 2020; 21: 121 30. 4. kalra s, bajaj s, unnikrishnan ag, et al. therapeutic experience of saxagliptin as first add-on after metformin in indian type 2 diabetes patients: a noninterventional, prospective, observational study (ontarget-india). indian j endocr metab. 2019; 23: 312 7. 5. zang l, han y, chen l, et al. comparison of the effectiveness and safety of vildagliptin add-on to metformin versus other oral dual anti diabetes agents in patients with type 2 diabetes: the china prospective diabetes study. diabetes ther. 2019; 10: 1391 405. 6. gullapalli h, desai s. comparison of efficacy and safety of metformin and vildagliptin versus metformin and glimepiride in patients of type 2 diabetes mellitus. natl j physiol pharm pharmacol. 2018; 8: 521 5. 7. yavropoulou mp, pikilidou m, kotsa k, et al. efficacy and tolerability of vildagliptin as first line treatment in patients with diabetes type 2 in an outpatient setting. j diabetes metab disord. 2015; 14: 118. 8. nathan dm. clinical practice. initial management of glycaemia in type 2 diabetes mellitus. n engl j med. 2002; 347: 1342 9. 9. riddle mc. a verdict for glimepiride: effective and not guilty of cardiovascular harm. diabetes care.2019; 42: 2161 3. 10. mathieu c, degrande e. vildagliptin: a new oral treatment for type 2 diabetes mellitus. vasc health risk manag. 2008; 4: 1349 60. 11. hayashi t, murayama h, shinfuku y, et al. safety and efficacy of vildagliptin: 52-week post-marketing surveillance of japanese patients with type 2 diabetes in combination with other oral antidiabetics and insulin. expert opin pharmac other. 2020; 21: 121 30. 12. sarkar bs, mukherjee s, maji m, et al. a prospective observational study to compare the efficacy and adverse effects of glimepiride and vildagliptin added to metformin in type 2 diabetes mellitus. j clin diagn res. 2019; 13: oc09 oc11. 13. cohen cm, davis c, shalev v, et al. effectiveness of vildagliptin as add-on to metformin monotherapy among uncontrolled type 2 diabetes mellitus patients in a real-world setting. j diabetes. 2018; 10: 68 72. 14. li-nong ji, chang-yu pan, ju-ming lu, et al. efficacy and safety of combination therapy with vildagliptin and metformin versus metformin up-titration in chinese patients with type 2 diabetes mellitus: study design and rationale of the vision study. cardiovascular diabetology. 2013; 12: 118. kareenmulla et al efficacy, safety of vildagliptin-metformin versus glimepiride-metformin vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 68 15. hye-soon kim, doo-man kim, bong-soo cha, et al. efficacy of glimepiride-metformin fixed dose combination vs metformin uptitration in type 2 diabetic patients inadequately controlled on low dose metformin monotherapy: a randomized, open label, parallel group, multicenter study in korea. j. diabetes investigation. 2014; 5(6): 701 708. 16. manuel gonzale, guerrero romero, rafael violanteortiz, et al., efficacy of glimepiridemetformin combination versus glibenclamidemetformin in patients with uncontrolled type 2 diabetes mellitus. j of diabetes and its complications. 2009; 23(6): 376379. how to cite this article: bingi p, kareemulla s, himabindu b, khan ds, ramu d, mahammed rafi s. a comparative study on efficacy and safety of vildagliptin-metformin versus glimepiride-metformin in type 2 diabetes mellitus patients. indian j pharm drug studies. 2022: 1(2) 63-68. funding: none conflict of interest: none stated lenin et al. effectiveness of drum stick leaves vol 1 | issue 1 | apr jun 2022 indian j pharm drug studies | 26 original article evaluate the effectiveness of phytochemical, physicochemical and mineral analysis of moringa oleifera (drum stick leaves) santhadani lenin1, sujatha ramasamy1, deivasigamani revathy1 from, department of nutrition and dietetics, n.k.r govt. arts college for women, namakkal, tamil nadu, india correspondence to: santhadani lenin, department of nutrition and dietetics, n.k.r govt. arts college for women, namakkal, india. email: alphaomegabiovision@gmail.com abstract moringa oleifera lam (moringaceae) is one of the most adaptable plants in the world. m. oleifera is used in nature to treat a variety of diseases and is now available without a prescription in the form of an herbal infusion. they are thought to cure a variety of ailments in the native medicine system. the presence or absence of such plant synthesized primary and secondary metabolites determines the values of food and medicine. the aim of this analysis was to assess the phytochemical elements, physicochemical properties, and nutritional values of dried m. oleifera leaf powder using a qualitative test. m. oleiferais used medicinally due to the presence of active phytochemical constituents such as alkaloids, flavonoids, hormones, phenols, and carbohydrates. ash (6.18%) , moisture (70.27%), fiber (20.26%), carbohydrates (40.40%), protein (27.73%), fat (2.24%), tannin (22.16%) , phytates (0.37g/100g), vitamin c (0.86 mg/g), beta-carotene (18.21 mg/100g), and folic acid (0.95 mg/100g) are among the physicochemical properties studied.mineral research that may be involved has been examined, and its wide-ranging activities have been held responsible. moringa leaves have been shown to be a good source of dietary nutraceuticals as well as essential characteristics for potential nutritional and technological applications key words: moringa oleifera lam, phyochemical, physiochemical, mineral, dietary nutaceutical erbal and organic drugs have a wide variety of chemical compositions. this can be so extreme that it can result in therapy failure or poisoning, so it's understandable that different samples of the same natural substance will have drastically different reactions. m. oleiferais a type of vegetable shrub that grows to a height of 5–15 metres and has a 30 cm diameter soft and brittle stem [1]. moringa leaves are round, pinnately tripled, and small round or oval in shape. the fruit is long and angular, with triangle-shaped sides; the drumsticks are 15–45 cm long and contain about 20 seeds [1]. moringa thrives in moist tropics or hot, dry climates, and can thrive in less fertile soils. it is also droughtresistant [2]. moringa is a tropical and subtropical plant that originated on the indian subcontinent and has since spread throughout the world. plant sections that function as cardiac and circulatory stimulants include leaves, roots, seeds, barks, fruits, flowers, and immature pods. they also have antipyretic, antiepileptic, anti-inflammatory, and anti-ulcerative properties [3]. other important plant properties include antispasmodic [4], diuretic [5], antihypertensive [6], cholesterol reduction [7], antioxidant, anti-diabetic, hepato-protective [8], antibacterial and antifungal [9]. phytochemicals are chemical compounds found naturally in plants. they are caused by the plant's colour and organoleptic properties [10]. chemicals that may have biological significance but are not known as important plant nutrients are often referred to as it. while phytochemicals are available as a dietary supplement, the potential health benefits of phytochemicals are derived from the ingestion of the entire plant [11]. natural ingredients, either as pure compounds or as standardized plant extracts, have limitless possibilities for new medicines [12]. some are responsible for colour and other organoleptic effects, such as the deep purple of blueberries and the odour of garlic [13]. chemicals that may have biological significance (e.g., antioxidants) but are not defined as essential nutrients are commonly referred to as "essential nutrients" [14]. appropriate methodologies for accurate diagnosis, standardization, and quality assurance of herbal drugs are critical for the future development of herbal drug pharmacognosis [15]. verifying the origin of a drug, evaluating its worth and purity, and determining the nature of adulteration are all part of the drug evaluation process. phytonutrients found in naturally grown herbs and plants are extremely beneficial to our bodies and overall health. the most common and useful phytonutrients are natural minerals (such as zinc, iron, calcium, copper, and other elements) and vitamins (such as vitamins a, b, c, d, e, pp, and others) [16]. the aim of this study was to see the effectiveness of the phytochemical, physicochemical, and mineral analysis of m. oleifera. h lenin et al. effectiveness of drum stick leaves vol 1 | issue 1 | apr jun 2022 indian j pharm drug studies | 27 materials and methods sample collection and processing: the plant came from the erode district. indoors, the plant was air-dried and powdered with a mortar and pestle. for further research, the powdered sample was placed in an airtight jar. phytochemical screening: normal techniques were used to conduct preliminary phytochemical investigations for secondary metabolites on a powdered sample of m. oleifera leaves [17] [18]. alkaloids, flavonoids, hormones, terpenoids, anthroquinones, phenols, saponins, tannins, carbohydrates, oils, and resins were among the metabolites examined. analysis includes complete ash value [19], moisture content [20], fiber [18], total carbohydrate [21], proteins [22] [23], fat [24], tannin [25], phytates [26], vitamin c [27]. the normal method was used to evaluate iron forms (ii) and (iii) (vogel, 1961). mineral analysis: the mineral analysis was carried out, according to [23]. to avoid detergent absorption, the glassware and polyethylene containers used for examination were washed with tap water, then soaked in 6n hno3 solution overnight and rinsed with ultrapure water several times. precisely weighted (2.0 g) crop specimens were moved to a silica crucible and ashed for 3 hours at 450 c in a muffle furnace, followed by the application of 5 ml of 6 m hcl to the crucible. the acid solution containing the crucible was then digested on a hot plate to obtain a clean solution. in 0.1 m hno3 solution, the final residue was dissolved and made up to 50 ml. flame and graphite furnace atomic absorption spectrophotometers are used to examine the plant specimens (aa 6300, shimadzo, japan). metal content has been determined using an airacetylene burn. in flame mode, the instrument was operated under the following conditions: acetylene 1.8 l/min, air 15 l/min, inert argon gas flow and temperature parameters, as defined by the manufacturer. the absorption wavelength is given for determining the material, along with its linear working range and correlation coefficient of calibration graphs. the standard deviation value from triplicate measurements is used to round off the results. result and discussion the sample yielded significant diagnostic characters that could be useful in assessing the validity and detecting crude drug adulteration. herbal medicine is thought to have been used to cure many illnesses in the history of human medicine. herbal drugs have the advantage of having fewer side effects over time and being safe to use. they are also less expensive and more readily available than formulated medications [28]. the dried flower of m. oleifera l was analyzed for phytochemicals, physicochemicals, and minerals. phytochemical analysis: the qualitative phytochemical study of m. oleifera leaves was carried out. active phytochemicals such as alkaloids, flavonoids, steroids, phenols and carbohydrates. these active phytoconstituents are present in aqueous extract of m. oleifera. the presence of alkaloid shows cream color precipitate, flavonoids show reddish-brown colour precipitate, steroids show green colour formation, phenols show deep blue to black colour formation and carbohydrates show blue colour precipitate. preliminary qualitative screening is helpful in detecting bioactive concepts and could lead to drug development and manufacturing [29]. alkaloids are plant compounds that act as repellents for predators and insects. alkaloids have been shown to have microbiocidal properties, and their key anti-diarrheal activity is likely due to their effects on the small intestine, as well as anti-hypertensive properties [30]. several alkaloids are effective in the treatment of hiv infection and aids-related intestinal infections [12] (table 1) table 1 phytochemical analysis of m. oleifera leave phytochemicals observations extracts distilled water alkaloids mayer’s test wagner’s test cream colour reddish brown solution/ precipitate present flavonoids lead acetate test h2so4 test yellow orange reddish brown / orange colour precipitate present steroids liebermannburchard test violet to blue or green colour formation present terpenoids salkowski test reddish brown precipitate absent arthroquinone borntrager’s test pink colour absent phenols ferric chloride test lead acetate test deep blue to black colour formation white precipitate present saponin stable persistent absent tannin brownish green / blue black absent carbohydrates yellow/brownish /blue /green color present oils & resins filter paper method absent flavonoids are powerful water-soluble antioxidants and free radical scavengers that protect cells from oxidative damage and have anti-cancer properties [31]. flavonoids, which contain hydroxyl groups, are thought to be responsible for the radical scavenging effects of most plants. phytochemicals such as tannins, saponins, and steroid-glycosides were found to be in relatively low concentrations. tannins may be a good way to help your kidneys [19]. tannins have also been shown to have antiviral, antibacterial, and anti-parasitic properties [10]. as an adjuvant, saponins are used in the production of vaccines. physicochemical analysis: the importance of physicochemical research is that it aids in the identification of different constituents or groups of constituents that often lead to the lenin et al. effectiveness of drum stick leaves vol 1 | issue 1 | apr jun 2022 indian j pharm drug studies | 28 discovery of a connection between structure-activity and the drug's probable mechanism of action. the physicochemical analysis revealed that m. oleifera leaf powder was manufactured to a high purity level and of good quality, as determined by the who [32]. ash (6.18 percent), moisture (70.27%), fiber (20.26%), carbohydrates (40.40 %), protein (27.73%), fat (2.24%), tannin (22.16%), phytates (0.37g/100g), vitamin c (0.86 mg/g), betacarotene (18.21 mg/100g), and folic acid (0.95 mg/100g) are all examined as a consequence of the physicochemical evaluation is investigated. in order to detect adulterants and improper drug handling, it is important to decide the physicochemical parameter. since mineral matter can cause a pharmacological effect [33], ash values are important quantitative standards [34]. they are also a criterion for evaluating the identity and purity of crude drugs, particularly in the form of powder [35]. in addition to lowering cholesterol and triglycerides, dietary fibres protect against cancer and digestive disorders. more information regarding a crude drug's total ash also represents the care taken in preserving products, as well as the purity of both crude and prepared drugs [35]. determination of iron forms: iron is needed for the development of hemoglobin, the normal functioning of the central nervous system, and the oxidation of starch, protein, and fat. the amount of iron in both dried and fresh leaves is determined. fresh leaves have a ferrous content of 2.57 0.02 and a ferric content of 1.23 ± 0.03. in dry leaves, ferrous is 2.09 ± 0.04 and ferric is 0.96 ± 0.02. in comparison to both, the test iron form for fresh leaves is higher. iron is essential for haemopoiesis, infection control, and cell-mediated immunity [36]. mineral analysis: mineral elements are essential for the proper functioning of all cells because they serve as structural tissue components, as well as components of body fluid and vital enzymes in major metabolic pathways. heavy metal contamination of medicinal plant materials can cause chronic or acute poisoning. as a result, it has become important to ensure the heavy metal content of all starting materials, as well as other inorganic elements that are needed. the presence of heavy metals was determined using elemental analysis, and the results for m. oleifera leaf powder in ppm are iron (fe) 0.548, copper (cu) 0.075, manganese (mn) 0.038, zinc (zn) 0.138, nickel (ni) 0.133, cobalt (co) 0.547, lead (pb) 0.486, aluminum (al) 1.984, vanadium (v) 1.096, chromium (cr) 0.048, molybdenum (mo) 0.137, mercury (hg) 0.145, arsenic (as) 0.052, cadmium (cd) 0.098.the most common nutritional deficiency has been described as iron deficiency anemia, which is estimated to affect more than one billion people worldwide [37]. reduced work capacity, behavioural and cognitive function impairments, and decreased infection tolerance are all consequences of iron deficiency [27]. it's also essential for cell growth and repair, bone development, and kidney function (table 2). table 2 mineral analysis for m. oleifera leaves elements analyzed quantity (in ppm) iron (fe) 0.548 copper (cu) 0.075 manganese (mn) 0.038 zinc (zn) 0.138 nickel (ni) 0.133 cobalt (co) 0.547 lead (pb) 0.486 aluminum (al) 1.984 vanadium (v) 1.096 chromium (cr) 0.048 molybdenum (mo) 0.137 mercury (hg) 0.145 arsenic (as) 0.052 cadmium (cd) 0.098 this is essential for maintaining the body's acidalkaline balance [38]. minerals such as cadmium, nickel, and lead are thought to be found in trace quantities. cadmium and lead in high concentrations are unsuitable for bodywork and unappealing. the study lays the groundwork for further isolation and characterization of the bioactive constituents present in the leaves of this plant, thanks to its therapeutic properties. magnesium is a part of chlorophyll and is an important mineral element in the treatment of ischemic heart disease and bone calcium metabolism [39]. zinc is a part of more than 50 enzymes in the body that play a role in immune system function [40]. an estimated 20% of the world's population is at risk of insufficient zinc intake [41]. conclusion the presence of phytochemicals, physico-chemicals, minerals and iron forms in leaves has been demonstrated in this research, which supports both their nutritional and ethno-medicinal benefits for human health. alkaloids, flavonoids, phenols, steroids and carbohydrates were present in high concentrations in m. oleifera leaves. the leaves still had a good amount of ash, moisture, fiber, carbohydrates, protein, fat, tannin, phytates, vitamin c, betacarotene and folic acid. in moringa oleifera leaves have a high nutritional value and are high in the mineral portion needed for good health. the sample's physicochemical characters were analyzed for standardization so that future research could be conducted on samples that were found to be equivalent based on these characters, ensuring the sample's scientific analysis. references 1. roloff a, weisgerber h, lang u, et al. moringa oleifera lam., 1785. sea. 2009; 10(10). 2. anwar f, latif s, ashraf m, et al. moringa oleifera: a food plant with multiple medicinal uses. phytother res. 2007; 21: 17-25. 3. pal sk., mukherjee, pk, saha bp. studies on the antiulcer activity of m. oleifera leaf extract on gastric ulcer models in rats. phytother res.1995; 9: 463-465. lenin et al. effectiveness of drum stick leaves vol 1 | issue 1 | apr 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component of leaves stalks and stems of sweet potatoes (ipomeabatatas poir). food chem.2000; 68: 359-367 38. okaka jc, akobundu ent, okaka can. food and human nutrition, an integrated approach. o. j. c. academic pub. enugu, nigeria.2006; 39. hotz c, brown kh. international zinc nutrition consultative group (izincg) technical document no. 1. assessment of the risk of zinc deficiency in populations and options for its control. food nutr bull. 2004; 25: s94-s203. how to cite this article: lenin s, ramasamy s, revathy d. evaluate the effectiveness of phytochemical, physicochemical and mineral analysis of moringa oleifera (drum stick leaves). indian j pharm drug stud. 2022; 1(1): 26-29. funding: none; conflict of interest: none stated http://www.depkes.go.id/article/view/2053/kemitraanpemerintah-dan-swasta-dalampengendalian-diabetesmelitus-di-indonesia-.html http://www.depkes.go.id/article/view/2053/kemitraanpemerintah-dan-swasta-dalampengendalian-diabetesmelitus-di-indonesia-.html http://www.depkes.go.id/article/view/2053/kemitraanpemerintah-dan-swasta-dalampengendalian-diabetesmelitus-di-indonesia-.html soares et al hsa-hctz interactions based on glucose concentrations vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 56 original article effect of glucose concentrations on the hsa-hydrochlorothiazide interaction – a study using circular dichroism and molecular docking marilia amável gomes soares1, otávio augusto chaves 2, antônio augusto fidalgo neto2, dilson silva1, dari cesarin-sobrinho3, célia martins cortez1 from,1postgraduation in medical sciences, rio de janeiro state university, brazil, 2institute of chemistry, federal rural university of rio de janeiro correspondence to: celia martins cortez, rio de janeiro state university, postgraduation in medical sciences, av. prof. manuel de abreu, 444. cep 20551-170 rio de janeiro, rj brazil. email: ccortezs@ime.uerj.br abstract diabetes mellitus is a chronic disease characterized by hyperglycemia and is accompanied by disturbances in the metabolism of carbohydrates, lipids, and proteins. it requires a lot of attention, as its complications l ead to serious health damage and even death. hydrochlorothiazide (hctz) is a diuretic that is well-tolerated in antihypertensive treatments, it reduces blood volume and, therefore, decreases peripheral vascular resistance. however, there are some comments in the literature on the possibility that high blood concentrations of blood glucose (glu) induce tolerance and cause adverse metabolic effects, which might generate clinical complications. this study aimed to present results by using circular dichroism (f ar and near uv) to study the interaction between human serum albumin (hsa) and hydrochlorothiazide in the absence and presence of glucose at 37ºc. methodsexperimental data were correlated for theoretical analysis through molecular docking simulations. th e ability of hydrochlorothiazide to bind with hsa was verified in normoglycemic (containing glucose 80 mg/dl) and hyperglycemic (containing glucose 320 mg/dl) solutions. far-uv analysis showed that hctz caused little perturbation on the secondary structure of albumin for the three hsa: hctz ratios (1:12, 1:24, and 1:48). we observed a strong disturbance in the secondary structure of albumin was for glu in hyperglycemic concentration, with a maximum reduction of 12.66% at 222 nm. an increase in the blood glu level causes a functional perturbation in the binding of hctz with hsa. hctzhsa binding is spontaneous and causes weak perturbation on the secondary and tertiary structure of albumin, however, at high glu concentration, the perturbation increases which makes the albumin structure unstable. key words: diabetes mellitus, glucose, hydrochlorothiazide, human serum albumin, cd-spectroscopy, molecular docking n the study performed by soares. et al we saw the interaction of hsa with hydrochlorothiazide (hctz) in both the absence and presence of glucose using uv-vis absorption and spectro fluorescence spectroscopy at 37 ºc. the study also demonstrated competitive binding using warfarin and digitoxin, markers for sudlow i and sudlow iii sites, respectively. as this drug was used in the treatment of hypertensive diabetic patients, we were interested in verifying if blood glucose (glu) concentration could interfere with this drug-protein interaction. for this, hsa solutions containing glu 80 mg/dl and 320 mg/dl were used named normoglycemic and hyperglycemic solutions, respectively. diabetes mellitus (dm) is a chronic disease characterized by high blood sugar levels, resulting from reduced or non-insulin production and/or reduced tissue sensitivity [2]. hyperglycemia is accompanied by disturbances in the metabolism of carbohydrates, lipids, and proteins. it is a disease that requires a lot of attention, as its complications can cause serious health damage and even death, especially among young people [3] [4]. by its diuretic effect, hctz reduces blood volume and, therefore, decreases peripheral vascular resistance, being in general a well-tolerated drug in antihypertensive treatments [5] [6] [7]. however, there are some comments in the literature on the possibility that high blood concentrations of glu induce tolerance and cause adverse metabolic effects, which might generate clinical complications [8] [9] [10] [11]. g however, some studies disagree with these opinions [12], suggesting longterm use and high doses (>50 mg/day) as factors that may be behind the adverse effects of hctz [13] [14]. the comparison of our results of the experiments using spectrofluorescence and uv-vis spectroscopy showed that the microenvironment disturbance around aromatic amino acid residues of hsa by its interaction with hctz was considerably greater in hyperglycemic solution than in normoglycemic solution [15]. the fluorescence quenching experiments showed that this drug binds to albumin forming a complex in the absence that might generate a conformational change in albumin and a reduction in the number of sites available for drug binding in the iia subdomain. i soares et al hsa-hctz interactions based on glucose concentrations vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 57 as the biodistribution and effects of drugs depend on their ability to bind to plasma proteins, especially albumin, which is the main drug transporter, it becomes relevant to enlarge the study on disturbances caused by elevated blood glucose in the hsa structure and in the hctz-hsa interaction. circular dichroism (cd) spectroscopy is a widespread technique for checking perturbation on the secondary (far-uv) and tertiary (near-uv) structures of macromolecules upon ligand binding [15]. it is known that hsa exhibits two negative bands in the far-uv: one at 208 nm (π-π* transition) and the other at 222 nm (n-π* transition), which are characteristic of the protein's structural units [16]. complementing spectroscopic analysis, which allows obtaining experimental data on the binding affinity of a ligand with hsa and theoretical analysis using molecular docking explains the binding ability at the molecular level, which helps in understanding the experimental data [17] [18]. aiming to complement the results obtained in the previous work [1], we studied the interaction of hsa-hctz in the presence and absence of glu through the circular dichroism technique (far and near uv) also at 37 ºc. current and previous data were correlated to theoretical analysis through molecular docking simulations, including the evaluation of the main glu conformation (open or cyclic) in the protein binding pocket. material and methods materials and equipment hctz (6-chloro-3-3, 4dihydro2h-1,2,4-benzothiadiazole-7-sulfonamide1,1dioxide), dglucose, hsa, warfarin, digitoxin as well as phosphate buffer solution (pbs) were purchased from sigma-aldrich chemical company, st. louis, usa. there was no further purification. methanol (spectroscopic grade) was obtained from vetec, química fina ltda, rio de janeiro, brazil. a millipore milli-q system (merck kgaa, darmstadt, germany) was used to obtain purified water.circular dichroism (cd) spectra were measured on a jasco j-815 spectrophotometer coupled to a jasco pfd425s15f thermostatic cuvette door with 0.1 ºc accuracy, with 3 channels, one for steady-state fluorescence, another for the cd and the third for the uv-vis (jasco easton, md, usa) [19]. circular dichroism (cd) measurements the circular dichroism (cd) analysis was performed using 3.0 ml of hsa solution (10-6 m, in pbs, ph=7.4) in the absence and presence of hctz (1:12, 1:24, and 1:48 hsa-hctz solution, in pbs). the effect of the glu concentration on hsa-hctz cd spectra were evaluated in solutions containing 80 mg/dl and 320 mg/dl glu. all cd spectra were recorded to both far-uv (200-250 nm, for secondary structure) and near-uv (260-320 nm, for tertiary structure) [20, 21]. the intensity of the signals in the cd spectra was expressed as molar residue ellipticity (mre, in deg cm2 dmol-1), defined according to the following equation [21]. , (3) where θ, n, l, and cp are the observed ellipticity (mdeg), a number of amino acid residues (585 to hsa) [22], optical cuvette length (1.0 cm), and molar hsa concentration (10-6 m), respectively. in order to calculate the quantitative loss of the helical structure of the protein due to hctz binding, the mre values at 208 and 222 nm were obtained according to the equations [23]. , (4) , (5) where 𝑀𝑅𝐸208 and 𝑀𝑅𝐸222 are the significant ellipticity molar residues (deg.cm2/dmol) at 208 nm and 222 nm, respectively. molecular docking simulations the crystallographic structure of hsa was obtained from protein data bank (pdb) with access code 1n5u [22l]. the chemical structure of hctz and glu (open and cyclic forms) were built and minimized in terms of energy using the functional density theory (dft) by becke-3-lee yang parr (b3lyp) with the standard base set 631g*, available in the spartan'14 software (wavefunction, inc.) [24]. figure 1 far-uv cd spectra for hsa-hctz (a) and hsa-glucose (b) at 37 ºc, respectively. near-uv cd spectra for hsahctz (c) and hsa-glucose (d) at 37 ºc, respectively. [hsa]= 1.00 × 10-6 m and [glucose] = 80 and 320 mg/dl soares et al hsa-hctz interactions based on glucose concentrations vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 58 figure 2 far-uv cd spectra for hsa-hctz in the presence of glucose, 80 mg/dl (a) and 320 mg/dl (b) at 37 ºc. near-uv cd spectra for hsa-hctz in the presence of glucose, 80 mg/dl (c) and 320 mg/dl (d) at 37 ºc. [hsa] = 1.00 × 10-6 m and [glucose] = 80 and 320 mg/dl. molecular docking studies were performed using gold 5.7 software (cambridge crystallographic data centre ccdc) [25]. hydrogen atoms were added to the albumin structure according to the data inferred by the program on the ionization and tautomeric states. it is known that hsa presents three main binding pockets for different ligands (sites i, ii, and iii, which are identified through trp214, tyr-411, and tyr-161 residues [23] [26] [27]. in order to identify the main amino acid residues involved in the interaction hsa-hctz, as well as to evaluate the main possible binding site, a spherical 10 å radius around trp-214, tyr-411, and tyr-161 residues was defined, and molecular docking simulations were carried out [28]. furthermore, the theoretical evaluation of the glu effect (in the open and cyclic forms) on hsa-hctz binding was simulated, as well as the docking calculations were carried out to hctz and glu (in both forms) at the same time under the same spherical radius described above. a more positive docking score value indicates better interaction, due to the negative sum of a series of energy terms involved in the protein binding interaction process. the number of genetic operations (crossing, migration, and mutation) in each docking run was defined as 100,000. the scoring function used was “chemplp”, which is the standard function of the gold 5.7 software. the figure of the best docking pose for each case was generated with pymol delano scientific llc software [29]. results and discussion three different cases were analyzed by cd measurements: (i) evaluation of the albumin structure upon hctz binding, (ii) evaluation of the albumin structure upon glu binding, and (iii) glu effect on hsa-hctz cd spectra. figure 1 a shows that in the far-uv, hctz caused little perturbation on the secondary structure of albumin for the three investigated hsa: hctz ratios (1:12, 1:24, and 1:48). the content of α-helix (%) at 208 and 222 nm (table 1) confirms that hctz caused little perturbation in the secondary structure of albumin, since, for the 1:48 ratio, the maximum reductions of 3.47% and 3.79% at 208 and 222 nm, respectively, were found [30]. in the absence of hctz, glu at normoglycemic concentration also caused a weak disturbance, with a maximum reduction of 3.45% at 222 nm. but a relatively strong disturbance in the secondary structure of albumin was observed for glu in hyperglycemic concentration, with a maximum reduction of 12.66% at 222 nm (figure 1b and table 2). thus, high concentrations of glu in diabetic patients can significantly disrupt the secondary structure of albumin, possibly destabilizing the interaction forces (e.g., hydrogen bonding) responsible for protein stability [31]. soares et al hsa-hctz interactions based on glucose concentrations vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 59 table 1 α-helix % for hsa without and in the presence of hctz in pbs solution at 37 ºc ratio hsahctz hsahctz (208 nm) hsahctz (222 nm) reduction % (208 nm) reduction % (222 nm) 1:0 54.50 51.49 1:12 54.04 50.40 0.84 2.11 1:24 52.92 50.27 2.90 2.37 1:48 52.61 49.54 3.47 3.79 in addition, the near-uv cd spectra for hsahctz reveal weak signals in the range 290-305 nm, which associates with the amino acid residue trp-214 (figure 1c). this indicates that the binding of hctz to the subdomain iia, which is located at the primary site for this drug in hsa [1], does not significantly perturb the protein tertiary structure, as demonstrated by the slight increase in cd signals. these results suggest that there is an increase in hydrophilicity in the trp-214 environment, the same trend observed in the results of steady-state fluorescence quenching [32]. verifying the effect of glu in pure hsa solution in figure 1d, we observed that the cd signals in the trp-214 region show a significant decrease, mainly in the hyperglycemic condition, indicating an increase in the tertiary structure of the protein by an increase in hydrophobicity. this may be due to a collapse transition which can directly impact the capacity of ligands binding to hsa [33]. these results are relevant since they are suggesting that high blood glu levels cause conformational changes in the albumin structure, which can decrease the ability to bind not only to hctz but also to other drugs [34]. table 2 α-helix % for hsa without and in the presence of glucose in pbs solution at 37 ºc. [glucose] hsa-glucose (208 nm) hsa-glucose (222 nm) reduction % (208 nm) reduction % (222 nm) 0 55.22 52.12 80 mg/dl 52.68 50.32 4.60 3.45 320 mg/dl 47.26 45.52 14.42 12.66 table 3 α-helix % for hsa-hctz in the presence of glucose (80 and 320 mg/dl) in pbs solution at 37 ºc 80 mg/dl glucose 320 mg/dl glucose ratio hsa-hctz 208 nm 222 nm reduction % (222 nm) 208 nm (222 nm) reductio n % (222 nm) 1:0 60.5 0 58.6 9 61.4 6 58.9 1 1:12 58.7 3 56.0 6 4.48 54.0 6 51.5 2 12.54 1:24 58.9 1 55.6 6 5.16 51.9 0 51.0 7 13.30 1:48 59.4 0 54.8 8 6.49 53.2 5 50.7 9 13.78 the same trend was also observed in the region of the spectrum associated with phe–255 (at 270 nm) and tyr–275 (at 282 nm) residues. the same kind of analysis was conducted for hsahctz in the solutions containing glu 80 and 320 mg/dl. figures 2a and 2b indicate moderate and strong perturbations in the secondary structure of hsa for normoglycemic and hyperglycemic conditions, respectively [35]. the α-helix content at 222 nm (table 4) clearly shows a higher tendency of perturbation on the secondary structure upon hctz addition in the presence than in the absence of glu (for hsa: hctz = 1:4, maximum reduction of 6.49% and 13.78% at 222 nm, in 80 and 320 mg/dl, respectively). these results are related to spectrofluorimetry results that suggested a conformational change in the hsa structure caused by glu in hyperglycemic concentration as responsible for the marked decrease in the binding capacity of hctz to has [1]. table 4 α-helix % for hsa-hctz in the presence of glucose (80 and 320 mg/dl) in pbs solution at 37 ºc. 80 mg/dl glucose 320 mg/dl glucose ratio hsa-hctz 208 nm 222 nm reduction % (222 nm) 208 nm 222 nm reduction % (222 nm) 1:0 60.5 0 58.6 9 61.4 6 58.9 1 1:12 58.7 3 56.0 6 4.48 54.0 6 51.5 2 12.54 1:24 58.9 1 55.6 6 5.16 51.9 0 51.0 7 13.30 1:48 59.4 0 54.8 8 6.49 53.2 5 50.7 9 13.78 for the near-uv cd spectra of hsa-hctz in the presence of glu, unusual behavior can be seen (figures 2c and 2d). in the hyperglycemic condition, the tertiary contacts decreased in phe and tyr regions, due to the decreased αhelical content in the glu concentration range under study, which may be responsible for the structure loss [32]. but an opposite effect is found in the trp-214 region (290-305 nm). overall, the presence of glu destabilized the protein structure, and this destabilizing effect of glu becomes more evident with the increase in the drug concentration. theoretical evaluation of the binding ability to identify the possible main binding sites (sites i, ii, or iii, located in subdomain iia, iiia, and ib, respectively) [36], as well as the amino acid residues involved in the hsahctz interaction, and the impact of glu addition (in open and cyclic forms) in the binding ability, molecular docking calculations were performed with gold 5.7 software. soares et al hsa-hctz interactions based on glucose concentrations vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 60 from literature, it is known that in a plasma medium, dglucose is found essentially as a mixture of two anomers (roughly one-third of α-d-glucopyranose and about twothirds as β-d glucopyranose), with practically no furanose forms [37] and the interconversion between open-chain aldehyde and ring forms is fast and affected by the medium conditions, being energetically favorable in the cyclic than the open form (e.g. for glu, about 99.98% is cyclic in water) [38]. thus, all molecular docking calculations for glu were carried out only assuming β-d-glucose in both cyclic and open forms. table 5 shows the docking score values (dimensionless) for the best docking pose at the three main binding sites of the hsa structure available for interaction hsa-hctz, hsa-glu, and hsa-glu.hctz. table 5 docking score values (dimensionless) for the interaction of hsa with hctz and glucose at sites i-iii. sample site i site ii site iii hsa-hctz 58.3 51.1 40.8 hsa-open-glu 56.6 43.5 43.6 hsa-cyclic-glu 47.6 41.8 39.6 hsa-open-glu.hctz 64.6 41.2 50.4 hsa-cyclic-glu.hctz 49.9 39.0 41.8 the site i had the highest docking score value, the results of molecular docking suggested the subdomain iia as the main binding site for hsa-hctz, hsa-open-glu, and hsacyclic-glu, confirming results from spectrofluorimetric [1]. although glu in its open form is in a smaller proportion than its cyclic form, the results of molecular docking suggested a higher docking score for the open than for the cyclic form (56.6 and 47.6, respectively), probably due to the greater flexibility of the open-glu to adapt to the protein-binding pocket. in addition, if hctz and glu simultaneously bind to the same protein pocket, the results of molecular docking also suggested site i as the main region for this interaction. furthermore, according to the theoretical analysis, the interaction hsaopenglu.hctz is more favorable than hsa-open-glu and hsa-hctz (docking score values: 64.6, 56.6, and 58.3, respectively). on the other hand, cyclic-glu showed the opposite trend, decreasing the binding capacity of hctz to the same binding pocket. experimental data also indicated a negative effect of glu on the hsa-hctz interaction [1], which may probably be associated with the fact that glu is in its pyranose form. according to the molecular docking analysis (figure 3 and table 3), hydrogen bonding and van der waals interactions are the main intermolecular forces involved in hsa-hctz, hsa-glu, and hsa-glu.hctz interactions (for both open and cyclic forms of glu). as an example, the hydrogen atom of the -nh3+ group of lys194, lys-198, and lys-443 residues is a potential donor for hydrogen bonding with the sulfonamide group of hctz at distances of 2.10, 3.50, and 3.70 å, respectively. on the other hand, the oxygen atom of gln-220, glu-449, and asp450 residues is a potential acceptor for hydrogen bonding with the nh-portion of the hctz structure at distances of 2.10, 1.60, and 1.60 å, respectively. in addition, the amino acid residue arg221 also interacts with the sulfonamide group of hctz via hydrogen bonding at a distance of 1.80 å. finally, van der waals interactions between hctz and amino acid residues trp-214 and val-342 at distances of 3.10 and 2.80 å, respectively, have also been observed. figure 3 (a) best docking poses for the interaction of hsa-hctz at sites i, ii, and iii. the zoom representation shows the main amino acid residues involved in the interaction hsa-hctz at a site i. best docking pose for the interaction between hsaglucose (b) and hsa-glu.hctz (c), respectively for glucose in the open form at a site i. best docking pose for the interaction between hsa-glucose (d) and hsa-glu.hctz (e), respectively for glucose in the cyclic form at the site i. amino acid residues, hctz, and glucose in the open and cyclic forms are in cyan, purple, beige and orange colors, respectively. hydrogen, oxygen, nitrogen, chloro, and sulfur are in white, red, dark blue, green, and yellow, respectively. soares et al hsa-hctz interactions based on glucose concentrations vol 1 | issue 2 | jul sep 2022 indian j pharm drug studies | 61 conclusion circular dichroism confirms the decrease in the binding ability of hctz to albumin with an increase in glu concentration due to the perturbation of the albumin structure. the binding hctz-hsa is spontaneous and causes weak perturbation on the secondary and tertiary structure of albumin, however, at high glu concentration, it was observed an increase of perturbation on these structures increases, conducting the albumin structure to an instability condition and difficulty the bind of hctz to the protein binding pocket. there is just one primary binding site for hctz in subdomain iia (the site i), and in this binding pocket of hsa, hydrogen bonding and van der waals interactions as the key forces for the association. overall, the experimental and theoretical results suggest that the increase in the blood glu level can cause functional perturbation in the 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its analyses and the influence of diet: are routine glycemia measurements reliable enough? j endocrinol metabol. 2019; 9: 63 70. 39. kuriyan j, konforti b, wemmer d. the molecules of life. 1st ed., taylor & francis group, 2013. how to cite this article: marilia amável gomes soares, otávio augusto chaves, antônio augusto fidalgo neto, dilson silva, dari cesarin-sobrinho, célia martins cortez. effect of glucose concentrations on the hsahydrochlorothiazide interaction – a study using circular dichroism and molecular docking. indian j pharm drug studies. 2022: 1(2) 56-62. funding: none conflict of interest: none stated mandumpala & kumar energy drinks and long term cardiovascular risks vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 88 review article potential effects of excessive energy drinks consumption in young people on long-term cardiovascular risks akhil kumar1, janice jacson mandumpala2 from, 1,2pharm d intern, department of pharmacy practice, 1jayamukhi college of pharmacy, telangana, 2nirmala college of pharmacy, muvattupuzha, kerala, india. correspondence to: janice jacson mandumpala, pharm d intern, department of pharmacy practice, nirmala college of pharmacy, muvattupuzha, kerala, india, pin – 686661 email: janice.jacson@gmail.com abstract energy drinks have been recently associated with adverse outcomes. one of the prominent issues being considered is the link between the risk of developing cardiovascular diseases and energy drink consumption. the increasing concentration of caffeine accompanied by sugars, taurine, etc. are mainly responsible for these risks. in the long term, the children and the youth are vulnerable to the dangers of energy drink consumption. attention needs to be given to the adverse cardiovascular effects in this population. regulatory actions and future restrictions on the sale and consumption of these drinks can bring about positive attitudes in the minds of people and influence their patterns of energy drink consumption. this review gives a comprehensive picture of the various aspects related to energy drinks consumption, cardiovascular risks, risk in the younger population and regulatory actions to be implemented. keywords: energy drinks, cardiovascular diseases, risk, youth literature review is a critical analysis of academic publications and scientific articles in order to comprehend theories and thoughts regarding a specific issue. the goal of the literature review is to explore different literature pertaining to a subject, collect and synthesise information, conduct a critical analysis of the obtained data to discover recent knowledge and gaps, and summarize the literature [1]. in this review of literature, the aim of the researcher is to collect and synthesize information from various secondary resources about the adverse effects of consuming large amounts of energy drinks among the young generations and shed light on its long-term cardiovascular risk potential. energy drinks appeared in europe and asia in the 1960s. the introduction of 'red bull' in austria in 1987 and in the united states in 1997 resulted in a massive spike in the sale of high caffeine content energy drinks. as they have grown in popularity, their consumption has risen at an exponential rate. energy drinks have risen in popularity as evidenced by their increased consumption patterns in the younger population. young males primarily consume energy drinks for, psychoactive, stimulant and performance-enhancing drug effects [2, 3]. there are no specific restrictions or limitations on the sale and consumption of energy drinks making them accessible to all age groups. though there are extensive studies suggesting a positive association between consumption of energy drinks and improved athletic performance there are cardiovascular risks associated with it. young, caffeine-deficient or caffeinesensitive pregnant women, competitive athletes, and people with underlying cardiovascular disease are the most vulnerable to these risks [4]. energy drinks role of ingredients: caffeine and taurine are the two most common stimulant components in energy drinks. caffeine has always been attributed to the majority of the adverse consequences associated with energy drink intake. caffeine is found in energy drinks in quantities ranging from 70 to 200 mg per 500 ml can. it stimulates the sympathetic system indirectly by binding to the adenosine receptor/gprotein on the surfaces of cardiac cells. this results in an increase in circulating noradrenaline and adrenaline levels. noradrenaline raises blood pressure and heart rate, stimulates the release of glucose from energy stores and improves blood supply to skeletal muscle. the positive inotropic and chronotropic effects on the cardiovascular system are due to these related mechanisms. caffeine in large quantities, especially in younger people, can disturb nervous system functioning, just as high sugar content makes an individual prone to overweight, obesity, dental erosion, and tooth decay. caffeine's effects are highly dependent on the dose consumed, and individual responses are quite variable, a dose of a mandumpala & kumar energy drinks and long term cardiovascular risks vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 89 50-100 mg can be stimulating, while a dose of 500 mg can lead to serious health issues. furthermore, athletes consuming energy drinks prior to extended physical activity should be warned about caffeine's diuretic effect that can exacerbate dehydration caused by sweat [5]. taurine is a sulfur-containing amino acid that influences a variety of biological and physiological processes in the human body, including important cardiovascular effects, platelet aggregation, and central nervous system neuromodulation. animal studies have suggested that taurine can lengthen the qt interval in a dose-dependent manner. sugars in energy drinks can also affect the cardiovascular system as consuming dietary calories is known to cause an increase in heart rate, cardiac output, and pulmonary ventilation rate. sucrose, glucose and high fructose corn syrup are easily absorbed sugars that are available in large quantities in energy drinks, which are the sources of ‘rapid boosters’. ginkgo biloba, guarana, ginseng, theophylline vitamins, l-carnitine, etc. are also among the other constituents present in variable concentrations in energy drinks [6, 7]. cardiovascular effects of energy drinks myocardial infarction, aortic dissection, arrhythmias, qt prolongation, and sudden death are the cardiovascular adverse events linked to caffeinated energy drink use and the most common adverse being arrhythmias hypertensive patients and hypertension-risk groups, such as patients with kidney failure, morbidly obese individuals, patients with valvular heart disease, pregnant women, and patients with hypertension-related complications or congenital or acquired vascular abnormalities, can face cardiovascular risk from energy drink consumption [8]. risk of supraventricular and ventricular arrhythmias: caffeine promotes cardiac adenosine, which inhibits cardiac fibroblast formation in vitro and hence hinders remodelling after myocardial infarction, which may be a contributing factor in the development of atrial fibrillation. caffeine is pro arrhythmic at very high doses because it increases intracellular calcium, which facilitates atrial automaticity and post depolarization-induced triggered activity. energy drinks have also been linked to ventricular arrhythmias and sudden cardiac death in some cases [9–11]. it is recommended that healthy people restrict their energy drink use to one per day. mixing energy drinks with alcohol is also a potential source of threat as it can lead to synergistic proarrhythmic effects [12]. thus, amplifying the arrhythmic activity and increasing the risk of adverse cardiovascular events. risk of prolonged qt interval: ingestion of caffeine poses a risk of qt interval prolongation. other energy drink constituents, like ginseng, have been associated with qt prolongation in a young patients, leading to torsades de pointes [13]. ephedra, have been linked to a variety of cardiovascular issues, prompting its removal from the market [14]. risk of myocardial ischaemia or infarction: evidence suggests a risk of myocardial infarction in energy drink consumers [15]. the plausible explanation for this adverse event is the increase in mean arterial blood pressure, increase in platelet aggregation and impairment in endothelial function after consumption of energy drinks [16]. they all contribute to the development of long-term hypertension, increased coagulability, sudden cardiac death, cumulative vascular load, dyslipidemia, atherosclerosis and other life-threatening conditions. other risks: the other uncommon cardiovascular risks associated with energy drink consumption are aortic dissection and dilated cardiomyopathy. caffeine-induced elevated blood pressure is one likely explanation for energy drinks-related aortic dissection. future studies linking energy drinks consumption and aortic dissection are needed [17]. energy drinks and the younger population: higher occurrence of energy drinks associated with adverse events in children and adolescents is attributed to their relative caffeine naivety and elevated levels of caffeine in their system. as per the recommendation of the american academy of pediatrics and the national federation of state high school associations, consumption of energy drinks is prohibited in children and adolescents, its consumption should be restricted before, after or during physical activity. the 2015 dietary guidelines advisory committee has also addressed the threats pertaining to energy drinks consumption, highlighting the sensitivity of children and adolescents to the negative health effects of caffeine, as well as the scarcity of data on caffeine use in this vulnerable cohort [18]. table 1: possible cardiovascular manifestations of energy drink abuse in adolescents possible cardiovascular manifestations of energy drink abuse in adolescents tachycardia atrial fibrillation or flutter ventricular arrhythmias qt prolongation st-segment elevation myocardial infarction hemodynamic failure sudden cardiac death cumulative cardiovascular load effective commercial marketing, peer influence, and a lack of knowledge of the possible hazards associated with energy drinks consumptions are the few reasons for its increased popularity among young adults and adolescents. the propensity mandumpala & kumar energy drinks and long term cardiovascular risks vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 90 among young people to use alcohol-mixed energy drinks, particularly during social events, is a major public health concern. energy drink consumption can also precipitate the onset of substance use disorder in people who are predisposed to such tendencies [19]. also, there are concerns being raised about consuming energy drinks with illicit substances like marijuana and amphetamines [20]. it is proven that caffeine administration raises blood pressure and lowers heart rate. similar findings have been reported in both preand post-pubertal boys and girls. there are gender differences in cardiovascular responses to caffeine which usually emerge after puberty. the metabolism of caffeine is altered by fluctuating steroid hormones that are present during the pubertal phase of boys and girls leading to varied cardiovascular responses. according to studies, estradiol decreases cytochrome p450 function, resulting in an increased concentration of caffeine in the systemic circulation. another possible explanation is that these gender differences are linked to variations in caffeine consumption patterns as the younger population ages. studies have also documented evidence of changes in caffeine metabolism with changes in the phases of the menstrual cycle in post-pubertal girls [21]. caffeine if consumed in large quantities can also lead to exacerbation of underlying cardiac conditions [22, 23]. prevention of cardiovascular diseases: cardiovascular diseases are one of the leading causes of mortality globally. diet is a combination of carbohydrates, fats and protein that have an impact on human health. the link between nutrition and health is complicated, and attempts to delay or prevent chronic diseases like cardiovascular diseases are of particular importance because chronic diseases, and specifically cardiovascular diseases, remain the largest cause of death and disability globally [24]. cardiovascular prevention should include a multimodal behavioural intervention focused on health education, physical activity, and psychological risk factors. this critical stage in cardiovascular prevention begins with changing one's smoking habits. half of all preventable deaths in smokers are attributable to cardiovascular diseases, which are caused by smoking. smoking cessation has been shown to have immediate and longterm benefits. to achieve the goal of quitting smoking, health care practitioners must provide an intensive programme that includes professional assistance for improving patients' motivation and, in certain cases, pharmacological aids. regular physical activity and aerobic exercise training have also been linked to a lower incidence of cardiovascular diseases and nonfatal coronary events in both healthy people and those with coronary risk factors and cardiac patients. a heart-healthy diet plays a crucial role in preventing cardiovascular disease. observational studies provide the majority of evidence supporting the impact of healthy eating habits on cardiovascular risk. saturated fatty acids should account for 10% of total energy intake, with polyunsaturated fatty acids replacing them as much as possible; transunsaturated fatty acids should account for 1% of total energy intake; 200 g fruit/day; 200 g vegetables/day; fish at least twice a week; alcohol consumption should be limited to two glasses (20 g/day alcohol) for men and one glass (20 g/day alcohol) for women. the amount of energy consumed should correspond to the amount of energy required to achieve a healthy weight. weight loss in overweight (bmi 25–29.9 kg/m2) and obese (bmi.30 kg/m2) patients is significant for cardiovascular protection since it is linked to improvements in cardiovascular risk variables such as blood pressure, dyslipidemia, and insulin resistance. prevention is critical in diabetic patients to lower the risk of microvascular and cardiovascular problems. another important goal in the prevention of cardiovascular diseases is reducing lipid levels. increased plasma cholesterol and ldl cholesterol, as well as hypertriglyceridemia and low hdl cholesterol, are major risk factors for cardiovascular diseases [25]. integration of these preventive methods in conjunction with decreased consumption of energy drinks can be a valuable strategy. therefore, practising good cardiovascular risk prevention practices can yield better life outcomes, reduce economic burden and also decrease the burden on the expanding healthcare system. regulatory requirements the food and drug administration (fda) has primary jurisdiction over energy drink safety, labelling, and ingredients. state and local governments can exercise jurisdiction over public health and safety to control the sale of these products and safeguard consumers through their legislatures and agencies [26]. regulatory actions on the manufacturing and sale of highenergy drinks by the fda and the consumer product safety commission are needed, considering the serious health issues it imposes. in addition to this, medical vigilance can also be implemented and health education can be imparted to parents and young children about the inherent risks associated with the consumption of energy drinks [27]. the rapid introduction of caffeine in non-traditional food, beverages and energy drinks makes it necessary to implement dietary assessment tools to account for evolving caffeine sources and novel components in the food supply chain. energy drinks consumption is associated with adverse events, poison centre visits, emergency visits and hospitalisations, a systematic collection of these data along with an understanding of the various risk factors can be a useful source for determining the risk of toxicity among the youth and other populations [28]. literature gap after a thorough literature review, it can be understood that there is a lack of large epidemiological studies that correlate the mandumpala & kumar energy drinks and long term cardiovascular risks vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 91 direct association between cardiovascular events and energy drink consumption. evaluation of energy drinks over a long period of time and in a systematic manner and reducing overall caffeine consumption, particularly among children, should be a priority. future studies exploring the adverse outcomes of energy drink consumption, and its interaction with various food products other than alcohol and illicit substances are recommended. future studies should reflect on the association of other ingredients in energy drinks and their contribution to cardiovascular risk. research should also be extended to identify the exact reason that leads to adverse events – rapid intake, the volume of intake or intake with other ingredients. individual case reports can be a valuable source to stage a platform for formulating a hypothesis, and testing of hypothesis on a larger target population can yield valuable scientific results. references 1. paul j, criado ar. the art of writing literature review: what do we know and what do we need to know? int bus rev. 2020, 29: 10.1016/j.ibusrev.2020.101717 2. reissig cj, strain ec, griffiths rr. caffeinated energy drinks-a growing problem. drug alcohol depend. 2009, 99: 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| 97 review article current and emerging pharmacotherapies for zika virus: a comprehensive review queeny wilcy noronha1, thanshifa fathima1, ramdas bhat2, a r shabaraya3 from 1 ug student, department of pharmacy practice, 2 assistant professor, department of pharmacology, 3 principal and head of department of pharmaceutics, srinivas college of pharmacy, valachil, post farangipete, mangalore, karnataka, india-574143 corresponding to: ramdas bhat, assistant professor, department of pharmacology, srinivas college of pharmacy, valachil, mangalore -574143 karnataka, india. email: ramdas21@gmail.com, tel.: +91 7795772463 abstract zika virus is a single-stranded rna virus that belongs to the flaviviridae family and was first identified in the zika forest of uganda in 1947. since 2007, there have been several outbreaks of zika virus in various countries, with the most significant outbreak occurring in brazil in 2015. the virus is primarily transmitted by infected aedes mosquitoes, but it can also be sexually transmitted, transmitted through blood transfusions, and from mother to child during pregnancy. while most people who become infected with zika virus experience mild or no symptoms, there is a potential for severe outcomes, including neurological complications like guillain-barre syndrome and congenital birth defects like microcephaly. this article provides an overview of zika virus, its transmission, symptoms, and potential impacts on public health. despite no specific antiviral treatment available for zika virus, there are ongoing efforts to develop vaccines and treatments. the potential for sexual transmission of zika virus has also led to recommendations for safe sexual practices, particularly for pregnant women or women who may become pregnant. continued research is needed to better understand the virus and its potential impacts on public health, including long-term effects and the potential for the virus to be transmitted through other routes. the emergence of zika virus as a significant public health concern highlights the need for continued vigilance and research to mitigate the potential impacts of this and other emerging infectious diseases. given the threat of zika virus and its ability to spread across international borders, global collaboration and coordinated responses are essential to address emerging infectious disease threats. it is essential to continue investing in research and development to reduce the impact of emerging infectious diseases on public health. key words: zika virus (zikv), mosquito, rna, world health organization (who), infection n rna flavivirus known as the zika virus (zikv) was first discovered in uganda, africa, in 1947. for many years, the virus was not seen as a serious threat to public health; nevertheless, in 2007, an epidemic was discovered in the yap islands in the pacific [1]. further breakouts occurred in french polynesia in 2015 after which they expanded to other pacific islands and, starting in 2016, migrated quickly into americas [2]. the world health organization (who) declared a public health emergency in 2016 due to possibility zikv infection during pregnancy might have catastrophic consequences for the infant, such as microcephaly and neurological impairment [3]. despite a decline in transmission since 2017, the virus is still present and outbreaks are still a possibility [4]. the emergence of zikv in south america has been the subject of extensive research, with previous reviews covering this topic [5]. nonetheless, the emphasis of this review is on recent discoveries from 2019 to 2020, including updates on epidemiology, transmission, immunogenicity and host variables, clinical characteristics, and potential therapies [6]. as our understanding of the virus grows, ongoing research is assisting in the discovery of new strategies for zikv prevention and treatment. nonetheless, the risk of outbreaks and the terrible side effects caused by zikv infection highlight the ongoing need for research into this virus and the development of effective treatments [7]. epidemology human infection incidents were few and uncommon before 2007. serological testing and viral isolation were the main sources of human infection reporting, which suggested extensive dispersion in africa and southeast asia but no significant outbreaks [8-10]. in the island of yap, there was an event in 2007, which resulted in 49 confirmed cases and 59 probable cases over the course of a four-month period [1113]. with 294 confirmed cases over a 10-week period, french polynesia had another significant outbreak in 2013 [14]. the virus entered the americas in 2014. cases of locally acquired infection were found on easter island, while instances in northeastern brazil were discovered in 2015 [15-16]. a mailto:ramdas21@gmail.com bhat r et al. zika virus: a comprehensive review vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 98 the 2013 chikungunya virus epidemic was similar to the rising frequency of the zika virus in the americas [17]. because most infected persons do not seek medical attention, it is challenging to assess the number of zika cases [18]. while colombia has reported more than 25,000 suspected cases, brazilian officials believe that there have been close to 1.5 million infections since the outbreak started [19-22]. figure 1: areas of locally acquired zika virus infection, june to february, 2016, adapted from world health organization situation report,19 february 2016 [7] clinical manifestations the majority of zika virus patients don't exhibit any symptoms. if so, symptoms are often mild (rash, fever, conjunctivitis, muscle and joint pain, malaise, and headache), start 3–14 days after infection, and last 2–7 days. although other arboviral and non-arboviral conditions might also present with same symptoms, a laboratory confirmation of zika virus infection is necessary [23]. complications zika virus infection during pregnancy causes microcephaly and other congenital deformities in the foetus, such as limb contractures, high muscle tone, eye abnormalities, and hearing loss. congenital zika syndrome is the umbrella term for these clinical features. the risk of congenital deformities following zika virus infection during pregnancy is unknown; it is estimated that 5-15% of babies born to mothers who contracted the virus while pregnant show signs of zika-related problems. congenital defects can develop after an infection, whether it is symptomatic or not. preterm birth, stillbirth, and foetal death are other complications of zika infection during pregnancy [24]. treatment the current gold standard of therapy for zika virus (zikv) infection is symptomatic treatment as there is no particular antiviral vaccine or medication available. it is important to take analgesics and antipyretics carefully to avoid side effects such hepatopathy, allergies, and nephropathy. as clinical diagnosis and serological analysis can be imprecise, aspirin administration should be avoided to prevent bleeding issues in individuals who have been incorrectly diagnosed with zikv infection [25]. a typical symptom of rashes is severe pruritus, which may be controlled by avoiding hot showers and overusing soap as well as using enough skin moisturisers. if these efforts are unsuccessful, calamine or menthol-based cooling creams can be utilised, and older antihistamine medications may offer comfort owing to their calming effects. as it is uncertain how well corticosteroids would treat this ailment, they should be avoided [26]. the diagnosis of guillain-barré syndrome (gbs) should be made when the patient exhibits areflexia, clinical progression, and progressive weakening affecting two or more limbs over the course of up to four weeks. because there is a possibility of respiratory muscle paralysis, patients with suspected gbs should be closely watched in intensive care units. plasmapheresis and hyperimmune intravenous immunoglobulin (ivig) are two treatment options for gbs that, although being pricey bhat r et al. zika virus: a comprehensive review vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 99 treatments, shorten the time it takes for a patient to recover [27]. virology and pathogenesis the flaviviridae family, which also contains other medically relevant mosquito-borne viruses including denv, wnv, and yellow fever virus, is home to the positive-sense, singlestranded rna virus known as the zika virus [28]. a polyprotein that is broken down into 10 proteins, including the capsid, envelope, and seven nonstructural proteins, is encoded by its 10,794-nucleotide genome. it also includes two noncoding sections at its 5' and 3' ends. both the african and asian subspecies of the virus originated in east africa in the late 19th or early 20th century, and both have since migrated to numerous regions, including the pacific islands and the americas [29]. many genomic regions were shown to be under strong negative selection pressure in recent research on the molecular evolution of the zika virus, which might have resulted in recombination events and the elimination of deleterious polymorphisms [30]. to ascertain the effect of this discovery on the virus's transmission, zoonotic maintenance, and epidemiology, more research is required. the axl receptor tyrosine kinase and other entry and adhesion factors let the virus invade host cells through cellular receptors and infect keratinocytes, immature dendritic cells, and fibroblasts [31]. zika virus antigens were exclusively found in the nucleus of infected cells, unlike other flaviviruses, suggesting a unique replication location that needs more research. cellular autophagy boosts zika virus replication in skin fibroblasts [32]. diagnosis the overlap with other arboviruses makes it difficult to reliably diagnose zika virus infection based only on clinical signs. because of this, a precise diagnosis requires laboratory testing. patients with an acute fever, rash, myalgia, or arthralgia who have recently gone to places where the zika virus is still being transmitted need to get tested for the virus as well as for chikv, denv, and zika virus. a pcr-based diagnostic and a serologic assay, both of which are commercially accessible and authorized for use in emergency situations, are both available. based on the clinical data supplied by the healthcare practitioner, the laboratory chooses the proper test.serologic testing is not advised during the acute period of sickness; instead, molecular amplification tests, such as rt-pcr, are the method of choice. nonetheless, if samples test negative for the zika virus by rt-pcr, serologic testing can be taken into consideration. due to cross-reactivity with other flaviviruses, such as denv, serologic testing has limits in terms of specificity. thus, employing a different testing method, positive serologic test results should be verified. the kind of sample used can also alter the likelihood of detection, and additional specimen types, such urine and saliva, are being examined for their potential diagnostic value [33]. viruria may last longer than viremia, and saliva and urine may be acceptable substitutes, particularly when drawing blood is difficult. according to one study, zika virus rna can be found in urine up to 20 days after viremia has been eliminated. as a result, when the zika virus is clinically suspected, rt-pcr urine testing should be considered. similarly, salivary rt-pcr testing has been shown to improve the rate of infection diagnosis during the acute phase, but blood remains the preferred sample. large reference laboratories typically conduct zika virus testing, and it is best to contact local public health organisations before testing to organise sample collection [33]. prevention and control of zikv mosquitoes pose the most risk for zikv infection, hence it is important to manage and limit their breeding grounds in order to stop the virus from spreading [34]. mosquito nets, insect repellents, and closing cracks and gaps can all help with this. larvicides should be used with insecticides that are suggested by the who pesticide evaluation system. insect repellents should not be used to infants less than two months; however, mosquito nets can shield them from bites [35]. the centers for disease control and prevention advises using mosquito repellents with active chemicals such picaridin, deet, eucalyptus oil, ir3535, oil of lemon, and para-menthane-diol, which are safe for use by expectant and lactating moms. nevertheless, children under three years old should not use repellents that include para-menthane-diol, lemon oil, or eucalyptus oil. flying insect foggers with tetramethrin and cypermethrin and indoor mosquito-killing sprays with imidacloprid and -cyfluthrin can both kill mosquitoes [36]. before receiving blood transfusions, it is advisable to get tested for zikv infection to stop blood-borne illness. pregnancy should be avoided in high-risk locations until the virus has been completely eradicated, or increased caution should be used because zikv infection has been associated to microcephaly [37]. the spread of zikv can also be stopped by using a variety of vector control techniques. a technique that works well is giving mosquitoes a bacterium that regulates their number. to manage mosquito populations, wolbachia, an intracellular bacterium, can be used as a biopesticide [38]. toxorhynchites splendens mosquito larvae can be used since the adults consume honeydew, fruit, and nectar while the larvae do not feed on blood but rather the bhat r et al. zika virus: a comprehensive review vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 100 larvae of other mosquito species [39]. using sterile males to cause infertility in wild, fertile aedes species mosquito females is another successful tactic [40]. conclusion the zika virus is a newly emerging disease that, while less dangerous to adults than sars and mers, can cause foetal defects if contracted during pregnancy. it is one of the torch illnesses, which can result in congenital abnormalities. to manage potential zika-related problems in different countries, medical community knowledge must be increased, vector control strategies improved, and disease surveillance systems expanded. references 1. dick gw, kitchen sf, haddow aj. zika virus (i). isolations and serological specificity. transactions of the royal society of tropical medicine and hygiene. 1952 sep 1;46(5):509-20. 2. duffy mr, chen th, hancock wt, et al. zika virus outbreak on yap island, federated states of micronesia. new england journal of medicine. 2009 jun 11;360(24):2536-43. 3. musso d, nilles ej, cao-lormeau vm. rapid spread of emerging zika virus in the pacific area. clinical microbiology and infection. 2014 oct 1;20(10): o595-6. 4. krow-lucal er, de andrade mr, cananéa jn, et al. association and birth prevalence of microcephaly attributable to zika virus infection among infants in paraíba, brazil, in 2015–16: a casecontrol study. the lancet child & adolescent health. 2018 mar 1;2(3):205-13. 5. gulland a. zika virus is a global public health emergency, declares who. bmj. 2016 feb 2; i657. 6. musso d, ko ai, baud d. zika virus infection—after the pandemic. new england journal of medicine. 2019 oct 10;381(15):1444-57. 7. baud d, gubler dj, schaub b, et al. an update on zika virus infection. the lancet. 2017 nov 4;390(10107):2099-109. 8. gorry c. cuba confronts zika: all hands-on deck. medicc review. 2016; 18:6-10. 9. mret d. al. 2009 zika virus outbreak on yap island, federated states of micronesia. n. engl. j. med.; 360:2536-43. 10. haddow ad, schuh aj, yasuda cy, et al. genetic characterization of zika virus strains: geographic expansion of the asian lineage. plos neglected tropical diseases. 2012 feb 28;6(2): e1477. 11. dick gw, cocina sf, haddow aj. zika aislamientos de virus y especificidadserológica. transactions of the royal society of tropical medicine and hygiene. 1952; 46:509-20. 12. campos gs, bandeira ac, sardi si. surto de vírus zika, bahia, brasil. emerg infect dis. 2015; 21:1885-6. 13. simpson di. zika virus infection in man. transactions of the royal society of tropical medicine and hygiene. 1964;58(4):335-8. 14. macnamara fn. zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria. transactions of the royal society of tropical medicine and hygiene. 1954 mar 1;48(2):139-45. 15. fagbami ah. zika virus infections in nigeria: virological and seroepidemiological investigations in oyo state. epidemiology & infection. 1979 oct;83(2):213-9. 16. smithburn kc. neutralizing antibodies against arthropod-borne viruses in the sera of long-time residents of malaya and borneo. american j hygiene. 1954;59(2):157-63. 17. darwish ma, hoogstraal h, roberts tj, et al. a seroepidemiological survey for certain arboviruses (togaviridae) in pakistan. transactions of the royal society of tropical medicine and hygiene. 1983 jan 1;77(4):442-5. 18. olson jg, ksiazek tg. zika virus, a cause of fever in central java, indonesia. transactions of the royal society of tropical medicine and hygiene. 1981 jan 1;75(3):389-93. 19. basarab m, bowman c, aarons ej, cropley i. zika virus. bmj. 2016 feb 26; i1049. 20. cao-lormeau vm, roche c, teissier a, et al. zika virus, french polynesia, south pacific, 2013. emerging infectious diseases. 2014 jun;20(6):1085. 21. tognarelli j, ulloa s, villagra e, et al. a report on the outbreak of zika virus on easter island, south pacific, 2014. archives of virology. 2016 mar;161(3):665-8. 22. nelwan ej, pohan ht. dengue convalescent rash in adult indonesian patients. acta med indones. 2014 oct 1;46(4):339-40. 23. musso d, ko ai, baud d. zika virus infection — after the pandemic. longo dl, editor. new england journal of medicine. 2019 oct 10;381(15):1444–57. 24. plourde ar, bloch em. a literature review of zika virus. emerging infectious diseases. 2016 jul;22(7):1185. 25. simmons cp, farrar jj, van vinh chau n, wills b. dengue. new england journal of medicine. 2012 apr 12;366(15):1423-32. 26. ansar v, valadi n. guillain-barré syndrome. primary care: clinics in office practice. 2015 jun 1;42(2):189-93. 27. lanciotti rs, kosoy ol, laven jj, et al. propriedadesgenéticas e sorológicas do vírus zika associado a umaepidemia, estado da micronesia, 2007. emerg infect dis. 2008;14(8):1232-9. 28. kreil tr, berting a, kistner o, et al. west nile virus and the safety of plasma derivatives: verification of high safety margins, and the validity of predictions based on model virus data. transfusion. 2003 aug;43(8):1023-8. 29. gatherer d, kohl a. zika virus: a previously slow pandemic spreads rapidly through the americas. journal of general virology. 2016 feb 1;97(2):269-73. 30. faye o, freire cc, iamarino a, et al. molecular evolution of zika virus during its emergence in the 20th century. plos neglected tropical diseases. 2014 jan 9; 8(1): e2636. 31. hamel r, dejarnac o, wichit s, et al. biology of zika virus infection in human skin cells. journal of virology. 2015 sep 1; 89(17):8880-96. 32. buckley a, gould ea. detection of virus-specific antigen in the nuclei or nucleoli of cells infected with zika or langat virus. journal of general virology. 1988 aug; 69(8):1913-20. 33. musso d, ko ai, baud d. zika virus infection—after the pandemic. new england journal of medicine. 2019 oct 10;381(15):1444-57. 34. fu kw, liang h, saroha n, et al. how people react to zika virus outbreaks on twitter? a computational content analysis. american journal of infection control. 2016 dec 1;44(12):17002. 35. labib ba, chigbu di. pathogenesis and manifestations of zika virus-associated ocular diseases. tropical medicine and infectious disease. 2022 jun 15;7(6):106. bhat r et al. zika virus: a comprehensive review vol 1 | issue 3 | oct – dec 2022 indian j pharm drug studies | 101 36. kazmi ss, ali w, bibi n, nouroz f. a review on zika virus outbreak, epidemiology, transmission and infection dynamics. journal of biological research-thessaloniki. 2020 mar 4;27(1). 37. vraga ek, bode l. using expert sources to correct health misinformation in social media. science communication. 2017 oct;39(5):621-45. 38. burd i, griffin d. the chasm between public health and reproductive research: what history tells us about zika virus. journal of assisted reproduction and genetics. 2016 apr; 33:439-40. 39. benelli g, jeffries cl, walker t. biological control of mosquito vectors: past, present, and future. insects. 2016 oct 3;7(4):52. 40. singh rk, dhama k, malik ys, et al. zika virus–emergence, evolution, pathology, diagnosis, and control: current global scenario and future perspectives–a comprehensive review. veterinary quarterly. 2016 jul 2;36(3):150-75. how to cite this article: ramdas bhat, queeny wilcy noronha, thanshifa fathima, a r shabaraya. current and emerging pharmacotherapies for zika virus: a comprehensive review. indian j pharm drug studies. 2022;1(3) 97-101. funding: none conflict of interest: none stated thaker et al. clotrimazole micro emulsion for topical drug delivery vol 1 | issue 1 | apr jun 2022 indian j pharm drug studies | 30 original article formulation and characterization of clotrimazole micro emulsion for topical drug delivery vamakshi thaker1, a.k. singhai2, dilip kumar tiwari3 from, 1m. pharmacy, 2professor and principal, 3asstt. professor, department of pharmaceutical, lakshmi narain college of bhopal (m.p.) india. correspondence to: vamakshi thaker, m. pharmacy, department of pharmaceutical, lakshmi narain college of bhopal (m.p.), india. email: vidhivamakshi@gmail.com abstract an antimycotic drug like clotrimazole is used for treating candida albicans and other fungal infections. clotrimazole seems to be a topical therapy treating vulvovaginal candidiasis, tineapedis, and oropharyngeal candidiasis. that is a synthetic azole antimycotic. by decreasing the production of ergosterol, this drug inhibits fungal growth. clotrimazole has become a medication of interest for a variety of illnesses, including sickle cell anemia, malaria, and some cancers, in addition to its antimycotic properties. it is mixed with other molecules to create clotrimazole compounds with enhanced pharmacological effectiveness. numerous novel pharmaceutical formulations enabling variable releases developed. clotrimazole is a welltolerated, little-adverse drug. however, certain immunocompromised individuals are developing treatment resistance. in this study, we discussed the pharmaceutical chemistry, use, and pharmacology of clotrimazole. key words: clotrimazole antifungal drug, topical drug delivery system, clotrimazole micro emulsion gel. lotrimazole is a synthetic imidazole derivative most often used to treat yeast, dermatophyte infections of the vagina, and skin. this drug works well against microsporum spp., candida spp, trichophyton spp., and malazzesia furfur in vitro (1). it also shows modest activity in vitro action against gram-positive bacteria and, at extremely high doses, activity against gram-positive bacteria trichomonas spp. clotrimazole shows successful results in individuals who already failed to get treated by certain other antifungal medications such as nystatin, and amphotericin b. in the case of trichomonal vaginitis, the results aren't promising. topical application of clotrimazole help treat skin infections caused by candida or dermatophytes (2). clotrimazole cream is equally efficient as whitfield's ointment or tolnaftate in the treatment of dermatophytoses, and even as efficient as nystatin inside the treatment of cutaneous candidiasis in clinical studies. clotrimazole topical formulations are typically well-tolerated, although in a few cases, local discomfort has prompted therapy discontinuation. (13). clotrimazole has a molecular formulation of c22h17cln2 as well as a molecular weight of 344•8 g mol−1. clotrimazole is in widespread use for the treatment of candida albicans and other fungal infections. its antimycotic properties were discovered in the late 1960s. as an active ingredient, it is marketed as a generic drug under various trade names and by various companies worldwide. in addition to its antimycotic activity, clotrimazole is used in the treatment of metronidazoleresistant trichomoniasis to relieve symptoms (15) and displays activity against certain gram-positive bacteria (16). it is a synthetic compound. figure 1: the chemical structure of clotrimazole (1[(2chlorophenyl) diphenylmethyl]-1h-imidazole) (1). materials and methods polyethylene sorbitan mono-oleate (psmo) and sorbitan monooleate (smo) acquired by p. c. drug center co. ltd. rci labscan limited has acquired isopropyl-alcohol (ipa) (7). these were bought from the orbit pharmaceutical, gujarat isopropyl palmitate (ipp). sigma aldrich bought fumed silica. c thaker et al. clotrimazole micro emulsion for topical drug delivery vol 1 | issue 1 | apr jun 2022 indian j pharm drug studies | 31 during the tests, distilled water was utilized. all chemical products were medicinal and utilized without further purification. pre-formulation study solubility solubility of clotrimazole determined in different oils, surfactants, and co-surfactant. clotrimazole was added in excess to oils, surfactants, and co-surfactant and stirred for 24 h on a magnetic stirrer. samples were centrifuged at 1500 rpm for 10 min after stirring, and the drug present in the supernatant get extracted at λ max 261 nm (8). pha standard solution of clotrimazole, 1mg/ml prepared. further, diluted with usp buffers of ph 1.2, 4.5, and 6.8, each up to 10ml (5). incubated the solutions for 2 hour at 37° c. to achieve an adequate solubility level aqueous samples were prepared with acetonitrile as a co-solvent at an effective final concentration of 10% (v/v). the samples were assayed for drug content by the validated hplc method. ftir analysistest solution dissolved in 50mg of the substance to be examined in ethanol (96%) r and dilute to 5ml with the same solvent. reference solution dissolves in 50mg of clotrimazole crs in ethanol (96%) r and is diluted to 5ml with the same solvent. plate thin layer chromatography f254 plate r. mobile phase concentrated ammonia r1, propanol r, toluene r (0.5:10:90 v/v/v) solubility of the drug in different solventssolubility is calculated using the following protocol. partially insoluble in water, soluble in ethanol (96%), and methylene chloride. in different oils such as oleic acid, lemon oil, olive oil, and methane oil, the solubility of clotrimazole was studied. the maximum solubility of clotrimazole in mentha oil was discovered among the oils tested. the analgesic and cooling effects of mentha oil itself are sensory. mentha oil is used for the oil phase for the clotrimazole microemulsion (6). stability based on visual identification microemulsion with clotrimazole remained as clear liquid for two months without the occurrence of phase separation or flocculation at room temperature and refrigerator temperature. the results of various studies performed on me gel were found to be satisfactory so, both were found to be stable for two months (4, 5). drug excipient studyaccurately weighed amounts of clotrimazole (100 mg), and each selected excipient (500mg) were placed in a 5ml glass vial and mixed thoroughly. closed vials containing blends were stored in ovens at 60°c and 40°c for 14 days. a standard clotrimazole sample without mixing with an excipient clotrimazole sample was kept under similar conditions. the amount of drug substance in blends was determined based on the expected drug to excipient ratio in the final formulation. duplicate samples of drug–excipient blends were analyzed after 14 days by validated hplc methods. preparation of ctm micro emulsion and ctm micro emulsion-based-gelsthe desired micro emulsion and micro emulsion-based gels with 1 percent w/w of clotrimazole have been selected. to obtain clotrimazole micro emulsions, the medicine has been dissolved. for clotrimazole-based gel, fumed silica dispersed in clotrimazole micro emulsions and produced. preparation of blank ctm micro emulsion and ctm micro emulsion gelmicro emulsion components in the preceding report, the region of micro emulsion chosen. the simple blending of ipp, 2:1 water and ipa, and 1:1 psmo and smo mixtures at 20% of concentrations produced two micro emulsions. 30% and 50%w/w for me1 and in 20%, 40% w/w for me2 1. afterwards, 2.0 % 5 % of fumed silica were added into me1 and me2 to obtain micro emulsionbased gel designated as mbg1-1 to mbg1-2 and mbg2-1 to mbg2-2 respectively. table 1composition of studied micro emulsion – based gel systems micro emulsion fumed 2.5% w/w silica fumed 5.0% w/w silica me 1 mbg1 1 mbg1 2 me2 mbg2 1 mbg2 2 characterization of blank ctm micro emulsion and ctm micro emulsion based gelimportant characters of ctm micro emulsion and ctm micro emulsion-based gel are • the look has been seen visually. • spreadability was achieved by the spreading of a low to high skin stretchable and retainable quantity of each formulation over the skin with sensational consideration in the + to +++ range. • dilution tests and conductivity measurements have established the kind of micro emulsions. • the dilution inspection should be carried out by dropping into the water of each micro emulsion, classified for miscibility or immiscibility. • the conductivity meter cm-115 was tested (orbit pharmaceutical, gujarat). determination of transmittance of clotrimazole micro emulsionsthe %transmittance was checked against distilled water using a uv-visible spectrophotometer at λ max 630 nm (1, 2). t % = antilog (2 absorbance) drug release kineticsthe drug release kinetic study was performed to find drug release mechanisms from dissolution parameters by using various kinetic model equations. the zeroorder, first-order, hixon crowell, korsmeyer peppas, and higuchi plot models were tested. thaker et al. clotrimazole micro emulsion for topical drug delivery vol 1 | issue 1 | apr jun 2022 indian j pharm drug studies | 32 methods for antifungal activityfollowing protocol was followed for measuring anti-fungal activity: • the cup-plate method was used for anti-fungal formulation. • candida albicans suspension was poured into sterilized dextrose agar media (cooled at 40ºc) and was mixed thoroughly. • 20ml of the above-mentioned suspension was poured aseptically in a pre-sterilized petri dish and was allowed to solidify. the surface of the agar plate was pierced via a sterile cork borer. • these wells were filled with an equal volume of the optimized batch of micro emulsion-based gel and marketed 1 % clotrimazole gel followed by incubation at 18-24ºc, for 72 h. • fungal growth was detected and the zone of inhibitions was measured using an antibiotic zone reader. results pre-formulation studies chemical propertiesit is a white powder or colorless crystalline powder. it has a melting point of 147-149° c. it is soluble in ethanol, acetone, and chloroform, but almost insoluble in water. it is odorless, tasteless, and subject to rapid decomposition in an acid solution. clotrimazole hydrochloride has a melting point of 159° c. phthe ph of clotrimazole in different solutions at initial and 2hrs. it was observed that, at ph 1.2, ph 4.5, and ph 6.8. at initial time 98.94 ± 1.55, 98.85 ± 1.02, and 99.58 ± 1.72, respectively. the value observed at 2 hrs of 98.25, 98.25, and 99.35, respectively. table 2ph observed time ph 1.2 ph 4.5 ph 6.8 initial 98.94 ± 1.55 98.85 ± 1.02 99.58 ± 1.72 2 hrs 98.25 98.25 99.35 ftir analysisthe ftir spectra of the clotrimazole and optimized clotrimazole microemulsion gel were recorded with kbr on an infrared spectrophotometer as shown in the figure. solubility in different solventsclt experimental solubility values in buffers рн 2.0 and 7.4, 1-octanol and hexane expressed in molarity (s) in the temperature range (293.15313.15) к. the temperature dependences of the drug solubility in the studied solvents are shown in the figure 3, 4. stated that studied solvents the compound solubility increased at higher temperatures. clotrimazole is stable in the buffer solution ph range of 1.2 7.5, but it degrades in strongly acidic and basic media and at high temperatures (9, 10). stabilityoptimized micro emulsion & micro emulsion gel were subjected to a stability study for two months at room temperature and refrigeration conditions (2-8°c). during the period of storage, the me was subjected for % transmittance, % assay & ph, while i gel was subjected for % transmittance, % assay, ph, consistency & viscosity (physical). results are shown in table 3 table 3result of stability study of micro emulsion at room temperature test initial time 1month 2month transmittance % 99.6 ± 0.06 99.6 ± 0.21 99.4 ± 0.18 assay % 99.1 ± 0.26 98.8 ± 0.15 98.82 ± 0.13 ph 5.50 ± 0.19 5.50 ± 0.14 5.50 ± 0.14 result of stability of micro emulsion gel at room te mperature assay % 99.26 ± 0.68 99.2 ± 0.28 99.2 ± 0.26 ph 6.10 ± 0.25 5.92 ± 0.38 98.82 ± 0.13 transparency transparency & clear transparency & clear transparency & clear thaker et al. clotrimazole micro emulsion for topical drug delivery vol 1 | issue 1 | apr jun 2022 indian j pharm drug studies | 33 viscosity very good very good very good result of stability stud y of micro emulsion at refrig eration temperature transmittance % 99.9 ± 0.10 99.5 ± 0.10 99.4 ± 0.09 assay % 99.6 ± 0.22 99.1 ± 0.30 99.0 ± 0.30 ph 5.46 ± 0.22 5.4 ± 0.20 5.42 ± 0.20 result of stability of m icro emulsion gel at refriger ation temperature assay % 99.6 ± 2.42 98.9 ± 1.23 98.6 ± 1.13 ph 5.99 ± 0.20 5.99 ± 0.14 5.97 ± 0.12 transparency transparency & clear transparency & clear transparency & clear viscosity very good very good very good figure 4 ftir spectroscopy of clotrimazole drug. drug excipient chemical compatibilitythe total number of drug excipient blends in the study may be very high; therefore, excipient rank-ordered with their solubility for clz were selected primary screening. for example, oils such as capryl 90, lauroglycol 90, and capmul mcm c8 exhibiting higher solubility for clotrimazole were selected. as summarized in the table, every excipient clotrimazole had degraded approximately 5-15% in 14 days at both storage conditions. the rate of degradation increased with an increase in temperature. a similar degradation peak of clotrimazole was evident in chromatograms of all samples. the representative chromatograms of the sample stored at 60o c for 14 days are shown in the figure. which shows a well-resolved degradation product of clotrimazole. in the solution state, the stability of clotrimazole is ph-dependent. preparation of ctm micro emulsion & ctm me gel following steps were followed in preparing ctm micro emulsion and ctm me-based gel: the micro emulsions and micro emulsion based-gels which had desirable appearance were selected and were added with 1% w/w of clotrimazole. the drug was dissolved in micro emulsions to obtain clotrimazole micro emulsions. fumed silica was dispersed in clotrimazole micro emulsions for the preparation of clotrimazole micro emulsion-based gel. preparation of blank micro emulsions and micro emulsion based-gelsfollowing steps were followed in preparing blank micro emulsion and me-based gel: micro emulsion components in the preceding report, the region of micro emulsion was chosen. the simple blending of ipp, 2:1 water and ipa, and 1:1 psmo and smo mixtures at 20% of concentrations produced two micro emulsions. 30% and 50% w/w, respectively for me1 and in those of 20 %, 40 %, and 40 % w/w, respectively for me 2 1. afterwards, 2.0 % and 5 % of fumed silica was added into me1 and me2 to obtain micro emulsion-based gel designated as mbg1-1 to mbg1-2 and mbg2-1 to mbg2-2, respectively. table 7physical properties of clotrimazole micro emulsions and micro emulsion based-gels characteristics of clotrimazole micro emulsions and micro emulsion-based gelsfollowing characteristics were observed: 1%w/w clotrimazole was incorporated in me1, me2, andmbg2-2, and me1-c, me2c, and mbg2-2-c, were thaker et al. clotrimazole micro emulsion for topical drug delivery vol 1 | issue 1 | apr jun 2022 indian j pharm drug studies | 34 obtained respectively. no significant visual changes were observed. however, conductivity values of clotrimazole-loaded samples were low in comparison to their blank counterparts, while ph and spreadability showed remarkable change. the samples were water-in-oil type; therefore, clotrimazole located in the external oil phase, that resulted in lower conductivity. the rheological behaviour ofme1-c andme2-c still showed as newtonian flow, also the mbg2-2-c still were shear-thinning like their blank counterparts. the viscosity of mbg2-2-c was raised slightly in comparison to its blank counterpart. characteristics of blank micro emulsions and micro emulsion-based gelsthe obtained micro emulsions (me1 and me2) were clear, pale yellowish liquids with little smell of alcohol and were immiscible with water. the results of dilution and conductivity exhibited that both me1 and me2 were waterin-oil types since their hlb value was 9.65. figure 5 characteristics clotrimazole micro emulsions and micro emulsion based-gel table 8characteristics of blank micro emulsions and micro emulsion-based gels figure 6characteristics of blank micro emulsions and micro emulsion based-gels figure 7a: clotrimazole formulation assay-based results 7b: clotrimazole formulation software based results thaker et al. clotrimazole micro emulsion for topical drug delivery vol 1 | issue 1 | apr jun 2022 indian j pharm drug studies | 35 the ph of the formulations fa and fb was found to be 6.6 ± 0.07 and 6.1 ± 0.4respectively. the ph has been adapted to the appropriate physiological ph of 6.1-6.6. in usp, clotrimazole results were determined to be not less than 90.0 percent and not more than 110.0 percent 20. ”test findings using the established analytical technique. the f2 formulation of f2 drugs discovered 98.9 ± 0.46% whereas, the f3 formulation produced 100.3 ± 0.71 %. fig. shows fb as an optimal formulation based on the desired assessment.researchers also determined that clotrimazole is often a suitable phenomenon for hydrogel integration. it also shows that the solubility problem of hydrophobic drugs also isn't addressed by alcoholic content. a hydrogel without any hard solvents that may irritate can be produced by co-solvents. it was discovered to be compatible with solvents as carbomer was used as a gelling agent. the approach devised does not require the removal of polymer over the day and so saves time. has an increased propensity and is much more patient adaptive, therefore more clinical studies are necessary. drug release kinetics studythe kinetic research on drug release. the formulation of hydrogel based on micro emulsion is an effective promoter of the localization of clotrimazole to the skin. it was shown that the drug permeability of the optimized formula based on micro emulsion (in vitro) was below (92.04 percent) its optimal hydrogel formulation based on micro emulsion (ex vivo) (96.12 percent ). this might be because of the drug partitioning into the oil phase of the hydrogel-based on micro emulsion that lowers drug release. antifungal activitythe values of the mean zone of inhibition (in vitro antifungal activity) of optimum micro emulsion-based hydrogel batch and marketed formulation (15). for topical antifungal medicines, such as clotrimazole, effective formula is required. their thermodynamically and isotopically stable characteristics are caused by surfactants and co-surfactants that lower interfacial tension from the oil to the water phase. table 8drug kinetics release study discussion clotrimazole o/w micro emulsion by the titration technique throughout their experiment. surfactants and co-surfactants have been combined and applied wisely to the water drop. the medicine was dissolved in the oil phase and stirred continually into the aforementioned solution. the solution allowed for clear and transparent liquid micro emulsion to be formed. the phase titration technique was used to manufacture clotrimazole loaded o/w micro emulsion. surfactants and co-surfactants have been combined and applied wisely to the water drop. the drug was dissolved in the oil phase and stirred continually into the mentioned solution (14). all located clear and transparent liquid solutions. the greater clotrimazole solubility in the oil phase is essential since clotrimazole is a low water-soluble medicine. in different oils including oleic acid, lemon oil, olive oil, and mentha oil, the solubility of clotrimazole was studied (4). the maximum solubility of clotrimazole in mentha oil has been discovered among the oils that have been tested. the analgesic and cooling effects of mentha oil itself are sensory. mentha oil has thus been used for the oil phase for the clotrimazole micro emulsion (11, 12). the maximal solubility of clotrimazol in tween 80 was shown. tween 80 was therefore used as the surfactant for the formulation of clotrimazole. micro emulsion for other co-surfactants including ipa and ethanol, clotrimazole demonstrated the highest solubility of propylene glycol. the skin is well permeated by propylene glycol. the co-surfactant is, therefore propylene glycol. nevertheless, the further development of clotrimazole as a pharmaceutical is an area of intense research at present. there are prospects both for its exploitation in new indications and for thaker et al. clotrimazole micro emulsion for topical drug delivery vol 1 | issue 1 | apr jun 2022 indian j pharm drug studies | 36 the development of new formulations. a scaffold based on clotrimazole is being used as a pharmacophore in the design and synthesis of novel antimalarial drugs that are cheap and easy to synthesize [22]. palladium–clotrimazole complexes that exhibit enhanced cytotoxicity against tumour cell lines, in comparison with clotrimazole alone, are under investigation as novel antineoplastic agents [23]. indeed, several other metal – clotrimazole complexes, such as ruthenium– clotrimazole and platinum –clotrimazole, also display promising antineoplastic characteristics [24]. there is also intense interest in using clotrimazole and its metabolite as lead compounds in the strategic design of novel treatments for sickle cell disease, on the basis that they can reduce erythrocyte dehydration in vivo by inhibiting the so-called gardos, calcium-dependent potassium channel that malfunctions in this disease. conclusion in conclusion, clotrimazole is an effective, safe, and welltolerated drug with unusual chemistry that is widely used in the treatment of skin, vulvovaginal and oropharyngeal fungal infections. it is sold in most developed countries worldwide under a variety of trade names, and a large number of clotrimazole formulations are available. although emerging resistance to clotrimazole may limit the future use of this drug in certain patient subpopulations, in the general population, its widespread use is likely to continue for the foreseeable future. the ongoing development of clotrimazole as a pharmaceutical is currently focused on finding new clinical indications for the drug, its use as a lead compound in structure-based drug design studies, and the optimization of formulated products to enhance drug delivery. new approaches to the formulation of clotrimazole include a buccal bio-adhesive film containing clotrimazole, which was found to inhibit oral candidiasis for up to 6 h (18), and a thermos-sensitive vaginal gel formulation formed by complexation of clotrimazole with beta-cyclodextrin, which has been shown to reduce the release rate of clotrimazole in comparison with standard preparations (17). this type of slow-release formulation may exhibit increased efficacy over other vaginal delivery systems, as traditional vaginal creams, pessaries and tablets tend to have short residency times in the vagina due to the natural cleansing process that takes place there. the use of liposomes containing clotrimazole may also provide increased residency in the vagina, thereby improving gel formulations for treatment (19). rs 100 nano-capsules have recently been studied in the treatment of c. albicans and c. glabrata, and these have been reported as more active than free clotrimazole alone (20). given the scale of the current market for vulvovaginal clotrimazole preparations, novel formulations that can demonstrate an advantage over pre-existing preparations could potentially attract a large revenue stream. nano-fiber mats for oral applications are also superior in efficacy and have reduced toxicity over lozenges and powders in current use, although further pharmaceutics investigations are needed (21). references 1. carbone. c, martins-gomes. c, silva. am, et al. clotrimazoleloaded mediterranean essential oils nlc: a synergic treatment of candida skin infections. pharmaceutics. 2019; 11(5):231. 2. blokhina. s, sharapova. a, ol’khovich. m., et al. experimental solubility of clotrimazole and some thermodynamic aspects of dissolution in different solvents. j emerg med. 2019; 682. 3. mehta d. p., rathod h. j., shah d.p. et al. design, development and characterization of microemulsion based hydrogel of clotrimazole for topical delivery system. em med j. 2019; 1-10. 4. carbone c., fuochi v., zielińska a., et al. dual-drugs delivery in solid lipid nanoparticles for the treatment of candida albicans mycosis, colloids and surfaces b. biointerfaces. 2019; 10:70. 5. victor midlej, felipe rubim, wilmer villarreal, et al. martinsduarte, maribel navarro, wanderley de souza, marlene benchimol, zinc-clotrimazole complexes are effective against trichomonas vaginalis, parasitology j appl microbiol. 2019;1-11. 6. vainstein, mário lettieri teixeira, ricardo josé alves, alexandre meneghello fuentefria. chloroacetamide derivatives as a promising topical treatment for fungal skin infections: mycologia. j chromatogr sci. 2019; 1-12. 7. chaiyakarn pornpitchanarong, kanokwan singpanna, theerasak rojanarata, et al. catechol-bearinghyaluronic acid coated polyvinyl pyrrolidone/hydroxyl propyl-βcyclodextrin/ clotrimazole nanofibers for oral candidiasis treatment, key engineering materials.curr pharm des. 2019; 163-168. 8. ali a., muhammad i. n., hasan s. f., et al. development and pharmaceutical evaluation of clotrimazole loaded topical hydrogel formulation. mol cell endocrinol. 2018; 675-81. 9. atefeh zare, zeinab moshfeghy, mohammad m.z, et al. vaginal cream versus placebo on bacterial vaginosis: a randomized clinical trial, j of herbal medicine. j pharm sci. 2018;11:3-9. 10. kaur a, jyoti k, baldi a, et al. self-assembled nanomicelles of amphiphilic clotrimazole glycyl-glycine analogue augmented drug delivery apoptosis and restrained melanoma tumour progression materials sci. and engin. int j pharm. 2018; 89:75-86. 11. machado d.e., perini j.a., menezes e, et al. clotrimazole is effective for the regression of endometriotic implants in a wistar rat experimental model of endometriosis, molecular and cellular endocrinology. j.mce. 2018; 476:17-26. 12. cota b.r., vega p.p.c., navarrete j.m, et al. efficacy and safety of eberconazole 1%otic solution compared to clotrimazole 1% solution in patients with otomycosis. am j otolaryngo. 2018; 17:307-312. 13. gagini t, vegas l.c., villarreal w, et al. metal–azole fungistatic drug complexes as antisporothrix spp. agents. j chem. 2018; 13641-13650. 14. cavalheiro m, costa c, silva-dias a, et al. a transcriptomics approach to unveiling the mechanisms of in vitro evolution towards the fluconazole resistance of a candida glabrata clinical isolate, antimicrobial agents and chemotherapy. am j diol. 2018; 10-17. 15. cudmore s.l., delgaty k.l., hayward-mcclelland s.f., et al. treatment of infections caused by metronidazoleresistant trichomonas vaginalis. clin microbiol rev. 2004; 17:783-93. thaker et al. clotrimazole micro emulsion for topical drug delivery vol 1 | issue 1 | apr jun 2022 indian j pharm drug studies | 37 16. alsterholm m, karami n, faergemann j. antimicrobial activity of topical skin pharmaceuticals – an in vitro study. acta derm venereol. 2010; 90:239-45. 17. bilensoy e, rouf m, vural i, et al. thermo-sensitive vaginal gel formulation for the controlled release of clotrimazole via complexation to beta-cyclodextrin. j control release. 2006; 116:107-09. 18. singh s, jain s, muthu m, et al. preparation and evaluation of buccal bio-adhesive films containing clotrimazole. aaps pharmacy tech. 2008; 9:660-67. 19. vanic z, skalko-basnet n. nano pharmaceuticals for improved topical vaginal therapy. can they deliver? eur j pharm sci. 2013; 50:29-41. 20. santos s.s., lorenzoni a, pegoraro n.s., et al. formulation and in vitro evaluation of coconut oil-core cationic nano-capsules intended for vaginal delivery of clotrimazole colloids. surf b bio-interfaces. 2014; 116:270-76. 21. tonglairoum p, ngawhirunpat t, rojanarata t, et al. fastacting clotrimazole composited pvp nanofibres for oral candidiasis application. pharm res. 2014; 11291-11091. 22. gemma s, campiani g, butini s, et al. design and synthesis of potent anti-malarial agents based on clotrimazole scaffold: exploring an innovative pharmacophore. j med chem. 2007; 50:595-98. 23. navarro m, pena n.p., colmenares i, et al. synthesis and characterization of new palladium clotrimazole and palladium chloroquine complexes showing cytotoxicity for tumor cell lines in vitro. j inorg biochem. 2006, 100:152-57 24. robles-escajeda e, martinez a, varela-ramirez, et al. analysis of the cytotoxic effects of ruthenium ketoconazole and ruthenium clotrimazole complexes on cancer cells. cell biol toxicol. 2013; 29:431-43. how to cite this article: thaker v, singhai a.k, tiwari d.k. formulation and characterization of clotrimazole micro emulsion for topical drug delivery. indian j pharm drug studies. 2022;1(1):30-37. funding: none conflict of interest: none stated chandur & shabaraya a containing lycopene phytosomes for improved absorption vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 82 original article characterization of liquid oral containing lycopene phytosomes for improved absorption viresh k chandur1, ramkrishna shabaraya a2 from 1* asso. prof. 2 prof. principal, department of pharmaceutics, srinivas college of pharmacy mangalore. correspondence to: viresh k chandur, asso. prof. department of pharmaceutics, srinivas college of pharmacy, mangalore. tele: 9742565141, email: viresh.chandur2009@gmail.com abstract objective: owing to life style modifications and use of nutraceuticals present study was aimed to develop stable and controlled release oral liquid containing lycopene phytosomes for better therapeutic activity. tomatoes are consumed raw or in the processed forms like ketchup, sauce, soup and salad. the global lycopene market size was valued at $107.2 million in 2020, and is projected reach $187.3 million in 2030, registering a cagr of 5.2% from 2021 to 2030. it has several medicinal properties, which has increased its demand in the nutraceutical market. method: extracted lycopene was complexed with phospholipid (1:1) to form phytosomes and subjected to pre and post evaluations of liquid oral nanosuspension (lps1 to lps4) containing different concentration of sodium carboxymethylcellulose, and hydroxy propyl methyl cellulose k4m results: acceptable results were seen for pre evaluation for ftir, dsc compatible studies and post evaluation like ph(4.4 to 5.1), viscosity (15.2 to 42.2 cp), drug content (96.5 to 98.8%), specific gravity (1.1042 to 1.1062), particle size(450.5 to 594.5nm) and zeta potential (-21.2 to -23.2) respectively. conclusion: overall lps3 was found to be better and stable formulation containing 10ml of 1% hpmc k4m as suspending agent. anti-oxidant property was found comparatively same for plain and complexes of lycopene, difference was found for duration of antioxidant activity which can be correlated with in-vitro dissolution studies. key words: lycopene, liquid oral, anti-oxidant, nutraceutical, phytosomes. ecent studies have an impact on how people feel about using dietary supplements, and consumer interest in self-care is rising [1]. the potential of complementary and alternative medicine—these so-called "community-based lifestyle interventions"—to prevent diseases has been the subject of a growing body of scientific research over the past ten years. one of the active components derived from a natural source is lycopene [2]. lycopene, a chemical with a high lipophilicity, has a variety of pharmacological effects. its bioavailability is however constrained by its poor water solubility and significant presystemic metabolism. due to their poor oral bioavailability, many barriers prevent the medical use of medications derived from herbs, especially those that include polyene chains with 35–40. carbon atoms in their chemical structure. the use of phytosomal technology is one approach to address these more recent issues. lycopene and the consumption of foods containing lycopene may impact the risk of cancer or cardiovascular disease, according to encouraging results from epidemiological, cell culture, and animal research, but more clinical trial data is required to support this idea [3]. a unique herbal formulation called a phytosome, which resembles a tiny cell, combines the bioactive phytoconstituents of an herb extract complexed with phospholipids echnique is a ground-breaking approach for dramatically ito create lipid-compatible molecular complexes. this phytosome tmproving bioavailability, substantially increasing clinical benefit, assuring delivery to the tissues, and maintaining nutrient safety. over traditional botanical extracts, phytosomes offer superior pharmacokinetic and pharmacodynamic behaviour. this method made use of the complex formation that can occur when phospholipid molecules interact with herbal extracts or their constituents to create a lipid-compatible molecular complex that is soluble in both a lipid environment and water [4]. delivering an effective level of the active components is necessary for any herbal product to be effective. this problem is solved by the phytosome technology, which significantly increases the bioavailability of phytomedicines [5]. materials & methods tomatoes were collected from local market at mangalore of karnataka in the month of january 2018 and authenticated by pilikula nisarga dhama (botanical garden) mangalore. natural or synthetic phospholipids, such as phosphatidylcholine, were obtained from hi media, aprotic r mailto:viresh.chandur2009@gmail.com chandur & shabaraya a containing lycopene phytosomes for improved absorption vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 83 solvent, such as dioxane or acetone, n-hexane, ethanol were obtained by merck chemicals. preparation of oral nanosuspension containing lycopene phytosomes: this preparation is majorly includes two steps. extraction and preparation of lycopene phytosomes was done as per aghel n et al., 2011 and jain s et al., 2019 respectively [6, 7] and composition of suspension containing lycopene phytosomes was prepared as per table 1. table 1: composition of liquid orals ingredients lps1 lps2 lps3 lps4 lycopene phytosomes equivalent to 10mg / 5ml. 10mg / 5ml. 10mg / 5ml. 10mg / 5ml. simple syrup 10 ml 10 ml 10 ml 10 ml glycerin 10 ml 10 ml 10 ml 10 ml aqueous solution of na-cmc (1%) 5 ml 10 ml aqueous solution of hpmc k4m (1%) 5 ml 10 ml methyl paraben (%) 0.09 0.09 0.09 0.09 propyl paraben (%) 0.01 0.01 0.01 0.01 purified water 50 ml 50 ml 50 ml 50 ml preparation of lycopene phytosomal suspension dosage form: to lower the interfacial tension between liquid and air, the powdered form of the medication complex was completely moistened with syrup and glycerin solution. the wetted material was then slowly added to, with continuous triturating, the suspending agents such as sodium carboxy methyl cellulose (na-cmc) or hydroxy propyl methyl cellulose k4m in the aqueous medium containing specified preservatives. in order to create four different suspension formulations, lps1, lps2, lps3, and lps4, 5 and 10 millilitres of 1% aqueous na-cmc and hpmc k4m solution, respectively, were used. lastly, a continuous trituration process was used to raise the suspension to the final volume using filtered water in order to produce a uniform product. the production quality of all four suspension formulations was then assessed in accordance with established guidelines. [8]. formulation of lycopene phytosome liquid orals fig 1: lycopene phytosomal liquid orals. evaluation of lycopene phytosomes suspensions: evaluation of suspension: all the four suspension dosage form (lps1, lps2, lps3 and lps4) were evaluated for ph, viscosity, drug content, drug release studies and stability study of the final suspension was carried out. ph: using common buffer solutions, such as ph 4 and 7, the ph metre was calibrated. the ph of the suspension was tested after it had been dissolved in 50.0 ml of distilled water in an amount of about 5 ml. with the use of a systronic digital ph metre, the ph of the samples was determined. viscosity: using a brookfield viscometer, the sample's viscosity was measured (dv e model). a tiny volume holder was used to hold the necessary amount of suspension, and the lv4-27 spindle with a 100 rpm rotational speed was employed. centipoises (cp) and the associated percent torque value were recorded. specific gravity: relative density, or specific gravity, is the ratio of the density (mass of a unit volume) of a substance to the density of a given reference material. specific gravity usually means relative density with respect to water [9]. determination of drug content: accurately 5ml of formulation from different batches was measured and transferred to 100 ml volumetric flask. to this 50-70ml of 0.1 n hcl was added and sonicated for 30 min. volume was adjusted to 100ml. complete dispersion of contents was ensured visually and the dispersion was filtered using whattman filter paper. from this solution, 1 ml of sample was withdrawn and diluted to 10ml with 0.1 n hcl. contents of lycopene was measured at maximum absorbance at 471nm using jasco uv spectrophotometer [10]. particle size: particle size and zeta potential of the optimized lycopene phytosomes were done using malvern particle size and zeta analyzer (malvern panalytical version 7.13) [11]. in-vitro release studies: the drug release study was carried out using usp type ii paddle type apparatus at 37 ± 0.5ºc and at 50 rpm using 900 ml of 0.1 n hcl (ph 1.2). lycopene suspension equivalent to 10 mg of lycopene was used for the test. sample solution (5 ml) was withdrawn at predetermined time intervals, filtered through a 0.45 μm membrane filter, diluted and suitably analyzed by uv spectrophotometric jasco at 471 nm. fresh dissolution medium was replaced immediately after withdrawal of the test sample to maintain sink condition. the dissolution studies were carried out for a period of 2 h. and further dissolution medium ph was raised to 6.8 and dissolution studies was carried out for 10 h [12]. anti-oxidant activity by dpph method: dpph solution, 1mmol/l, was prepared by dissolving 31.54 mg of dpph in 95% v/v buffered methanol (40ml of 0.1 mol/l acetate buffer chandur & shabaraya a containing lycopene phytosomes for improved absorption vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 84 ph 5.5 with 60 ml of methanol) and made up to 50 ml with buffered methanol. the different concentrations of lycopene and lycopene phytosomes such as 0.5 mg, 1 mg, 2mg, 4mg and 8 mg. were made up to 4 ml with distilled water. 1 ml of dpph (1mmol, 3.953x10-10 μg/ml) was added to each test tube, shaken and the mixture was kept at 300c for 30 min. the 471 nm is a measuring absorbance of the resulting solution. the effect of ascorbic acid (vitamin c) on dpph was also assessed for comparison with that of lycopene phytosomes. a buffered methanolic dilution (0.2, 0.4, 0.6, 0.8, 1.0 ml) of 1 mg/ml ascorbic acid was made to 4 ml with distilled water. 1 ml dpph radical (1nmom/l) was added to each test tube and same procedure as in dpph scavenging experiment was followed. the absorbance measured for the control solution (buffered methanol with dpph) was in the range 0.500 ± 0.040. antiradical activity was expressed as inhibition percentage (1%) and calculated using the following equation: inhibition percentage = [(abs control – abs sample)/ abs control] x 100 [13]. stability studies: in order to determine the change in evaluation parameters like physical appearance, drug content, in vitro drug release profile on storage, stability studies of optimized batch was carried out at accelerated storage conditions at temperature 40±2˚c and 75±5% rh in a humidity chamber (rotek) as per ich q1c guidelines for 6 months. sample were withdrawn after each month and evaluated for changes in physical appearance, drug content and in vitro drug release profile [11]. fig 2: viscosity of liquid orals. results and discussion satisfactory attempt to formulate phytosomal oral suspension dosage form for controlled delivery of lycopene using polymer like sodium cmc and hpmc k4m showed reproducible results of the executed experiments, it can be concluded that: the lycopene phytosomal liquid oral suspension dosage form can be prepared by best use of suspending agents like sodium cmc and hpmc k4m. the prepared liquid orals were evaluated and showed to be uniform and stable in terms of ph of the liquid oral formulations was found to be in acceptable range for gastric absorption (ph 4.5); best to be absorbed by oral route, viscosity of liquid oral dosage forms were found to be satisfactory and shows the ease of pour ability from the container. different concentration of suspending agents showed noticeable change in viscosity. sodium cmc showed better suspending property than hpmc k4m. specific gravity found to be near to one which is essential for a stable suspension and uniformity in the drug content as given in table 2. particle size and zeta potential: particle size (450.5nm to 467.1nm), zeta potential (21.7 to – 23.2) and poly dispersity index (0.515 to 0.591) of all the respective suspension dosage form were up to the acceptable values for the stable suspension. table 3 and fig 3. table 2: ph, viscosity and specific gravity of lycopene suspension code ph viscosity 100 rpm specific gravity drug content % cp % torque lps1 4.4 29.5 12.6 1.1058 98.8 lps2 4.5 42.2 18.0 1.1062 97.8 lps3 4.9 15.2 10.2 1.1042 96.5 lps4 5.1 22.3 11.6 1.1052 97.2 table 3: particle size and zeta potential of lycopene suspension. code avg. particle size (nm) zeta potential poly dispersity index (pdi) product quality lps1 464.8 21.7 0.515 good lps2 450.5 21.9 0.578 good lps3 467.1 23.2 0.521 good lps4 594.4 0.591 good fig 3: particle size distribution of lycopene phytosome lps3 chandur & shabaraya a containing lycopene phytosomes for improved absorption vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 85 antioxidant activity: the antioxidant activity of the lycopene and phytosomal complex was compared taking ascorbic acid as standard; the results are given in fig 4. antioxidant activity of the extracted lycopene and lycopene phytosomal complex was performed and it found to be for lycopene 8 to 60 % antioxidant activity with increase in concentration to that of lycopene phytosomal complex between 9 to 61 % compared to the standard antioxidant ascorbic acid. the anti-oxidant activity was found comparatively same for plain and complexes of lycopene, difference was found for duration of antioxidant activity which can be correlated with in-vitro dissolution studies. fig 4: anti-oxidant activity by dpph method of tomato extract and phytosomes containing lycopene. in-vitro drug release studies: the percentage release of the drug during dissolution studies showed less than 14% release in first 2 hours in the acidic ph and more than 95% of release within 10 hours of all the formulations in ph 6.8 for lps2 containing 10 ml of 1% sodium cmc for 12 hours compared to hpmc k4m which sustained up to 10 hours.fig 5. fig 5: dissolution studies of lycopene suspensions stability studies: stability studies conducted for suspension was found to be stable throughout the period stating that formulations are stable. by enhancing its therapeutic usefulness, phytosomes demonstrated potential technology for maintaining the stability of the components that are lightsensitive. alternative dosage forms are advised because liquid dosage forms choose to shorten their shelf life. table 4: stability studies of best formulations lps2 code lps2 at 25±2o c / 65±5% rh* and at 40±2o c / 75±5% rh** for 6 months month ph viscosity %cdr at the end of 12 h remark 1 4.50* 42.23* 98.06* stable 4.60** 41.23** 98.01** stable 2 4.50* 42.20* 98.00* stable 4.65** 41.00** 98.00** stable 3 4.50* 42.23* 98.00* stable 4.60** 41.20** 98.00** stable 4 4.60* 42.40* 96.58* stable 4.60** 41.21** 96.40** stable 5 4.60* 42.31* 96.21* stable 4.65** 42.00** 96.11** stable 6 4.65* 42.24* 95.86* stable 4.60*8 41.23** 95.12** stable conclusion for the reproducible outcomes of the carried out studies, a satisfactory attempt has been made to manufacture phytosomal oral suspension dosage forms for controlled delivery of lycopene utilising 10 ml of 1% polymers like sodium cmc and hpmc k4m. in order to include stable goods into oral liquid dosage forms, lycopene complexes with phospholipids were successfully created into phytosomes. this was a good innovation in the field of nanotechnology. the ph, specific gravity, particle size, and zeta potential of an elegant suspension, among other characteristics, were all found to be within acceptable limits. compared to the commercially available traditional dosage form, the therapeutic concentration of the food component could be sustained for a period of 12 hours (syrup). acknowledgement: authors are thankful to vision group of science and technology, govt. of karnataka, and srinivas college of pharmacy mangalore for providing laboratory facilities to carry out the research work. references 1. hyunjeong p, young‑jun k, youngjae s. estimation of daily intake of lycopene, antioxidant contents and activities from tomatoes, watermelons, and their processed products in korea. appl biol chem, 2020; 63(50): 1-11. 2. muhammad i, fereshteh g, iahtisham ul-haq, et al. lycopene as a natural antioxidant used to prevent human health disorders. antioxidants 2020; 9(706): 1-27. 3. muhammad a, shabbir h, abdul rk. development and validation of hplc assay of lycopene in different matrices. world journal of applied chemistry, 2020; 5(2): 26-33. 0 100 % in h ib it io n concentration anti-oxidant activity by dpph method of tomato extract and phytosomes containing lycopene. std lycopene phytosomal lycopene 0 20 40 60 80 100 120 0 5 10 15 % c d r time (h) dissolution studies of lycopene suspensions lps3 lps4 lps1 lps2 chandur & shabaraya a containing lycopene phytosomes for improved absorption vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 86 4. fatima m. saeed k. analysis and estimation of lycopene extracted from tomatoes. bio scientific revi. 2020;2(2):23-32. 5. ying h, hong c, yuhao s, et al. increased blood alpha-carotene, all-trans beta-carotene and lycopene levels are associated with beneficial changes in heart rate variability: a cvd-stratified analysis in an adult population-based study. nj. 2021;20(43):1-10. 6. aghel n, ramezani z , amirfakhrian s, isolation and quantification of lycopene from tomato cultivated in dezfoul, iran, j of natural pharmaceutical products 2011; 6(1): 9-15. 7. jain s, ancheriya r, srivastva s, soni shankar lal, mukesh s, formulation and characterization of cefixime phytosomes for oral drug delivery, ajprd. 2019; 7(5): -65-73 8. ravi, viresh c, ramakrishna s, sanjay, design and characterization of phytosomal nano carriers for enhanced rutin delivery, am. j. pharmtech res. 2015; 5:(4)1-13. 9. beny b, prakash rao b. preparation and evaluation of oral liquid sustained delivery of metformin hcl, j. pharm. sci. & res. 2021; 13(1): 64-69. 10. anjali k, mohanan s. formulation and evaluation of liquid oral suspension of paracetamol using newly isolated and characterized hygrophila spinosa seed mucilage as suspending agent. asian j pharm clin res, 2018; 11(11): 43741. 11. umasri n, vijayalaxmi s, kousar b, et al. lantana camara: an herbal extract oral suspension formulation and evaluation for its anti-tussive activity. ejbps, 2017; 4(12): 743-50. 12. parthy m. malyadri t, saibabu c. formulation development and characterization of eplerenone insitu oral gels. int j indig herbs drugs 2021; 6(3):69-78. 13. jain s, dhanotiya c, malviya n. physicochemical characterization and determination of free radical scavenging activity of rutin-phospholipid complex ijpsr, 2012; vol. 3(3): 909-13. how to cite this article: viresh k chandur, ramkrishna shabaraya a. characterization of liquid oral containing lycopene phytosomes for improved absorption. indian j pharm drug studies. 2023: 2(2) 82-86. funding: none conflict of interest: none stated shinde et al in vitro pharmacological investigation of fruit of acacia nilotica vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 33 research article in vitro pharmacological investigation of fruit of acacia nilotica pradnya shinde1, shraddha kadav2, ashwini kamble3, ashpak tamboli4 from, 1,2,3sahyadri college of pharmacy, methwade, sangola, maharashtra, 4associate professor, hod, pharmaceutical chemistry, sahyadri college of pharmacy, methwade, sangola, maharashtra. correspondence to: pradnya shinde, vidyanagar, akluj road, malshiras, solapur, maharashtra, india email: pradnyashinde6859@gmail.com abstract the famous medium-sized acacia nilotica tree, often known locally as "babul" or "kikar," is widely distributed throughout tropical and subtropical regions. it has a wide variety of possible medical applications and antioxidant action. this study's goal was to ascertain the fruits of acacia nilotica's invitro anti-inflammatory and antioxidant activities. acacia nilotica hydroalcoholic extract's invitro antiinflammatory and anti-oxidant activities were assessed. the significant activity of inhibition of protein denaturation shown a maximum of 76% inhibition at 500 µg/ml concentration. from the hydroalcoholic extract 400 g/ml showed a maximum of 99.85% of dpph radical scavenging activity. key words: protein denaturation, acacia nilotica, dpph, free radical scavenging ndia's variety of medicinal plants is its true source of wealth. since the beginning of time, numerous varieties of medicinal herbs and their preparation have been used. developing nations have a diverse range of medicinal plants that could be used to create novel biologically active compounds. drug development from plants must inevitably entail a multidisciplinary strategy in terms of a contemporary research project. mineral, plant, and animal products were the primary sources of medicines for a very long time [1]. there is evidence that almost all ancient civilizations used herbs for the treatment of illnesses and for reviving bodily systems. herbal medicines are typically made from a single extract, a fraction of that extract, or a combination of fractions and extracts from various plants. these substances must be carefully standardized for safety, efficacy, and costeffectiveness [2–4]. along with other areas of human activity, the study of medicine and related sciences has advanced quickly. the body's antioxidant defence mechanisms are only effective when the number of free radicals is within the normal physiological range; otherwise, oxidative stress, which can lead to tissue damage and consequent illnesses [5, 6]. material and methods method of extraction weighed 45 gm of powdered drug of fruits of acacia nilotica and it was extracted in soxhlet apparatus by using hydroalcoholic (viz. water: ethanol) solvent. soxhlet apparatus filled with powder of acacia nilotica fruit then added the solvent system into it and started heating at 55 0 c after completing one cycle of the assembly. stopped heating when solvent became colourless then extract was collected and air dried. invitro anti-inflammatory activity inflammation is the body's natural defence mechanism and is characterized by pain, swelling, heat generation, and loss of function in the wounded area. the location and severity of the damage will determine how much function the afflicted part loses. the body's reaction to an accidental cut is comparable to the reaction to other types of tissue injury brought on by burns from heat, radiation, bacterial, or viral attack since inflammation is the body's generalised defence mechanism. inflammation is a complicated process that is linked to pain and includes things like increased protein denaturation, increased vascular penetrability, and layer change. prostaglandin, kinins, and histamine are chemical mediators released by injured bodily tissue (cells) during inflammation. vasodilation and capillary permeability are increased as a result. this causes the blood flow to the injured location to increase. various in vitro and in vivo models are employed to research anti-inflammatory efficacy. acacia nilotica invitro model is assessed for the current study of anti-inflammatory activities. invitro protein denaturation inhibition bioassay chemicals used for the activity: bsa (bovine serum albumin), nacl, disodium hydrogen phosphate, potassium dihydrogen phosphate, methanol, standard diclofenac sodium. i shinde et al in vitro pharmacological investigation of fruit of acacia nilotica vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 34 preparation of reagent % bovine serum albumin: dissolve 5 gm. of bsa in phosphate buffer saline of ph 6.3 phosphate buffer saline of ph 6.3: adjust the ph of the solution to 6.3 by adding hcl after dissolving 0.68 grammes of potassium dihydrogen phosphate, 0.895 grammes of disodium hydrogen phosphate, and 3.50 grammes of sodium chloride. preparation of standard solution: the standard stock solution of diclofenac sodium in methanol was generated at a concentration of 10,000 mg/ml, and three different concentrations of 100, 200, and 500 mg/ml were prepared from this stock solution. test solution preparation: using methanol as the solvent, stock solutions of various fruit extracts containing 10,000 g/ml were created. three distinct concentrations of 100, 200, and 500 g/ml were made from this stock solution. procedure: the procedure for the current study's assay of protein denaturation inhibition is provided below. in this assay, 0.1 ml of test solution of acacia nilotica fruit extract of different concentrations (100 g/ml, 200 g/ml, and 500 g/ml produced in methanol) and 0.9 ml of bovine serum albumin (5 percent aqueous solution) were taken in different test tubes. in place of the test solution for the control, 0.1 ml of distilled water was employed. for 0.1 ml of diclofenac as is customary. instead of the test solution, sodium was used (different concentrations of methanol were made, namely 100, 200, and 500 g/ml). the mixture was incubated at 37 °c for 5 min, then test tubes were heated at 55 °c for 3 min, and finally allowed to cool. each test tube received 2.5 ml of phosphate buffer saline with a ph of 6.3 after being cooled. at 660 nm, the absorbance was spectrophotometrically quantified [14]. standardization: by employing standardised diclofenac sodium, the invitro serum albumin denaturation inhibition bioassay was standardised. fig. no. 1: images of anti – inflammatory activity calculation: the inhibition to the protein denaturation was measured as abs of control – abs of treated % inhibition= --------------------------------------- x 100 abs of control invitro anti-oxidant activity oxidation: the chemical reaction known as oxidation involves the transfer of an electron or hydrogen atom from a material to an oxidising agent. free radicals are produced as a result of the oxidation reaction. antioxidants: by preventing the start of oxidative chain reactions, antioxidants are substances that prevent or delay the oxidation of other molecules. the reducing agents are frequently antioxidants. dpph radical scavenging activity principle: due to its unpaired electron, the persistent free radical known as dpph (1, 1-diphenyl-2-picrylhydrazyl) has a rich purple color. when an antioxidant provides the dpph with an electron, the dpph's deep violet color decolorizes to a pale yellow non-radical form. when a solution of dpph is mixed with a solution of a substrate that can give off a hydrogen atom, at that point this gives rise to the reduced form with the loss of this violet shading. this change in colour of dpph and the subsequent drop in absorbance are observed spectrophotometric ally at a maximum of 517nm. chemicals used for the activity: dpph (2, 2-diphenyl-1picrylhydrazyl), ascorbic acid, methanol. preparation of reagents: 1. dpph solution: dissolve 0.025 mg of dpph in 1000 ml of methanol. prepare fresh solution while using. 2. standard solution (ascorbic acid solution): dissolve 100 mg of ascorbic acid in 100 ml of methanol. 3. test solution (extract solution): dissolve 50 mg of test sample in 50 ml of methanol. procedure: using a methanolic solution of dpph, the ability of acacia nilotica fruit extracts to scavenge free radicals was evaluated. a free radical is dpph. antioxidants convert dpph to the 517 nm-measured 2, 2diphenyl-1-picrylhydrazine. the common antioxidant utilised is ascorbic acid. the assay is carried out utilising the brand williams et al. method. the reaction combination used to assess this activity contains 3.9 ml of methanolic dpph solution and 0.1 ml of various extract solutions (containing 25, 50, 100, 150, 200, and 400 g/ml). instead of extract, 0.1 ml of ascorbic acid was utilised for the standard. moreover, 0.1 ml of methanol was employed for the blank preparation. this combination was incubated at room temperature for 30 minutes in the dark. 517 nm absorbance measurement. all extracts’ percentage dpph radical scavenging activity was evaluated and compared to the reference value [14]. shinde et al in vitro pharmacological investigation of fruit of acacia nilotica vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 35 fig. no. 2: images of antioxidant activity standardization: ascorbic acid was used to standardise the invitro antioxidant dpph free radical scavenging activity. calculation: the percent inhibition was measured as abs of control – abs of treated % inhibition= --------------------------------------- x 100 abs of control result invitro anti-inflammatory activity: invitro protein denaturation inhibition bioassay: bioassay for in vitro suppression of protein denaturation in vitro percentage (percent) inhibition of protein denaturation caused by heat of standard diclofenac sodium in table no.1 invitro antioxidant activity: invitro antioxidant activity: dpph free radical scavenging activity table no. 1: heat induced protein denaturation inhibition of standard diclofenac sodium standard drug concentration in μg/ml absorbance (%) inhibition diclofenac sodium 100 0.115 38.50 200 0.076 59.35 500 0.043 77.00 table no. 2: heat induced protein denaturation inhibition of different extracts of acacia nilotica fruits. plant extract concentration in μg/ml absorbance (%) inhibitin control 0.024 hydroalc oholic extract 100 0.014 41.66 200 0.011 54.16 500 0.006 76 table no. 3: percentage of dpph free radical scavenging activity of fruit extract of acacia nilotica. solution con. in μg/ml (%) inhibition plant extract 25 80.6 50 81.67 100 85.67 150 99.21 200 99.35 400 99.85 standard solution 99.87 graph no. 1: percentage inhibition of protein denaturation at different concentration of fruit extract of acacia nilotica graph no. 2: dpph radical scavenging activity of extract of acacia nilotica fruits discussion acacia nilotica contain number of phytochemicals such as alkaloids, flavonoids, phenols and terpenes which are responsible for anti-inflammatory and antioxidant activity. inflammation include events such as expansion of vascular permeability and increment of protein denaturation. inflammation leads to tissue injury which causes release of chemical mediator such as prostaglandins, kinins and histamine. these mediators leads to vasodilation which causes increase blood flow at the site of injury. antiinflammatory compounds gives inhibitory effect against inflammation by inhibiting release of chemical mediators the protein denaturation inhibition assay is being used for the first time in this work to evaluate the antiinflammatory 41.66 54.16 76 0 10 20 30 40 50 60 70 80 100 200 500 conc. of extracts percentage (%) inhibition percentage (%) inhibition 0 20 40 60 80 100 120 25 50 100 150 200 400 plant extract standard solutio 1 2 percentage (%) inhibition percentage (%) inhibition shinde et al in vitro pharmacological investigation of fruit of acacia nilotica vol 2 | issue 1 | jan – mar 2023 indian j pharm drug studies | 36 efficacy of the fruit extracts of the ethnomedical herb acacia nilotica. heat causes denaturation of proteins. evaluation of hydro-alcoholic extract's inhibition of protein denaturation. according to the results of the current investigation, percentage inhibition rises as concentration goes from 100 g/ml to 500 g/ml. in comparison to the other fruit extracts of acacia nilotica, these all exhibit the highest percentage inhibition of 76 percent at 500 g/ml concentration as shown in table no. 2. following that, the % inhibition of standard diclofenac sodium is compared to that of the hydro-alcoholic extract. at a concentration of 500 g/ml, diclofenac sodium exhibits a 77 percent inhibition in percentage as shown in table no. 1. ros include free radicals which is produced by oxidation process. but the excessive generation of free radicals leads to oxidative stress and causes various diseases. antioxidant compound reduces the generation of free radicals and prevent the disease caused by oxidative stress. the phenolics and flavonoids leads to prevention of ros formation by inhibiting oxidative enzyme. the dpph free radical scavenging assay is being used for the first time in this work to evaluate the anti-oxidant activity of the fruit extract of the ethnomedicinal herb acacia nilotica. the hydro-alcoholic and aqueous concentration's activity to scavenge dpph free radicals has come to an end. in comparison to the other fruit concentrates of acacia nilotica, the 400 g/ml concentration of the acacia nilotica fruit extract exhibits considerable dpph free radical scavenging activity of 99.85 percent as shown in table no. 3. the common antioxidant utilised is ascorbic acid nociceptive action it blocks the pain sensation caused by inflammation. protective extent of acacia nilotica: acacia nilotca provide effect against peripheral as well as cns inflammation. it shows antinociceptive action that it blocks the pain sensation caused by inflammation. it act as antiinflammatory by inhibiting formation of pain mediators i.e. prostaglandins and kinins at peripheral target sites. acacia nilotica shows the cns depressant activity which helps to treat pain attacks, anxiety. it increases the activity of gaba receptor which shows calming effect which used to treat anxiety, pain sensation and insomnia. conclusion in the current investigation, the hydroalcoholic extract's protein denaturation inhibition assay method is used to assess and confirm the invitro anti-inflammatory effect. these analyses lead to the conclusion that, when compared to the industry standard, diclofenac sodium, the hydroalcoholic extract significantly inhibits protein denaturation by 76% at a concentration of 500 g/ml. dpph radical scavenging activity is another method of confirming the in vitro antioxidant activity of acacia nilotica fruit. according to the results of this test, the hydroalcoholic extract exhibits 99.85 percent radical scavenging activity at a concentration of 400 g/ml. for this test, the standard antioxidant is ascorbic acid. from all of the research, it has been determined that the fruit extract of acacia nilotica may have antioxidant and anti-inflammatory properties. references 1. de pasquale c, pistorio ml, veroux p, et al. quality of life and mental health in kidney transplant recipients during the covid-19 pandemic. front psychiatry. 2021, 12: 10.3389/fpsyt.2021.645549 2. dahanukar sa, kulkarni ra, rege nn. pharmacology of medicinal plants and natural products. ijp. 2000, 32. 3. patwardhan b. ayurveda: the ‘designer’ medicine: a review of ethnopharmacology and bioprospecting research. indian drugs. 2000, 37. 4. kelly gs. nutritional and botanical interventions to assist with the adaptation to stress. altern. med. rev. 1999, 4. 5. sahoo n, manchikanti p, dey s: herbal drugs: standards and regulation. fitoterapia. 2010, 81: 10.1016/j.fitote.2010.02.001 6. cheeseman kh, slater tf: an introduction to free radical biochemistry. br med bull. 1993, 49: 10.1093/oxfordjournals.bmb.a072625 7. ajith ta, janardhanan kk. indian medicinal mushrooms as a source of antioxidant and antitumor agents. j. clin. biochem. nutr. 2007, 40: 10.3164/jcbn.40.157 8. sies h. oxidative stress: oxidants and antioxidants. exp. physiol. 1997, 82: 10.1113/expphysiol.1997.sp004024 9. zhang l, ravipati as, koyyalamudi sr, et al. antioxidant and anti-inflammatory activities of selected medicinal plants containing phenolic and flavonoid compounds. j agric food chem. 2011, 59: 10.1021/jf203146e 10. tang sy, whiteman m, peng zf, et al. characterization of antioxidant and antiglycation properties and isolation of active ingredients from traditional chinese medicines. free radic biol med. 2004, 36: 10.1016/j.freeradbiomed.2004.03.017 11. cai y, luo q, sun m, corke h. antioxidant activity and phenolic compounds of 112 traditional chinese medicinal plants associated with anticancer. life sci. 2004, 74: 10.1016/j.lfs.2003.09.047 12. dragland s, senoo h, wake k, et al. several culinary and medicinal herbs are important sources of dietary antioxidants. j nutr. 2003, 133: 10.1093/jn/133.5.1286 13. hendra r, ahmad s, oskoueian e, et al. antioxidant, antiinflammatory and cytotoxicity of phaleria macrocarpa (boerl.) scheff fruit. bmc complement altern med. 2011, 11: 10.1186/1472-688211-110 14. dafallah aa, al-mustafa z. investigation of the antiinflammatory activity of acacia nilotica and hibiscus sabdariffa. am j chin med. 1996, 24: 10.1142/s0192415x96000323 how to cite this article: pradnya shinde, shraddha kadav, ashwini kamble, ashpak tamboli. in vitro pharmacological investigation of fruit of acacia nilotica. indian j pharm drug studies. 2023; 2(1) 33-36. funding: none conflict of interest: none stated mhamane & adat cicer arietinum leaf anti-diabetic activity vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 70 original article in-vitro anti-diabetic activity of leaves of cicer arietinum supriya mhamane1, priyanka adat2 from, 1sahyadri college of pharmacy, methwade, sangola, maharashtra, 2assistant professor, pharmaceutical chemistry, sahyadri college of pharmacy, methwade, sangola, maharashtra, india. abstract chickpea, (cicer arietinum), also known as garbanzo bean or bengal gram, yearly plant of the pea family fabaceae, widely grown for its beneficial seeds. chickpeas are cultivated in more than 50 countries. largest areas under chickpeas are india, pakistan, ethiopia, turkey and mexico. this study incorporates the cicer arietinum leaves anti diabetic activity. it has traditional medicinal uses like anti-diabetic and it is also used in illness to optimize the taste. this work envisaged the anti-diabetic activity of leaves of cicer arietinum by using uv-visible spectrophotometer at 540nm. the maximum peak of inhibition of alpha-amylase is observed at 10µg/ml. key words: cicer arietinum, alpha amylase, enzyme inhibition, 3, 5-dinitro salicylic acid. iversity in the medicinal plants is the real wealth of india. since time immemorial many different types of medicinal herbs and their formulation have been used. therapy using medicinal plants is more esteemed than using synthetic chemicals shown by practical experience and several modern researches works. medicinal plants growing wild or cultivated are rich in the world, which forms a enormous natural and economical health which must be protected, increased for the development of economy, wealth of nation and health of people [1]. rich flora of medicinal plants in developing countries is potential source of new biologically active substances [2]. regarding modern research enterprise, drug development from plants must necessarily suggest a multi-disciplinary approach. as sources of many potent drugs, plants are used medicinally worldwide [3]. diabetes mellitus is not a single disease but is a group of metabolic disorders affecting a huge number of populations in the world. it is mainly characterized by chronic hyperglycemia, resulting from defects in insulin secretion or insulin action [4]. even though the cases of diabetes are increasing day by day, except insulin and oral hypoglycemic drugs no other way of treatment has been successfully developed so far. amylase and glucosidase are mainly used for evaluating the anti-diabetic activity of a particular drug [5]. access this article online received – 24th jan 2024 initial review – 02nd feb 2024 accepted – 08th feb 2024 quick response code materials and methods method of extraction: 50g of leaf powder has to be subjected to soxhlet extraction process using alcohol as the solvent. make sure that all the powder should be wet and the solvent passes from powder and settled to the bottom. due to heating to the apparatus at 550c the solvent system again goes at top of powder and by passing powder it again settle at bottom in rbf (round bottom flask) along with the chemical constituents which are dissolve in the solvent system. heat this apparatus upto the solvent becomes colorless. after that stop heating and keep for 15min for cooling. after cooling, collect all the extract in a clean beaker, then transfer some extract in a petri plate for air dry [6]. in vitro anti-diabetic activity: when blood glucose (sugar) is very high, then it is said to be diabetes (hyperglycemia). the main source of energy in our body is blood glucose, which is obtained from the food that we eat. insulin is a hormone secreted from beta cells of islets of langerhans in pancreas. with the help of insulin blood glucose enters in the cells and utilized as a source of energy [7]. α-amylase and α-glucosidase are carbohydrate hydrolyzing enzymes. α-amylase converts polysaccharides like starch, glycogen to disaccharides by breaking 1, 4-glycosidic linkage by hydrolysis and digest the carbohydrate. α-glucosidase converts this disaccharides to monosaccharides, which are responsible for postprandial hyperglycemia [8]. the agents / drugs that lower the abnormally high glucose (sugar) level in the blood [9]. __________________________________________________ correspondence to: supriya mhamane, korwali, tal. mohol, dist. solapur, maharashtra, india. email: supriyamhamane99@gmail.com. tel.: +91 8080244609. d mailto:supriyamhamane99@gmail.com mhamane & adat cicer arietinum leaf anti-diabetic activity vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 71 α-amylase inhibitory activity chemicals used in the assay: α-amylase, potato starch, sodium acetate trihydrate, glacial acetic acid, sodium hydroxide, 3,5-dinitro salicylic acid, sodium potassium tartarate, ethanol[10,11]. preparation of reagents 1. α-amylase solution: weigh 27.5mg α-amylase. take 250ml beaker and pour 100ml distilled water and weighed 27.5mg α-amylase. stir it for 2 min [10,11]. 2. sodium acetate buffer: weigh 2.72gm sodium acetate trihydrate and add it in 80 ml of distilled water. adjust the ph at 4.8 with glacial acetic acid. make up the volume at 100 ml with distilled water [12]. 3. starch solution (0.1% w/v): weigh 0.1gm potato starch and add it in 100ml sodium acetate buffer. stir to mix properly [10,11,15]. 4. 2n naoh: weigh 2gm naoh and add it in 25ml distilled water and stir it [13]. 5. 3, 5-dinitro salicylic acid reagent: weigh 1gm 3, 5-dinitro salicylic acid and add it in 50ml distilled water. weigh 30gm sodium potassium tartarate and add it in above solution by continue stirring. add 20ml 2n naoh and dilute this solution to 100ml with distilled water [10,11]. table 1: preparation of reagent for in-vitro anti-diabetic activity of leaves of cicer sarietinum. reagent quantity required α-amylase solution 0.5ml sodium acetate buffer [q.s] starch solution (0.1% w/v) 0.5ml 2n naoh [q.s.] 3, 5-dinitro salicylic acid reagent 1ml preparation of test solution: the stock solution of leaf extract was prepared of 100µg/ml by using ethanol and 70% aqueous ethanol as a solvent. for this, 0.001gm (1mg) semidried drug extract were added in 10ml of solvent. from this stock solution, 5 different concentrations of 2, 4, 6, 8 and 10µg/ml were prepared by pipetting 0.2, 0.4, 0.6, 0.8 and 1ml of stock solution and dilute it up to 10ml [14]. procedure: in this assay, 0.5ml of test solution of cicer arietinum leaf extract of different concentration such as 2µg/ml, 4µg/ml, 6µg/ml, 8µg/ml and 10µg/ml prepared in ethanol and hydro-alcohol were taken in different test tubes. then 0.5ml of α-amylase enzyme solution and 0.5ml of starch solution were added and allow standing for 3 minutes for reaction among the drug extract, enzyme and starch. then add 1ml of 3, 5-dinitro salicylic acid to stop the reaction. at the same time same procedure follow for control, instead of 0.5ml test solution add 0.5ml distilled water. then measure the absorbance at 540nm using uvvisible spectrophotometer [14]. figure 1: dilution of anti-diabetic activity calculation: inhibition of α-amylase were calculated by using following formula: % inhibition= × 10 results in-vitro anti-diabetic activity table 2: in-vitro anti-diabetic activity of cicer arietinum for ethanolic and hydro-alcoholic extract conc. (µg/ml) percentage inhibition (acarbose) (%) percentage inhibition (ethanolic) (%) percentage inhibition (hydro-alc.) (%) 2 66.78 12.89 1 4 69.31 29.92 3.84 6 82.78 38.13 6.47 8 84.12 54.02 27.88 10 99.86 99.64 80.1 graph 1: in-vitro anti-diabetic activity of cicer arietinum for ethanolic and hydro-alcoholic extract. discussion cicer arietinum plant shows presence of various plant constituents like alkaloids, glycosides, steroids, carbohydrates, mhamane & adat cicer arietinum leaf anti-diabetic activity vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 72 amino acids, inorganic elements. these constituents exhibit anti-diabetic activity [16]. alpha amylase converts polysaccharide into glucose and maltose. that’s why blood glucose level increases and causes the postprandial hyperglycemia. by inhibiting the alpha amylase enzyme we can prevent breakdown of carbohydrate into glucose and maltose [17,18]. alpha amylase inhibitory assay is firstly applied to study the anti-diabetic activity of the leaves of cicer arietinum. demonstration of the alpha amylase inhibition by ethanolic and hydro-alcoholic extract was done by this study. this study shows percentage inhibition of alpha amylase. ethanolic extract shows high percentage inhibition at 10µg/ml i.e. 99.64%. the percentage inhibition increases as concentration increases as shown in table and graph. hydroalcoholic extract shows high percentage inhibition at 10µg/ml i.e. 80.10%. the percentage inhibition increases as concentration increases as shown in table and graph [19,20]. conclusion these studies conclude that, the ethanolic and hydro-alcoholic extract of leaves of cicer arietinum shows anti-diabetic activity by alpha amylase inhibition assay. but the ethanolic extract has high percentage inhibition as compared with hydro-alcoholic extract. ethanolic extract at 10µg/ml shows high percentage inhibition i.e. 99.64%. the percentage inhibition increases as concentration increases from 2µg/ml to 10µg/ml as shown in table and graph. references 1. pradnya shinde et al. in vitro pharmacological investigation of fruit of acacia nilotica. indian j pharm drug studies. 2022; 3(3):111-115. 2. ali esmail al-snafi. the medical importance of cicer arietinum a review. res gate pub. 2016; 6(7):62-74. 3. karnail singh, kavita gahlot. pharmacognostical and pharmacological importance of cicer arietinum linn a review. rjpt pub. 2018; 11(10). 4. sameer suresh bhagyawant et al. chickpea (cicer arietinum l.) lectin exhibit inhibition of ace-i, α-amylase and α-glucosidase activity. pubmed publication. pmid: 30919768. 5. zoha malik et al. hypoglycemic potential of combined methanolic extract of nigela sativa (black cumin) and cicer arietinum (chickpea). res gate pub. 2022. doi:10.52700/pjbb. 6. karnail singh, kavita gahlot. pharmacognostic evaluation of stem and leaf of cicer arietinum linn. j pharmaco phytochemistry. 2018; 7(6):165-174. 7. https://www.niddk.nih.gov/health-information/diabetes/overview/ what-is-diabetes#:~:text=diabetes%20is%20a%20disease% 20that,to%20be%20used%20for%20energy. 8. madhusudhan telagari, kirankumar hullatti. in-vitro α-amylase and α-glucosidase inhibitory activity of adiantum caudatum linn. and celosia argentea linn. extracts and fractions. indian j pharmacol. 2015; 47(4):425–429. 9. janet l stringer, john a thomas. antidiabetic drug medicine. britannica. https://www.britannica.com/science/diuretic. 10. r. manikandan et al. phytochemical and in-vitro anti-diabetic activity of methanolic extract of psidium guajava leaves’, inter j curr microbiolo and applied sci. 2013; 2(2):15-19. 11. m amin mir et al. the anti-diabetic and spectral analysis of various extracts of lilium polyphyllum. res gate pub. 2020. 12. https://webstor.srmist.edu.in/web_assets/srm_mainsite/files/files/b i501%20advanced%20biochemistry%20and%20immunology.pd f 13. https://www.virusys.com/rsticketspro/9-sop-106-preparation-of2n-naoh/article 14. m amin mir et.al. the anti-diabetic and spectral analysis of various extracts of lilium polyphyllum. res square pub. doi: https://doi.org/10.21203/rs.3.rs-42363/v1 15. khandelwal k.r. practical pharmacognosy. nirali prakashan, 2007; 9-161. 16. ak jukanti, pm gaur, cll gowda et al. nutritional quality and health benefits of chickpea (cicer arietinum l.): a review. cambridge university. 2012. 17. mohammed hassanien am., usage of cicer arietinum as a local and eco-friendly natural coagulant in sewage treatment and its ability to increase the formation of floc process. doi: 10.21931/rb/2021.06.03.9 1939-1943. 18. cicer arietinum. rin. agricultural research service (ars), united states department of agriculture (usda). 2014. 19. cosmas parwada1, tatenda f parwada, justin chipomho, et al. evaluation of cicer arietinum (chickpea) growth performance and yield in different soil types in zimbabwe. j. current opinion crop sci., 2022; 3(1): 16-27. 20. ahm mahbubur rahman, m ismot ara parvin. taxonomic studies on the family fabaceae (weeds) at rajshahi university campus. plant. 2015; 3(3):20-25. doi: 10.11648/j.plant.20150303.11 how to cite this article: supriya mhamane, priyanka adat. in-vitro anti-diabetic activity of leaves of cicer arietinum. indian j pharm drug studies. 2024; 3(2):70-72. funding: none; conflicts of interest: none stated http://dx.doi.org/10.52700/pjbb https://www.niddk.nih.gov/health-information/diabetes/overview/%20what-is-diabetes#:~:text=diabetes%20is%20a%20disease% 20that,to%20be%20used%20for%20energy https://www.niddk.nih.gov/health-information/diabetes/overview/%20what-is-diabetes#:~:text=diabetes%20is%20a%20disease% 20that,to%20be%20used%20for%20energy https://www.niddk.nih.gov/health-information/diabetes/overview/%20what-is-diabetes#:~:text=diabetes%20is%20a%20disease% 20that,to%20be%20used%20for%20energy https://www.britannica.com/science/diuretic https://webstor.srmist.edu.in/web_assets/srm_mainsite/files/files/bi501%20advanced%20biochemistry%20and%20immunology.pdf https://webstor.srmist.edu.in/web_assets/srm_mainsite/files/files/bi501%20advanced%20biochemistry%20and%20immunology.pdf https://webstor.srmist.edu.in/web_assets/srm_mainsite/files/files/bi501%20advanced%20biochemistry%20and%20immunology.pdf https://www.virusys.com/rsticketspro/9-sop-106-preparation-of-2n-naoh/article https://www.virusys.com/rsticketspro/9-sop-106-preparation-of-2n-naoh/article https://doi.org/10.21203/rs.3.rs-42363/v1 mhamane s & shinde y anti-solar activity of malus domestica vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 174 original article in-vitro anti-solar activity of peel of malus domestica supriya mhamane1, yogesh shinde2 from, 1sahyadri college of pharmacy, methwade, sangola, maharashtra, 2assistant professor, pharmaceutics, sahyadri college of pharmacy, methwade, sangola, maharashtra. abstract hormone production is stimulated by sunlight and it can lead to the synthesis of vitamin d, it also stimulates the reconstruction of cells of the skin. melanin is the pigment that acts as a natural sunscreen for the skin. melanin is also stimulated by sunlight. sunburn, rashes, miliaria, itching, and other skin-related diseases can occur due to the excess harmful sun rays. this study determines the anti-solar property of the uv absorption of the peel of malus domestica. by using the soxhlet apparatus, the extract was obtained. for extraction of 25gm of the powdered drug, 100ml of solvent i.e. distilled water and methanol in the 2:5 ratio were used. the activity is determined by using uv-visible spectrophotometer in the range of 200–400 nm. the maximum absorbance was determined by conc. 6µg/ml at 205nm, good absorbance shows at conc. 4µg/ml at 207nm and moderate absorbance by conc. 2µg/ml at 207nm. keywords: malus domestica, uv spectrophotometer, anti-solar activity, uv-rays absorption, wavelength. he surface area of the skin is 1.5 to 2 m2. it is protective in nature. continuous exposure to sunlight can affect the skin and damage it. to protect the skin from this damage, it releases melanin [1]. excessive exposure to sunlight can cause rashes, sunburn, miliaria, itching, and other skin-related diseases. to protect the body from this, the sunscreens are used. based on the wavelength of uv light, it is uva, uvb, and uvc [2]. solar radiation affects the skin. uv-a and uv-b can cause sunburn, cutaneous degeneration, and cell skin cancer [3]. materials and methods peel of malus domestica was collected and shade dried. then it is powered by using a mixer grinder and passed from sieve no. 20. extraction method in the soxhlet apparatus, add 25gm of powdered drug and pour the solvent system in the ratio 2:5 of distilled water: methanol. pour solvent up to which three cycles were complete. then start the heating mantle at 550c and start the water supply to the condenser. the solvent pass from the powder and along with constituents present in the powder settles in the round bottom flask. keep this process up upto the solvent comes colorless [4]. qualitative tests 1) shinoda test: in a dry extract, add 5 ml of 95% ethanol and a few drops of conc. hydrochloric acid and 0.5gm of magnesium turnings. pink color shows the presence of flavonoids [5]. 2) lead acetate test: in a small quantity of extract, add lead acetate solution, the yellow-colored precipitate is formed [5]. _______________________________________ correspondence to: supriya mhamane, pharmaceutics, korwali, tal. mohol, dist. solapur, maharashtra, india. email: supriyamhamane99@gmail.com tel.: +91 8080244609 access this article online received – 12th june 2023 initial review – 17th june 2023 accepted – 28th june 2023 quick response code t mailto:supriyamhamane99@gmail.com mhamane s & shinde y anti-solar activity of malus domestica vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 175 fig.1: peel of malus domestica fig.2: peel powder of malus domestica fig.3: peel powder sieving fig.4: shinoda test fig.5: lead acetate test in-vitro anti-solar activity of peel of malus domestica procedure 10mg powdered drug was added in 100 ml of solvent i.e. distilled water: methanol in a 2:5 ratio to prepare a stock solution of 100µg/ml concentration. from this stock solution, 5 different concentrations of 2, 4, 6, 8, and 10 µg/ml were prepared by pipetting 0.2, 0.4, 0.6, 0.8, and 1 ml of stock solution and dilute it up to 10 ml. then observe the maximum absorbance and the wavelength at which maximum absorbance was observed photometrically by using a uv-visible spectrophotometer [6]. results and discussion the peel of malus domestica contains plant constituents such as flavonoids. this constituent shows anti-solar activity. uv radiation from the sunlight can cause the diseases like skin rashes, sunburn, miliaria, itching, and other skin-related diseases. to protect the skin from these diseases, anti-solar agents are used. anti-solar agents absorb the uv radiation which has 200 to 400 nm wavelength [1] (table 1-3). in-vitro anti-solar activity table 1: in-vitro anti-solar activity of methanolic extract of malus domestica of concentration: 2µg/ml s. no. wavelength nm. abs. 1 349.80 0.002 2 344.40 0.002 3 338.60 0.005 4 318.40 0.007 5 305.40 0.007 6 375.80 0.017 7 368.80 0.019 8 362.00 0.018 9 209.80 0.250 10 207.00 0.266 11 348.00 0.000 12 341.80 0.001 13 320.40 0.005 14 310.00 0.006 15 304.60 0.005 16 301.00 0.004 17 274.20 0.016 18 265.60 0.017 19 252.40 0.014 20 203.60 0.234 table 2: in-vitro anti-solar activity of methanolic extract of malus domestica of concentration: 4µg/ml s. no. wavelength nm. abs. 1 395.00 0.002 2 344.00 0.009 3 332.00 0.014 mhamane s & shinde y anti-solar activity of malus domestica vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 176 4 322.00 0.015 5 310.00 0.018 6 303.60 0.018 7 279.80 0.040 8 260.00 0.042 9 207.00 0.315 10 345.00 0.000 11 342.00 0.006 12 320.40 0.013 13 312.00 0.016 14 306.00 0.016 15 257.00 0.005 16 0.006 0.006 table 3: in-vitro anti-solar activity of methanolic extract of malus domestica of concentration: 6µg/ml s. no. wavelength nm abs. 1 378.40 0.016 2 371.00 0.018 3 354.40 0.022 4 349.00 0.025 5 346.80 0.024 6 342.20 0.024 7 334.40 0.026 8 332.00 0.028 9 327.40 0.027 10 324.40 0.028 11 320.80 0.032 12 319.00 0.031 13 314.00 0.030 14 308.00 0.036 15 302.20 0.045 16 386.20 0.056 17 380.40 0.065 18 278.40 0.063 19 373.20 0.065 20 368.20 0.065 21 363.60 0.063 22 207.80 0.561 23 205.00 0.568 24 375.60 0.015s anti-solar activity is first performed to study the absorption of uv radiation of the peels of malus domestica. estimation of the anti-solar activity by the methanolic extract was completed by this study. this study gives the range of wavelengths in which absorption of uv radiation was observed. the maximum absorption of uv radiation was found at a concentration of 2µg/ml, at 207nm i.e. 0.266. the maximum absorption of uv radiation was found at a concentration of 4µg/ml, at 207nm i.e. 0.315. the maximum absorption of uv radiation was found at a concentration of 6µg/ml, at 205nm i.e. 0.568. from 2µg/ml, the maximum absorption of uv radiation increases as concentration increases, which is as shown in (graph 1-4). the extract of 6µg/ml shows maximum absorption of uv radiation i.e. 0.568 as compared to the other concentrations [3] [7]. graph 1: in-vitro anti-solar activity of methanolic extract of malus domestica of concentration: 2µg/ml mhamane s & shinde y anti-solar activity of malus domestica vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 177 graph 2: in-vitro anti-solar activity of methanolic extract of malus domestica of concentration: 4µg/ml graph 3: in-vitro anti-solar activity of methanolic extract of malus domestica of concentration: 6µg/ml graph 4: comparison of in-vitro anti-solar activity of methanolic extract of malus domestica of concentration: 2µg/ml, 4µg/ml, 6µg/ml, 8µg/ml, 10µg/m mhamane s & shinde y anti-solar activity of malus domestica vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 178 conclusion the conclusion of this study shows that the methanolic extract of the peel of malus domestica shows anti-solar activity by determining its maximum absorption of uv radiation in a uv region i.e. 200nm to 400nm of wavelength. the extract of 6µg/ml shows maximum absorption of uv radiation i.e. 0.568 as compared to other concentrations. the maximum absorption of uv radiation was found at a concentration of 6µg/ml, at 205nm i.e. 0.568. from 2µg/ml, the maximum absorption of uv radiation increases as concentration increases. references 1. pawar d. b. et al, “study on anti-solar activity of ethanolic extract of leaves of ipomoea batatas linn”, world journal of pharmaceutical research 2014; 3(5):382-386. 2. rutuja sabne et al, “in-vitro anti-oxidant and uv protective activity of yellow flowers of tecoma stans l. juss”, international journal of chemistry, pharmacy & technology 2018 3(2):38-40. 3. natashya advaita et al, “in vitro evaluation of sun protection factor of vasconcellea pubescens fruit extract”, international conference on pharmaceutical research and practice, 2018;122-125. 4. karnail singh and kavita gahlot, ‘pharmacognostic evaluation of stem and leaf of cicer arietinum linn’, journal of pharmacognosy and phytochemistry 2018; 7(6): 165-174. 5. khandelwal k.r., ‘practical pharmacognosy’, nirali prakashan, 2007, 9-161. 6. m. amin mir et.al. “the antidiabetic and spectral analysis of various extracts of lilium polyphsyllum”, research square publication 2020;1-20 7. seyed alireza mortazavi et al, “an in-vitro evaluation of various rosa damascena flower extracts as a natural antisolar agent”, january 2004, international journal of cosmetic science 25(6):259-65. how to cite this article: supriya mhamane, yogesh shinde. in-vitro anti-solar activity of peel of malus domestica. indian j pharm drug studies. 2023; 2(4):174-178. funding: none conflict of interest: none stated https://www.researchgate.net/journal/international-journal-of-cosmetic-science-0142-5463 https://www.researchgate.net/journal/international-journal-of-cosmetic-science-0142-5463 hegde k et al. review on pharmacological potential of nephelium lappaceum l vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 90 review article a brief review on pharmacological potential of nephelium lappaceum l karunakar hegde1, thrupthi p rai2, ramkrishna shabaraya a3 from 1head of the department, 2pg scholar, 3prof. principal, department of pharmacology, srinivas college of pharmacy farangipete post, mangalore 574143. abstract nephelium lappaceum l. belongs to the sapindaceae family that grows in tropical and subtropical climates commonly known as rambutan. rambutan has a long history not only as a delicious and succulent fruit but also as a traditional medicine. the name "rambutan" comes from the malay-indonesian word "rambut," which means "hairy." because of this, it is occasionally referred to as "hairy litchi." the fruit is an ovoid berry that ranges in color from yellow to orange-red, or from brilliant red to maroon. its leathery skin is completely covered in spinterns of varying lengths. in asia, where it is commonly consumed fresh, canned, or processed and loved for its reviving flavor and unique appearance, rambutan is a significant commercial crop. researchers have discovered that rambutan fruit extracts include phytochemicals with antioxidant, antibacterial, antidiabetic, antiviral, anti-inflammatory, anti-cancer, anti-diarrheal, and cardiovascular activity. rambutan is a superfruit that can be used as a contemporary medicine to treat illnesses and promote good health. key words: nephelium lappaceum l., traditional, phytochemical, malay-indonesian, health he fruit known as nephelium lappaceum l. is a member of the sapindaceae family and is indigenous to tropical areas including indonesia, china, india, australia, malaysia, mexico, and thailand. the malay-indonesian word rambut, which means "hairy," is the source of the name for the rambutan fruit. n. lappaceum, m. cuspidatum var. eriopetalum, n. junglandifolium, n. maingayi, n. meduseum, n. ramboutan-ake, n. melanomiscum, n. reticulatum, and n. uncimatum are among the nine edible rambutan species that make up the nephelium genus, which has 22 species worldwide [1]. the fruit is an ovoid berry that ranges in color from yellow to orange-red to brilliant red to maroon. the flesh has a sweet to very mild sour flavor and is juicy and translucent white in color [2]. it is a significant commercial crop in asia and the fruit is valued for its reviving flavor and exotic appearance whether it is eaten fresh, preserved in syrup, or processed [3]. a grayish-brown tree with an evergreen canopy, nephelium lappaceum l grows to a height of 10 to 12 access this article online received – 29th may 2023 initial review – 15th june 2023 accepted – 27th june 2023 quick response code meters.the components of rambutan include total weight: 27.4%, peel: 13.2%, fruit: 11.7%, seeds: 2.53%, and seed coat: 60%1. the rambutan plant's various parts each have unique advantages and qualities. additionally, a test for antioxidant capacity found that rambutan fruit has an antioxidant level of 71.5%, which is equal to ascorbic acid [4]. since ancient times, the plant has been utilized in traditional medicine as a treatment for high blood pressure and diabetes. additionally, fruits have traditionally been a possible source of minerals and other nutrients [5]. as a result, this article provides an updated assessment of this significant plant with a focus on the traditional usage, phytochemistry, and pharmacological elements that will help researchers in the future find relevant scientific material (figure 1, table 1 and table 2). figure 1: rambutan fruit _______________________________________ correspondence to: karunakar hegde, head of the department, department of pharmacology, srinivas college of pharmacy farangipete post, mangalore email: khegde_sh2003@yahoo.com t mailto:khegde_sh2003@yahoo.com hegde k et al. review on pharmacological potential of nephelium lappaceum l vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 91 vernacular names [6] table 1: vernacular names of nephelium lappaceum l. hindi rambutan malayalam rambuttan bengali rambutan kannada rambutan telugu rambutan french ramboutan english rambutan taxonomical classification [5,7] table 2: taxonomical classification of nephelium lappaceum l. kingdom plantae subkingdom tracheobionta superdivision spermatophyta division magnoliophyta class magnoliopsida subclass rosidae order sapindales family sapindaceae genus nephelium l. species naphelium lappaceum l. habitat: a temperature range of 22–300°c and a maximum height of 700 m is deemed suitable for the growth and development of the rambutan in the majority of tropical asian locales. high rainfall regions with 2,000 to 5,000 mm of rain per year, distributed fairly consistently throughout the year, are typically regarded as appropriate, but supplemental irrigation may be necessary, particularly during the time from flower set to harvesting. although the rambutan can be grown effectively in a variety of soils, rich, well-drained, sandy loams or clay loams that are rich in organic matter produce the best growth and fruiting. alluvial soils made of basaltic rocks are the most frequently utilized forms of soil, however, crops grown on red lateritic soils typically succeed when fertilized appropriately using a mix of organic and inorganic fertilizers [8]. distribution: over 200 rambutan clones have been chosen for tropical asia, and malaysia has the widest variety of farmed and wild rambutan species. rambutan is grown in malaysia, thailand, the philippines, northern australia, sri lanka, india, madagascar, costa rica, the congo, and a few south american nations. the biggest rambutan producers in the world are thailand, malaysia, and indonesia. in indonesia, malaysia, australia, the philippines, and hawaii, rambutan farming is growing. in the indian states of tamil nadu, thrissur, pathanamthitta, kottayam, and ernakulum in kerala, nagerkoil, courtralam, the nilgiris, dakshina kannada and kodagu in karnataka, rambutan is grown in household gardens [1,9]. botanical description: evergreen rambutan trees can reach heights of 12 to 20 meters. the 12m tall, grayishbrown branching trees are vegetatively propagated. the lateral roots stay close to the soil surface while the main root penetrates several meters into the ground [5,9]. leaves: the leaves have 2 to 4 pairs of petiolate, alternating, and pinnate leaflets. the leaflets range in size from 5 to 28 cm and have an ovate to elliptical shape. the rachis maybe 7 to 30 cm long and 3-10 cm wide, with a complete edge, while the petioles are thick and 0.5 to 1 cm long. the inflorescence is upright, has numerous branches, and has numerous blooms [9]. flowers: small, 2.5–5 mm, apetalous flowers are produced in upright terminal panicles and are 15–30 cm broad. rambutan trees can be hermaphrodite, male, or female. only male flowers, which are apetalous and have 5-7 stamens and a primitive ovary, are produced by the male tree. a panicle can have between 300 and 500 blooms. the hermaphrodite trees produce hermaphrodite flowers with a bifid stigma, a bilocular ovary, and six inactive stamens. there are 500–800 flowers on a panicle. while the female tree only yields female flowers, the hermaphrodite trees also bear 0.5 to 0.8% male blossoms [9]. fruit: the fruit is a loose pendant cluster of 10–20 round to oval drupes that are 3–8 cm long and 3–4 cm in diameter. fruits have leathery, reddish, orange, or yellow skin that is coated with fleshy hair. the flesh (aril), which adheres to the seed, is 0.4–0.8 cm thick, transparent, juicy, acidic, subacidic, or sweet [1,9]. seeds: the seed is 2-3 cm long, oblong, and flattened. its color is shiny brown. the seed is soft, long to elliptical oval, 20 to 25 mm wide, and between 12 and 22 mm long [9]. chemical constituents a phytochemical investigation carried out on nephelium lappaceum l. epicarp revealed the presence of various phytoconstituents such as carbohydrates, alkaloids, steroids and sterols, glycosides, flavonoids, triterpenoids, tannins, proteins and amino acids and essential elements in the peel like mn, fe, zn, mg, k, na, ca [10,11,12]. it was determined that the main fatty acids in a rambutan seed are oleic, arachidonic, palmitic, stearic, gondoic, behenic, and hegde k et al. review on pharmacological potential of nephelium lappaceum l vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 92 palmitoleic. the chemical composition of the rambutan fruit pulp is proteins, carbohydrates, calcium, vitamin c, and iron. rambutan peel has a high content of phenolic compounds and antioxidant activity [11,12]. ellagic acid, corilagin, and geraniin from the peels and reported for their antioxidant activities. geraniin is the major constituent that exhibited much greater antioxidant activities [13,14]. pharmacological activities antioxidant activity: analyzing the activity of free radical scavengers allows for the measurement of antioxidant capacity. rambutan peel has similar free radical scavenging properties to geraniin [15]. geraniin, which constitutes the majority of the peel of the rambutan fruit, is why [16]. free radicals that might result in oxidative cell damage can be stabilized by antioxidants. the two antioxidant assays that receive the greatest attention are frap and the dpph radical scavenging assay. there are additional reports of other assays such as the lipid peroxidation assay, the galvinoxyl assay, and the beta-carotene bleaching assay [17]. antimicrobial activity: peel from a rambutan fruit contains antibacterial properties. salmonella typhi bacteria are actively inhibited by ethanol extract. the germs vibrio cholera, enterococcus faecalis, staphylococcus epidermidis, and pseudomonas aeruginosa were all susceptible to the effects of water, ether, and methanol extracts. the bacteria proteus vulgaricus and bacillus cereus were then susceptible to the chloroform extract's effects. compared to the seeds of the rambutan fruit, which only have activity against the microorganisms escherichia coli, s. aureus, p. aeruginosa, s. subtilis, and streptococcus pyogenes. the primary phenolic components found in the methanol extract of the rambutan peel, including geraniin, ellagic acid, quercetin, rutin, and chorilagin, shown inhibitory efficacy against bacteria at various doses and incubation durations [16,18]. anti-viral activity: geraniin is the substance that has an impact on denv-2 inhibition. the researchers concluded that geraniin compounds could prevent denv-2 from acting by interfering with the virus or by interacting with viral receptors that were important in viral penetration into cells but did not involve cellular receptors. to increase contact with virions, or viral particles, geraniin functions extracellularly. this interaction can lessen denv-2's ability to spread [19]. anti-osteoporosis: retinoic acid was incubated with the osteoporosis/op rat model in vivo to assess its capacity to inhibit op using histological, physicochemical, and serum biochemical indicators (serum calcium, phosphorus, alkaline phosphate/alp, and osteocalcin/ocn). rambutan peel was bigger than in the model group in the serum calcium and phosphorus of the positive and phenolic groups. rambutan peel was able to lower alp and ocn levels by 17.93% and 43.11%, respectively. these findings imply that rambutan peel can enhance retinoic acid-induced op rats' blood markers of bone metabolism [20]. anti-inflammatory activity: rambutan peel's phenolic content has been found to have anti-inflammatory properties. geraniin, catechins, and ellagic acid are the primary phenolic components that contribute to antiinflammatory effects. by lowering the synthesis of inflammatory mediators, or through free radicals and metal chelating action, phenolic substances have antiinflammatory effects [21]. anti-diabetic activity: in test animals, the presence of flavonoids such tannins, polyphenols, quercetin, catechins, and egcg can lower blood glucose levels by preventing the absorption of glucose, which in turn stimulates the release of insulin and indirectly overrides the antioxidant process. geraniin's capacity to stop formation accounts for its ability to lower blood glucose levels [22]. in rats given alloxan, the ethanol extract of rambutan and durian fruit peel can reduce blood glucose levels. rambutan fruit peels' ethanolic extract contains egcg, quercetin, and geraniin, all of which demonstrated antihyperglycemia activity [23]. anti-hypercholesterolemia: utilizing three different doses, examine the impact of decreasing blood cholesterol on rambutan peel extract. it was discovered that rambutan fruit peel extract significantly reduced blood cholesterol levels in rats, 60.75% more effectively than the positive control. this demonstrates how powerful an antihypercholesterolemia with herbal medicinal components the ethanolic extract of rambutan peel is [24]. anticancer activity: rambutan peel extract was found to have an antiproliferative impact on the cell lines mdamb0231 and mg-63 after 72 hours of incubation. polyphenol molecules are believed to have anti-cancer properties. according to some substances, biological reactive oxygen species (ros) can be neutralized. ros is a type of organism that fights cancer. while ros can be lowered by either natural or synthetic antioxidants, high ros reduction can prevent cancer [25]. antidiarrhoeal activity: when evaluated using a rat model of castor oil-induced diarrhea, the methanol extract of the seeds was found to have considerable antidiarrheal action. when compared to loperamide, the extract showed a substantial suppression of fecal dropping [26]. hegde k et al. review on pharmacological potential of nephelium lappaceum l vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 93 cardiovascular activity: according to a study, ethanol extract from rambutan bark had immediate impacts on rats' respiratory and cardiovascular responses. increases in mabp, systolic blood pressure, and heart rate were seen in the data, which showed a protracted cardiovascular response [27]. conclusion nephelium lappaceum l., a member of the sapindaceae family and commonly referred to as "rambutan," has been utilized for generations in traditional medicine. fruits have consistently been a possible source of minerals and other nutrients. numerous phytochemicals, including carbohydrates, alkaloids, steroids and sterols, glycosides, flavonoids, triterpenoids, tannins, proteins, and amino acids, have been found in nephelium lappaceum l.'s various parts. the pharmacological actions of nephelium lappaceum l. include antioxidant, antibacterial, antiviral, antiosteoporosis, anti-inflammatory, anti-hyperglycemic, anti-hypercholesterolemic, anticancer, antidiarrheal, and cardiovascular. every component of the rambutan has medicinal and nutritional value, in addition to having many additional uses. references 1. shahrajabian mh, sun w, khoshkharam m, et al. rambutan, a tropical plant with ethnopharmaceutical properties. agrociencia. 2020; 54(1): 121-25 2. mahmood k, fazilah a, yang ta, et al. valorization of rambutan (nephelium lappaceum) by-products: food and nonfood perspectives. int food res j, 2018; 25(3): 890-902 3. manaf yna, marikkar jmn, long k, et al. physicochemical characterisation of the fat from red-skin rambutan (nephelium lappaceum l) seed. j oleo sci. 2013;62(6):335-43 4. sholikhah amn, muhtadi. study of pharmacological activities and chemical content of rambutan (nephelium lappaceum l.) fruit peel extract: a systematic review. atlantis press, 2022; 251-60 5. sukmandari ns, dash gk, jusof wh, et al. a review on nephelium lappaceum l. res j 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and nephelium ramboutan-ake: a high potential supplement. molecules, 2021; 26(7005): 1-16 18. phoung nn, le tt, camp jv, et al. evaluation of antimicrobial activity of rambutan (nephelium lappaceum l.) peel extract. int j food microbiol, 2020: 1–9 19. ahmad sa, palanisamy ud, tejo b, et al. geraniin extracted from the peel of nephelium lappaceum binds to dengue virus type-2 envelope protein and inhibits early stage of virus replication. virol j, 2017:1-13 20. zhuang y, sun x, liu b, et al. effects of rambutan peel (nephelium lappaceum) phenolic extract on ranklinduced differentiation of raw264.7 cells into osteoclasts and retinoic acid-induced osteoporosis in rats. nutrients, 2020:1–14 21. li y, li z, hou h, et al. metal chelating, inhibitory dna damage, and anti-inflammatory activities of phenolics from rambutan (nephelium lappaceum) peel and the quanti fications of geraniin and corilagin. molecules, 2018:1-12 22. muhtadi, haryoto, sujono ta, et al. antidiabetic and antihypercholesterolemia activities of rambutan (nephelium lappaceum l) and durian (durio ziberhinus murr) fruit peel extract. j appl pharm sci, 2016: 190-4 23. muhtadia, primariantia au, sujonoa ta. antidiabetic activity of durian (durio zibethinus murr.) and rambutan (nephelium lappaceum l.) fruit peels in alloxan diabetic rats. procedia food sci, 2015; 3: 255-61 24. suhendi a, muhtadi. potential activity of rambutan fruit peel extract as antidiabetic and antihypercholesterol. icetea, 2015: 20-3. 25. emylia k, snz na, dasu\ki m. preliminary study on antiproliferative activity of methanolic extract of nephelium lappaceum peels towards breast (mda-mb-231), cervical (hela) and osteosarcoma (mg-63) cancer cell lines. health environ j, 2013: 66–79. hegde k et al. review on pharmacological potential of nephelium lappaceum l vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 94 26. morshed tmi, dash pr, ripa fa, et al. evaluation of pharmacological activities of methanolic extract of nephelium lappaceum l seeds. int j pharmacogn, 2014; 1(10): 632-9 27. srisawat r, puengpai s, nontamart n, et al. effects of the crude extract of the fruit rind of rambutan (nephelium lappaceum l) on blood pressure, heart rate and respiratory rate in anaesthetized male rats. planta medica, 2010; 76: 638-9 how to cite this article: karunakar hegde, thrupthi p rai, ramkrishna shabaraya a. a brief review on pharmacological potential of nephelium lappaceum l. indian j pharm drug studies. 2023; 2(3) 90-94. funding: none conflict of interest: none stated ashoka s m et al. a review on ayurvedic herb centella asiatica vol 2 | issue 1 | jan mar 2023 indian j pharm drug studies | 17 review article an update on the ayurvedic herb centella asiatica ashoka shenoy m1, vinisha seema dsouza2, ramkrishna shabaraya a3 from 1assi. prof., 2pg scholar, 3prof. principal, department of pharmacology, srinivas college of pharmacy farangipete post, mangalore 574143. correspondence to: vinisha seema dsouza, pg scholar, department of pharmacology, srinivas college of pharmacy valachil, farangipete post, mangalore, karnataka, india. email: vinishasdsouza19@gmail.com abstract plants have been demonstrated extraordinary source of medicine and recently focus on medicinal plant research has increased. for its traditional usage and medical benefits in the treatment of numerous ailments, centella asiatica is widely known. the utilization of this plant as a whole and isolated bioactive chemical are both commonly employed to cure a variety of human illnesses, according to published research. according to reports, centella asiatica has a range of pharmacological properties, including antibacterial, anticancer, wound-healing, neuroprotective, immunomodulatory, anti-inflammatory, hepatoprotective, insecticidal, and antioxidant properties. asiatic acid, asiaticosside, and madecassoside are among the several flavonoids and terpenoid chemicals found in centella asiatica, each of which is known for its pharmacological importance. the significant medicinal plant centella asiatica is identified and described as bioactive components, pharmacological characteristics, in vitro propagation, and traditional usage was all succinctly covered in the present review. keywords: centella asiatica, medicinal plants, triterpenes, terpenoids phytochemistry, pharmacology. or hundreds of years, malaysia and other countries of asia have employed the traditional herbal remedy centella asiatica (l) urban (umbelliferae) [1]. in malaysia, it is referred to as pegaga, and in america, gotu kola and pennywort. in addition to its typical usage as a medicine, it is consumed fresh as a salad, cooked as a vegetable, and blended as a beverage [2]. it is also utilized in nutraceutical products, making it a crucial commercial plant. even though its primary use has been to promote wound healing, this tropical plant has reportedly been used for a variety of medical conditions, including the treatment of wounds, asthma, ulcers, leprosy, lupus, vein diseases, memory improvement [3], as an antidepressant [4], antibacterial [5], antifungal [6], psoriasis [7], and anti-cancer agent. even though its primary use has been to promote wound healing, this tropical plant has reportedly been used for a variety of medical conditions, including the treatment of wounds, asthma, ulcers, leprosy, lupus, vein diseases, memory improvement, as an antidepressant, antibacterial, antifungal, psoriasis, and anti-cancer agent [1]. according to numerous studies, the triterpenes in this plant are thought to be its biologically active components and the presence of several triterpenes, including asiatic acid, madecassic acid, asiaticosside, and madecassoside is primarily responsible for this plant's medicinal properties [8]. triterpenes, which make up the majority of centella asiatica, have been identified as potential biomarker components [2]. asiatic acid was the only triterpene component proven to stimulate collagen formation in human fibroblasts in cell culture experiments. other studies have demonstrated that the plant's asiatic acid, madecassic acid, and asiaticosside, as well as their mixtures, were able to stimulate collagen synthesis in skin fibroblast culture whereas the plant's asiaticosside and madecassoside, respectively, stimulated type-i collagen and type-iii collagen. the same three triterpenes, asiatic acid, madecassic acid, and asiaticosside, were present in a titrated extract of centella asiatica, which increased the synthesis of fibronectin and collagen by 20–35% [9]. centella asiatica has been utilized in skin care products to improve the appearance of skin and restore skin firmness and elasticity because of its capacity to increase collagen [10]. when topical extract formulations were used on rats with experimental wounds, the epithelia grew more quickly and the rate of contraction during healing increased [11]. moreover, in patients with venous hypertension, centella asiatica extract increased capillary permeability and the microcirculation effect. moreover, the extract was observed to influence lipolytic activity, which increased the amount of cyclic adenosine monophosphate (camp) in human adipocytes and had a slimming effect on people [12]. the antioxidant ability was found to be neuroprotective and able to shield the rat brain from oxidative damage caused by aging [13]. several studies have revealed that centella asiatica extracts have antioxidant effects [14]. asiaticosside and f mailto:vinishasdsouza19@gmail.com ashoka s m et al. a review on ayurvedic herb centella asiatica vol 2 | issue 1 | jan mar 2023 indian j pharm drug studies | 18 flavonoids have reportedly been linked to the induction of antioxidant levels during the healing of wounds [15]. no reports are indicating that centella asiatica extracts can block uv rays. the purpose of this investigation is to assess the triterpene makeup of centella asiatica and its biological activity about the enhancement of its anti-inflammatory, antioxidant, lipolytic, and uv protection characteristics [16]. vernacular names are [17] – mandookaparni (hindi), gotukola (kannada), vallarai (tamil), kodagam (malayalam), bekaparanamu (telugu) and thankuni (bengali) (table 1). taxonomical classification table 1. taxonomical classification of centella asiatica [17] kingdom plantae division tracheophyta class magnoliopsida order apiales family apiaceae genus centella species c. asiatica habitat centella asiatica is found throughout tropical and subtropical regions of india up to an altitude of 600m. the plant is indigenous to southeast asia, india, sri lanka parts of china the western south sea islands, madagascar, south africa, the southeast usa, mexico, venezuela, columbia, and eastern south america [18]. botanical description stem: glabrous, striated, rooting at the nodes. flourishes extensively in shady, marshy, damp, and wet places such as paddy fields, and riverbanks forming a dense green carpet, and rather than clayey soil, the sandy loam (60% sand) is found to be the most fertile soil for its generation. seed: pendulous embryos which are laterally compressed [19]. leaves: 1-3 from each node of stems, long petioles, 2-6 cm long and 1.5-5cm wide, orbicular-reniform (figure 1). figure 1: centella asiatica leaf flowers: fascicled umbels, each umbel consisting of 3-4 white to purple or pink flowers, flowering occurs in the month of april-june (figure 2). figure 2: centella asiatica flower fruits: borne throughout the growing season in approx. 2 inches long, oblong, globular in shape, and strongly thickened pericarp (figure 3). figure 3: centella asiatica fruit phytochemistry c. asiatica contains a variety of amino acids, flavonoids, terpenoids, essential oils, alkaloids, and other compounds. most phytochemical studies focused on leaves, and the constituents differ depending on geographical distribution. for a very long period, c. asiatica has been utilized as a natural source of medicine. pentacyclic triterpenes, such as asiatic acid, madecassic acid, asiaticosside, and madecassoside, among others, are the primary active components of c. asiatica. to date, isoprenoids (sesquiterpenes, plant sterols, pentacyclic triterpenoids, and saponins) and derivatives of phenylpropanoid have been found as phytochemicals from c. asiatica (eugenol derivatives, caffeoylquinic acids, and flavonoids) [19]. pharmacological activity antiulcer activity: an acute toxicity study was carried out in which the animals were treated with the rhizome extract at a dose of 2 and 5 g/kg of c. asiatica leaf extracts and were kept under observation for 14 days. all the animals remain alive and did not manifest any significant visible signs of toxicity at these doses. at any point during the observation period, there were ashoka s m et al. a review on ayurvedic herb centella asiatica vol 2 | issue 1 | jan mar 2023 indian j pharm drug studies | 19 no unusual behaviors, macroscopic findings, symptoms, or body weight changes. the hematology and serum biochemistry parameters like triglycerides, creatinine, urea and hemoglobin, ast, alt, and alp of the extract-treated rats showed no significant change compared to the control normal rats. the oral lethal dose (ld50) for the male and female rats was larger than 5 g/kg body weight, suggesting that the extract is fairly safe even at these higher concentrations and had no acute toxicity [20]. anti-diabetic activity: alloxan monohydrate solution of 10 mg/ml was prepared in ice-cold citrate buffer (0.1m); the ph of the ice was kept at 4.5 and was administered to the rats within 5 mins at a dose of 50 mg/kg body weight intraperitoneally. the fasting blood sugar levels of each of the rats were checked every day with an autoanalyzer (glucometer) glucose kit. after 8 days, animals with fasting blood sugar levels of 250 mg/dl and above were considered to be diabetic and were used for the study and assigned into five groups of five rats each. group i served as the negative control and received tween 80 solution (solvent used to dissolve the extract) (10 ml/kg), group ii–iv received the c. asiatica extract at the dose of 250, 500, and 1000 mg/kg respectively while group v served as the positive control and received the standard reference drug glibenclamide (2 mg/kg) all by gastric gavage. the blood glucose levels of the rats were measured at 0, 1, 2, and 3 h after administration of the drug and extracts. blood samples were collected by tail snip and the blood glucose was measured with an autoanalyzer (glucometer) glucose kit. at the end of the experiment percentage reduction of the glucose levels of the rats at the 3rd hour was calculated [21]. anti-inflammatory activity: this study investigated the antipruritic and anti-inflammatory effect of centella asiatica extract in rats and anti-allergic in vitro using sheep (capra hircus) serum method and compound 48/80 induced mast cell degranulation method, compared with standard drug ketotifen fumarate. orally administered centella asiatica extract was tested for its ability to treat pruritis in rats. chlorpheniramine maleate was used as the standard medication, and carrageenan paw-induced inflammation was used to test the plant's ability to treat inflammation. the results show that the extracts of centella asiatica exhibited antiallergic, anti-pruritic, and anti-inflammatory activities [22]. cytotoxic and anti-tumor activity: the antioxidant activity of ae of centella asiatica was evaluated by its ability to scavenge dpph free radicals. the radical scavenging activity of the compounds can be measured by the decolorizing effect following the trapping of the unpaired electrons of dpph. the ae showed high antioxidant activity, with an ic50 value of 31.25 μg/ml. ascorbic acid and butylated hydroxytoluene (bht) produced ic50 values of 2.50 μg/ml and 7.58 μg/ml, respectively. previous research suggests that the presence of compounds with free hydroxyls may be the cause of the potent antioxidant action of polar extracts. since they contain a lot of hydroxyls that act as hydrogen donators, flavonoids have a perfect structure for scavenging free radicals in this context, making them significant antioxidant agents [23]. neuroprotective activity: the neuroprotective effect of c. asiatica and its major triterpene saponosides has been extensively studied through different experimental models on animals such as passive avoidance and elevated-plus labyrinth tests for memory enhancing effect. by administering the extract at doses of 100, 200, and 300 mg/kg (b.w.), the extract was tested in rats to see how it affected intra-cerebrovascular streptozocin-induced memory linked to the sporadic type of ad. oxidative stress markers like glutathione, superoxide dismutase (sod), and catalase (cat) were also measured. while a clear dose-dependent improvement was observed in memory-related behaviors in the rat group administered the extract at 200 mg/kg (b.w.) dose, a serious decrease in malondialdehyde (mda) and an increase in glutathione and cat levels was recorded, which led to a final suggestion by the authors that c. asiatica extract has a positive effect on memory that is also related to its remarkable antioxidant effect. the same research group subjected this extract to passive avoidance and spontaneous locomotor activity behavioral tests using pentylenetetrazole-(ptz-) induced memory loss in rats at 100 and 300 mg/kg (b.w.) doses. after the behavioral tests, the rat brains' mda and glutathione levels were assessed as oxidative stress markers, which are strongly linked to neurodegeneration. as a result, all test parameters significantly improved when the extracts were administered at the specified levels [24]. cardioprotective activity: laboratory-bred sprague–dawley rats of either sex weighing 200–350 g were selected. the rats were maintained under standard laboratory conditions at 25±2 °c, relative humidity. in group-i (control) animals, the percentage of left ventricular necrosis was reported to be 50.911.90. plvn was considerably and dose-dependently reduced in the treated groups. lipid peroxide concentrations were observed to be 27.571.42 nmol ml1 in serum and 109.242.77 nmol g1 in cardiac tissue in group-i animals. whereas in the treated groups, the lipid peroxide levels were found to be 17.68±0.91 nmol ml−1, 12.20±0.86 nmol ml−1, and 5.85±0.5 nmol ml−1 in serum [25]. skin protective activity: during clinical irradiation, centella asiatica may help avoid radiation-induced behavioral abnormalities. the plant extract exhibited radioprotective qualities, and the pretreatment of mice with it before gamma radiation exposure was successful in preventing radiationinduced liver damage. extractsd obtained under different extraction conditions show different extraction patterns, as well as different extraction yields, possibly due to the different polarities, the solubility of the solvents, and the effects of the extraction temperature [26]. ashoka s m et al. a review on ayurvedic herb centella asiatica vol 2 | issue 1 | jan mar 2023 indian j pharm drug studies | 20 memory enhancing activity: results of the preliminary screening showed that oral administration of c. asiatica for fifteen days enhanced the learning and memory in three months old mice as assessed by the radial arm maze test. spatial memory is frequently tested using the radial arm maze. the number of accurate entries increased dose-dependently during the radial arm maze test. it can be shown from this that the extract improved cognitive abilities in young adult mice [27]. wound healing effect: the effect is observed with a quicker wound contraction, probably owing to the stimulation of fibronectin and collagen i synthesis and matrix remodeling. these two are characteristic of the proliferative stage of the wound healing process. additionally, it has been discovered that c. asiatica is effective in maintaining connective tissue and bolstering veins that are weak. as a result, its use may be advantageous for the treatment of hypertensive microangiopathy and venous insufficiency. its oral supplementation has been demonstrated to promote fibroblast division after injury in addition to collagen synthesis and cellular proliferation. additionally, it might improve wound contraction and re-epithelialization in incision model wounds (p 0.001). c. asiatica has also been shown to improve the tensile strength of the newly formed skin of the wound in animal studies, which could lead to a decrease in the wound area and faster healing [28]. hepatoprotective activity: a safety evaluation using rodents to determine appropriate conditions to use. by conducting the 'single and 14-day repeated oral study in rodents', we confirmed that the non-toxic amount of ca-he50 was 2 g/kg/day. therefore, a study on the protective effect of ca-he50 on the liver using ca-he50 doses of 50, 100, and 200 mg/kg/day. due to the strain of metabolism on the liver, liver enlargement and edema frequently arise when liver failure is caused. the study found that when apap was used to cause liver damage, the weight of the liver decreased (p 0.05). when apap caused liver toxicity, a decrease in liver weight took place between six and twenty-four hours later and was accompanied by a rise in blood alt and ast values, which suggested liver cell lysis. we gave apap, just like in the first trial, and weighed the liver 24 hours later. the decrease in liver weight caused by apap in our study was likely due to liver cell injury (lysis) caused by apap toxicity. provision of 200 mg/kg ca-he50, however, prevented the apap-induced decrease in liver weight. thus ca-he50 helps to protect against liver injury [29]. anti-cancer activity: asiatic acid was evaluated for antiproliferative effect in lung cancer cells using mtt assay. oral administration of aa inhibited weight and tumor volume significantly in the lung cancer xenograft model. in a different study, asiatic acid demonstrated dose-dependently triggered apoptosis and lowered viability in human melanoma sk-mel2 cells. it reduced the focal adhesion kinase (fak) expression levels, and the probable mechanism of aa may be connected to the suppression of signal transduction carried out by fak. asiatic acid, asiaticoside, and madecassic acid was the major component of the titrated extract of c. asiatica, and asiaticoside reduces melanogenesis in b16f10 mouse melanoma by checking tyrosinase mrna expression [30]. anti-bacterial activity: methanol hot extract from c. asiatica leaves was taken to check the antibacterial activity which was assessed by zone of inhibition and minimum inhibitory concentration (mic) value (2 µg/disc) by disc diffusion method. in a study, it was discovered that essential oil extract had antibacterial activities against both gram-positive and gram-negative bacteria, including escherichia coli, pseudomonas aeruginosa, and shigella sonnei, with mic values ranging from 1.25 to 0.039 mg/ml. bacillus cereus and listeria monocytogenes 10403s were selected to study the antibacterial activity in c. asiatica under both normal and osmotic stress conditions. at 95% ethanolic extract, antibacterial activity was enhanced twice under osmotic stress conditions [31]. anti-fungal activity: the petroleum ether, ethanol, chloroform, n-hexane, and aqueous extract of c. asiatica showed activity against aspergillus niger and c. albicans with a zone of inhibition of 14, 16, 13, 13, and11 mm and 13, 15, 15, 11, and 9 mm, respectively. the control ketoconazole (10 g) demonstrated a 12 mm inhibition. when aspergillus flavus was exposed to an ethanolic extract of centella asiatica, penicillium citrinum showed the greatest antifungal activity (% mycelial inhibition = 26.3 mm).100% ethanolic extract of c. asiatica showed a zone of inhibition of 15.4 mm against a. niger [32]. antioxidant: c. asiatica extract and powder were evaluated for a reduction in oxidative stress in sprague-dawley rats. results showed a decrease in the generation of ros and oxidative stress in the rats. essential oil of c. asiatica extracted through steam distillation showed to be an excellent antioxidant for food containing lipids. its activity was quite comparable with the synthetic antioxidant butyl hydroxyanisole (bha). polyphenol, flavonoid, β-carotene, tannin, vitamin c, and dpph compounds are readily found in c. asiatica contributing to significantly higher antioxidant activity in the herb. crude methanolic extract on continuous supplementation for 14 days increased the level of antioxidant enzymes and the ascorbic acid level was reduced in lymphoma-bearing mice [33]. anti-depressant: compared to diazepam c. asiatica possesses an antianxiety effect but does not affect behavioral despair. total triterpenes and imipramine from c. asiatica were evaluated for antidepressant activity using a forced swimming test, the result showed a reduction in stillness duration and regulated amino acid levels. in another study, a decrease in corticosterone levels in serum and enhanced 5-ht, ne, da, and their metabolites 5-hiaa and mhpg in rat brains were observed. the standardized extract showed a reversal of ashoka s m et al. a review on ayurvedic herb centella asiatica vol 2 | issue 1 | jan mar 2023 indian j pharm drug studies | 21 physiological and behavioral changes following obx-induced depression in rats. a forced swim test was performed in male sprague-dawley rats treated with asiatic acid [34]. traditional uses: centella asiatica has traditionally been used to treat a variety of ailments. triterpene fractions, which make up the majority of c. asiatica's constituents, have a wide range of protective and therapeutic benefits. one of these effects is a significant influence on collagen synthesis and deposition during wound healing. several microcirculatory issues, skin inflammation (eczema, atopic dermatitis, leprosy, varicose ulcers, etc.), fever, intestinal issues, and genitourinary diseases are all treated with titrated extract of centella asiatica (teca). due to the presence of several saponin constituents, such as asiaticosside, asiatic acid, madecassic acid, and some other bioactive compounds, c. asiatica exhibits a wide range of pharmacological activities, including antibacterial, antidepressant, antiemetic, antineoplastic, antioxidant, antithrombotic, anxiolytic, gastroprotective, immunomodulatory, antigenotoxic, nerve regenerative, reproductive, and wound healing. conclusion centella asiatica is a well-known plant in indian medicine, and folk medicine believes it can help with heart and hepatic problems, ulcers, and other ailments. plant components including leaves, fruit, stem, and even roots are utilized for a variety of uses. chemically, centella asiatica includes amino acids, flavonoids, terpenoids, essential oils, and alkaloids among other physiologically active phytoconstituents. it is also important 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bacillus cereus and listeria monocytogenes under osmotic stress. aujt 2015;15(4):239-45. 32. . idris na, nadzir mm. antimicrobial activity of centella asiatica on aspergillus niger and bacillus subtilis. chem eng trans 2017; 56:1381-6. 33. chandrika ug, kumara pa. gotu kola (centella asiatica): nutritional properties and plausible health benefits. adv food nutr res 2015; 76:125-57. 34. kalshetty p, aswar u, bodhankar s, et al. antidepressant effects of standardized extract of centella asiatica l in olfactory bulbectomy model. biomed aging pathol 2012;2(2):48-53. how to cite this article: ashoka shenoy m, vinisha seema dsouza, ramkrishna shabaraya a. an update on the ayurvedic herb centella asiatica. indian j pharm drug studies. 2023; 2(1) 17-22. funding: none conflict of interest: none stated rai et al mindful-based stress reduction impact vol 1 | issue 2 | jul – sep 2022 indian j pharm drug studies | 38 review article advances in ocular drug delivery system for glaucoma management – a comprehensive review raju maski1, jitendra banweer2, megha mishra3, praveen tahilani3, gaurav goyanar4 from, 1m-pharma (pharmaceutics), 2sagar institute of research technology & science pharmacy,bhopal (m.p.), 3associate professor, sirts pharmacy college, bhopal (m.p.), 4department of pharmaceutical science, sage university, indore m.p. correspondence to: raju maski, m-pharma (pharmaceutics), sagar institute of research technology & science pharmacy, bhopal (m.p.). email: tahilanipraveen@gmail.com abstract in today’s world delivery of ophthalmic drugs remains challenging despite easy accessibility via the ocular surface. eye drops are easy and most widely used for drug delivery for treating ocular infections, particularly involving the internal segment having an additional benefit of avoiding first-pass metabolism there while passing through the systemic circulation. the challenges of drug administration through traditional methods involve improper patient education for drug installation techniques, compliance, adherence, and persistence. different dynam ic and static ocular barriers involved only permit limited drug delivery to the target ocular tissues. in this review, we described the development of welltolerated drug delivery systems that helps to overcome the factors limiting adequate drug delivery t o the glaucomatous patients targeting infected tissues with traditional techniques. keywords: drug delivery, glaucomatous, neuroprotection, ocular surfaces, ophthalmic drugs t is known that glaucoma is the second most common cause of blindness in the world. patients with glaucoma present with high ocular pressures that can cause optic neuropathy precipitating in the corresponding visual field loss [1]. recent studies showed there were about 60 million people having optic neuropathy secondary to glaucoma globally [2]. these studies have shown that primary openangle glaucoma (poag) leads to bilateral blindness in 9% of patients and unilateral blindness in 27% of patients, within 20 years of the first glaucomatous changes [3]. during 2010, an estimated 4.5 and 3.9 million people were diagnosed with bilateral blindness due to open-angle glaucoma (oag) and angle-closure glaucoma (acg), respectively. the number increased to 5.9 and 5.3 million in the year 2020 of bilaterally blind people due to oag and acg [2]. glaucoma is progressive and irreversible due to which visual field loss; however, attaining the normal range of the target intraocular pressure (iop) within alleviates the progression of visual field loss. the initial management strategy for ‘high-risk’ glaucoma suspect or a patient diagnosed with oag using topical anti-glaucoma drugs and lowering the iop. glaucoma is a slowly progressive pathology that can result in the loss of peripheral vision, decreased contrast sensitivity, and loss of visual acuity. due to the asymptomatic nature of the early phases of the disease most patients experience undiagnosed loss of vision until the advanced stages of the disease have occurred. thus the disease is known as the “silent thief of sight”. this indolent optic neuropathy is characterized structurally by a loss of retinal ganglion cells and optic nerve axons. glaucoma is the second leading cause of the world’s blindness with nearly 70 million cases worldwide and accounting for 12% of all cases of preventable blindness. it is estimated that by 2020, close to 4 million americans will have glaucoma with 50% undiagnosed and approximately 120,000 individuals developing blindness [4].in developing countries, where the access to adequate care and therapies is limited, people are going blind from a disease that can be successfully treated. patients in these countries may not have the ability to get to their clinics routinely for refills and exams. however, even in the us with ready access to medical care and pharmaceuticals, glaucoma continues to progress in many patients. often poor iop control is due to poor compliance and adherence to daily topical treatment regimens or inadequate, complex dosing regimens. despite effective monotherapy agents, data has shown that upwards of 40% of oag patients require combination therapy for iop reduction with close to 75% of glaucoma patients requiring adjunctive therapy after five years. the complexity, cost, and administration issues with multiple medications further reduce patient compliance and adherence. prescribing pharmacy claims data show the vast majority of patients do not take their i mailto:tahilanipraveen@gmail.com rai et al mindful-based stress reduction impact vol 1 | issue 2 | jul – sep 2022 indian j pharm drug studies | 39 topical medications or renew their prescriptions, resulting in patients regularly missing doses. retrospective populationbased data suggests a minority of patients consistently adhere to their topical medication. a sustained mode of delivery where the patient’s dependence on daily self-instillation is eliminated could dramatically improve these statistics [8]. various studies have highlighted the shortcomings of treatment regimens, drug efficacy factors causing short precorneal residence time, reduced absorption, and rapid turnover of lacrimal fluid, extensive nasolacrimal drainage, rapid blinking reflex, human factors compliance, and persistence [5-7]. methods for this review article we searched different electronic databases such as pubmed, google scholar. studies were included from 1975 to 2020 available in the english language only. ocular insertsfor the release of therapeutic drugs over a prolonged duration ocular inserts are sterile drugimpregnated microdevices placed in or around the eye. based on their physical and chemical properties, the inserts are classified into insoluble, soluble, or bioerodible [9]. the contact time of the drug to a few days increased the ocular surface after insertion, thereby increasing manifold bioavailability due to reduce washout by tears. pilocarpine ocular insertsin 1976, bensinger et al. demonstrated the use of a synthetic biosoluble matrix in the conjunctival cul-de-sac to increase the contact time of pilocarpine with the corneal tear film for intraocular pressure (iop) control. different doses ranging from 0.5 to 2 mg, 32 h post-insertion recorded with a significant reduction in the iop [10]. the iop had the maximum lowering of 6.25 ± 2.48 mmhg on the placement of 0.5 mg pilocarpine inset. while, a higher dose of 1.5 mg pilocarpine reduced the iop by 8.14 ± 0.96, 5 h post-placement. a significant reduction in the iop was noted at 32 hours after insertion of the 1 mg pilocarpine. soluble ophthalmic drug insertsas the soluble ocular drug insert (sodi) is an oval-shaped ocular insert made up of a copolymer of polyacrylamide, ethyl acrylate, and vinylpyrrolidone. maichuk [11] first reported and used to administer drugs including pilocarpine through the inferior culde-sac. the drug insert converts into a viscous polymer solution after 10 to 15-sec contact with the tear film followed by conversion to a polymer solution within 60–90 min of administration. ocusertocusert was one of the earliest models of ocular inserts developed by armaly and rao [12], made commercially available by alza corporation inc. ocusert releases the drug at a constant rate of 20 or 40 μg/h for an extended period of 7 days. pilocarpine was loaded in a polymer membrane system consisting of an inner layer of pilocarpine in alginate gel di-(ethylhexyl) phthalate for a release enhancer sandwiched between two outer layers of ethylene-vinyl acetate (eva) designed to release the drug at a predetermined constant rate. zimmerman et al. studied pilocarpine delivery in 40 patients for ocusert with a target release rate of 20 µg/h. initially, the mean iop was recorded to be 25.6 ± 5.6 mmhg. iop was reduced to 19.9 ± 3.9 mmhg by using pilocarpine-loaded ocusert. the study reported that the patients do not prefer the pilocarpine drops over the ocusert system. no side effects from the ocusert were noted [13]. pavan-langston et al. studied 29 patients who showed that pilocarpine-loaded ocuserts releasing either 20 or 40 μg/h of pilocarpine a satisfactory control of the iop. the ocuserts side effects were minimal or absent [14]. ocusert did not become a widely accepted method of drug delivery, although the clinical studies showed positive outcomes. this is because of the difficulty of device insertion, failure in satisfactorily controlling iop in many patients, ejection of the device from the eye, irritation during insertion, and the difficulty of device insertion [15]. poly (vinyl methyl ether-maleic anhydride) anhydride (pvmma) ocular insertspvmma and its alkyl monoesters are bioerodible polymers used for controlled timolol release in animals. the systemic effect of timolol is reduced by the polymer-assisted drug release. finne et al. [16] found a lower steady-state concentration (1.0 ± 0.1 ng/ml) in plasma three hours after administration and a peak concentration of timolol in tear fluid (64 ± 9 μg/ml). they also reported a 1.6-fold increase in timolol concentration in tears (104 ± 8 μg/ml) on the addition of disodium phosphate as a buffer. collagen shieldcollagen shields as postoperative corneal bandages developed by dr. svyatoslav fyodorov [17]. bloomfield et al. developed the drug delivery model for the collagen shields. a higher level of gentamicin in the tear film, tissues in rabbit eyes using wafer-shaped collagen inserts impregnated with gentamicin as compared with ointment, eye drops, and subconjunctival injection demostrated by bloomfield et al. [18]. collagen shields, were loaded with hydrophilic drugs in the collagen matrix by soaking a dry shield in the aqueous solution of the drug. the water-insoluble drugs are directly added to the shield during the manufacturing process. agban et al. [19] developed cross-linked collagen shields consisting of nanoparticles of titanium dioxide (tio2), zinc oxide (zno), polyvinyl pyrrolidone (pvp), and capped zinc oxide (zno/pvp) for controlled delivery of pilocarpine hydrochloride in glaucoma patients over a prolonged duration that undergoing animal trials. the results from the group show a sustained release of pilocarpine hydrochloride when crosslinked with zno/pvp nanoparticles for 14 days. ocufit srdeveloped by escalon ophthalmics inc. is a flexible rod-shaped silicon elastomer device designed for rai et al mindful-based stress reduction impact vol 1 | issue 2 | jul – sep 2022 indian j pharm drug studies | 40 retention in the conjunctival fornix for controlled release of drugs over long periods. the different models are a maximum of 1.9 mm in diameter and range between 25 and 30 mm in length. katz and blackman [20] later reported that expulsion of rod-shaped devices was significantly less frequent than that of oval, flat inserts. the insoluble ocufit had favorable properties of both long retention and sustained drug release. in 70% of the cases, the upper fornix placebo device was retained for two weeks or more. minidiscbawa et al. [21] developed the minidisc or ocular therapeutic system (ots) the miniature contact lens with a diameter of 4–5 mm with a convex and a concave face latter conforming substantially to the sclera of the eye. the minidisc is a polymer of hydroxyethyl methacrylate and ethylene glycol methacrylate. the size and shape of the ots allow easy placement of the device under either upper or lower lid without any foreign body sensation, distortion in vision, or decreased oxygen permeability. new ophthalmic delivery system (nods) nods is used for delivering drugs in precise amounts to the eye through the lower conjunctival sac using a water-soluble film loaded with the drug [22]. the device consists of 20µm-thick, 4 × 6 mm2 medicated flag, attached to a 0.7-mmlong paper-covered handle and a 3to the 4-µm-thick membrane. nods is manufactured using water-soluble polyvinyl alcohol (pva). greaves et al. [23] used radiolabeled nods loaded with pilocarpine nitrate to evaluate the pharmacokinetics and bioavailability in human subjects. topical bimatoprost ocular inserta bimatoprost-loaded insert consisting of a silicone matrix with a polypropylene backbone for sustained delivery to treat glaucoma underwent randomized phase ii clinical trial [24]. the diameter of the insert ranged from 24 to 29 mm and was placed between the upper and lower fornices. the bimatoprost ocular insert elutes the drug at a variable rate for six months, depending on the polymer-drug matrix properties. in 2016, de souza and colleagues developed an ocular insert with mucoadhesive properties developed from polymers of chitosan. the data from the in vitro studies showed sustained release of brimonidine tartrate. moreover, the authors highlighted the adherent properties of the chitosan-based polymer on the conjunctiva. they also confirmed the rate of constant release for a prolonged period of 30 days without an initial burst. the insert had biocompatibility with the surrounding ocular tissues [25]. patient education continues to be a significant challenge when it comes to the successful use of the inserts as it requires fine manual techniques to manipulate and place the insert. it was seen that the level of education and age continue to be the factors that govern the success of these devices when used for glaucoma [26]. hitoshi et al. studied the efficacy of ophthalmic inserts of timolol based on poly (2-hydroxypropyl methacrylate) and poly (2-hydroxyethyl methacrylate) polymers. the results from the study indicated that the prepared inserts resulted in a controlled release and an improved ocular bioavailability of timolol [27]. soak and releasewaltman and kaufman [31] first demonstrated the potential use of hydrophilic polymers of 2hydroxyethyl methacrylate (hema) for drug delivery using fluorescein stain. in 1974, hillman demonstrated the delivery of antiglaucoma drugs through soft contact lenses. he used polymers of vinylpyrrolidone soaked in 1% pilocarpine for drug delivery. he reported the system to be as efficacious as 4% pilocarpine topical eye drops [32]. microemulsion loaded lensesthe microemulsions for drug dispersal were favored due to the thermodynamic stability, high drug-loading capacity, ease of preparation, increased wettability, and easy tailoring of the drug release pattern. multiple groups have developed drug-loaded microemulsionincorporating contact lenses [48]. gulsen and chauhan encapsulated timolol in microemulsion stabilized within a silica shell using octadecyltrimethoxysilane (otms), followed by dispersion in a hydrogel lens. this model has shown sustained release of up to 8 days without affecting the transparency of the lens [33]. li et al. [34] developed contact lenses loaded with timolol, with oil-in-water-type microemulsions using a combination of ethyl butyrate, and pluronic f127. the group fabricated the microemulsion-laden gels, ethyl butyrate/water microemulsions stabilized by pluronic f127 surfactant, and subsequently polymerized after the addition of hema. vitamin e–loaded lenseschauhan et al. developed the technique using vitamin e as an in-situ transport barrier for timolol. the drug release was significantly increased by elevating the loading concentration of vitamin e from 10% to 40% in contact lenses [28]. the group demonstrated a quadratic increase in drug release duration in vitamin e loading. loadings of 10% and 40% vitamin e increased the release time of timolol by a factor of about 5 and 400, respectively. however, vitamin e loading in the lens led to an increase in lens sizes, a reduction in oxygen diffusion, and a significant decreament in ion permeability. film impregnation in contact lensciolino et al. [35] designed a latanoprost-eluting contact lens for treating glaucoma, manufactured by encapsulating the drug film enclosed in methafilcon lenses. these lenses have shown sustained release for up to 1 month in glaucomatous monkeys. the amount of drug delivered to the eye exceeded or was comparable to the delivered topical drops. contact lenses with polymer-drug films (40–45 mm in thickness) demonstrated an initial burst of latanoprost in the aqueous humor, a steady concentration was similar to the average hourly concentration rai et al mindful-based stress reduction impact vol 1 | issue 2 | jul – sep 2022 indian j pharm drug studies | 41 delivered from a drop of commercially available latanoprost solution [52]. enzyme-triggered timolol releasekim et al. [36] embedded nanodiamonds (nds) loaded with timolol in contact lenses. the nd–nanogel embedded contact lens acts as an enzyme trigger for the delivery of timolol. the nanogels sequester timolol before activating the lysozyme that causes chitosan degradation and subsequently allows sustained drug release. after 24-h treatment with lysozyme, the total steady drug release from the lens was 9.41 μg. intraocular implants intravitreal implantsintravitreal implants are devices capable of delivering drugs for a prolonged duration in the eye. although surgical implants present a viable option for longterm drug delivery, the invasive nature of the initial and subsequent surgical procedures to remove the implants does not make them a favorable choice for drug delivery. ozurdex is a degradable dexamethasone intravitreal implant produced by allergan, which was used to treat macular edema and noninfectious uveitis [53]. the device slowly degrades after implantation in the vitreous while delivering dexamethasone. the manufacturer conducts clinical trials of the implants loaded with brimonidine tartrate in the proprietary novadur poly (lactic-coglycolic acid) (plga) intravitreal polymer matrix platform for the management of geographic atrophy due to agerelated macular degeneration. topical daily ophthalmic brimonidine tartrate drops were prescribed for iop reduction and neuroprotective effect. if the novadur plga platform implants with brimonidine tartrate are approved, they can also be adapted for use in glaucoma patients [37]. subconjunctival inserts-the subconjunctival inserts were used as implants as a replacement for viscoelastic depot delivery injections. the vs101 ocular insert was one such insert developed by visci inc. in 2014 and later underwent a phase i/ii multicentric randomized control study to evaluate the safety and effectiveness of subconjunctival latanoprost insert in subjects with ocular hypertension or oag [38]. pcl-peg inserts-ng and colleagues used biodegradable microfilm synthesized by a combination of poly (lactide)/poly (ε-caprolactone) (plc) and poly (εcaprolactone)/poly (ethylene glycol) (plc/pcl-peg). the polymer was loaded with timolol maleate and inserted by conjunctival dissection [39]. the authors reported a decrease of 50.1% ± 8.5% in iop from baseline in primates with ocular hypertension, which was sustained for 140 days. ap-pcl inserts-alkoxylphenacyl-based polycarbonate polymers in combination with polycaprolactone (ap-pcl) were used by manickavasagam et al. [40] for sustaining delivery of brimonidine tartrate for three months. the major drawback attributed to the subconjunctival inserts is the requirement of a surgically invasive procedure which creates a small opening in the conjunctiva with a possibility of subconjunctival migration, infection in need of an operating room procedure for insertion/removal of the device. micro electro-mechanical system-the system termed as micro electro mechanical system (mems) works on the principle of bubble generation by electrolysis to mechanically push the loaded drug out of the reservoir. the device, currently in a preclinical development phase, also allows loading the drug multiple times [41]. saati et al. demonstrated the use of the mems pumping mechanism was based on electrolysis connected to a drug refill port a check valve to control delivery. the procedure is similar to the implantation of a glaucoma aqueous drainage device. liposomethe liposome-encapsulated drug was delivered as a solution as an eye drop. natarajan and colleagues used latanoprost-loaded egg-phosphatidylcholine liposomes for delivery. the liposomes remained stable for at least six months on storage at 4°c and at least one month at 25°c. a sustained release of 60% of latanoprost was achieved by two weeks in vitro. a high sustained iop-lowering effect was recorded in liposome-treated animals (4.8 ± 1.5 mmhg) compared with daily administration of topical latanoprost (2.5 ± 0.9 mmhg) beyond 90 days [42]. monem and colleagues used multilamellar vesicles (mlvs) as a vehicle for delivering pilocarpine. they reported neutral mlvs encapsulating pilocarpine hcl exhibited the most prolonged efficacy in the reduction of iop. they also reported negatively charged mlvs encapsulating pilocarpine hcl exhibited a significantly shorter period of drug action [43]. the group speculated that the frequency of administration of drug administration in humans would be reduced to half with the usage of mlv vehicles, thus promoting better compliance. polymeric nanoparticlesdue to their molecular-scale size, nanoparticles efficaciously deliver drugs in the anterior chamber in the posterior compartment via the bloodaqueous and the blood-retina barrier, respectively [44]. different types of nanoparticles were classified based on the origin of the constituent monomers and its emphasis was laid on effective drug loading on the nanoparticles through the process such as electrospraying and electrospinning. mehta et al. demonstrated a single-needle electrohydrodynamic process for adding a stable nanocoating to the contact lenses with timolol maleate. the in vitro studies showed biphasic release of the drug, with an initial burst release followed by sustained release [45]. chitosan-based polymeric nanoparticleschitosan, a 2amino-2-deoxy-beta-d-glucan, is being widely tested for rai et al mindful-based stress reduction impact vol 1 | issue 2 | jul – sep 2022 indian j pharm drug studies | 42 synthesizing nanoparticles for drug delivery [46]. the biodegradable, biocompatible, and mucoadhesive properties of chitosan make it highly suitable for delivering antiglaucoma drugs. li and colleagues [47] developed chitosan nanoparticles loaded with beta-adrenergic agent betaxolol, prescribed for lower iop. the ex vivo data published by the authors show a 1.75 times higher value compared with the topical eye drops. zhao and colleagues [49] have developed timolol maleate– loaded nanoparticles from glycosylated polymers of chitosan for ocular delivery. the authors reported an augmented transcorneal penetration due to high lipid solubility. the authors reported that the data from the in vivo experiments showed a sustained release over a significantly longer duration of time. mehta et al. used electrohydrodynamic atomization of timolol maleate–loaded pvp and poly (n-isopropyl acrylamide). the authors used the formulation approach for sustained timolol maleate release used the combination of chitosan, borneol and reported the biphasic and triphasic release, depending on composition [49]. poly (lactic-co-glycolic acid) nanoparticlesplga is a copolymer of cyclic dimers (1, 4-dioxane-2, 5-diones) of glycolic acid and lactic acid [50]. salama and colleagues [51] used plga nanoparticles for the delivery of brinzolamide subconjunctivally and reported the release of the drug was prolonged for a period of up to 10 days after a single dosage. khan and colleagues used plga-based nanoparticles for the delivery of forskolin, a natural extract with potent noradrenergic stimulatory action on adenylate cyclase [52, 53]. the authors reported a steady release of the drug from the plga polymer, with 90% release over 72 h compared with eye drops (96.6% release in 12 h). gelatin nanoparticlesthe ease of availability and high biocompatibility make gelatin a favorable polymeric vehicle for the delivery of antiglaucoma drugs to the eye. recently, shokry and colleagues reported the use of gelatin nanoparticles for delivering timolol maleate and reported increased mucoadhesion and transcorneal permeability due to its positive charge attracted to negatively charged lipid layers in the cornea [54]. the in vitro release studies showed a burst effect of timolol release followed by a sustained profile over a prolonged duration. the in vivo studies in the albino rabbits showed a sustained and higher efficacy in iop lowering. in another study, liao et al. [55] used silica-based mesoporous nanoparticles for pilocarpine with gelatin coating. the in vitro data showed a 36-day release profile of the gelatin-coated mesoporous nanoparticles with an efficacious in vivo ioplowering effect for 21 days. propoxylated glyceryl triacylate nanoparticlesjung et al. [56] developed a contact lens based on the principle of dispersing timolol-loaded propoxylated glyceryl triacylate (pgt) nanoparticles within the lens. timolol–pgt particles release the drug for an extended period (>30 days at room temperature) by hydrolysis of the ester bond. the bioavailability of timolol delivered through the contact lens showed 50% bioavailability as compared with only 1–2% through eye drops. pgt–ethylene glycol dimethacrylate nanoparticlesjung and chauhan [57] also developed a lens with highly crosslinked particles consisting of monomeric units with multi vinyl functionalities such as pgt and ethylene glycol dimethacrylate (egdma). the 3.5-nm nanoparticles encapsulated 48–66% of the drugs. the rate constant of ester hydrolysis was significantly less than those of the previous models developed by the same group, possibly due to steric effects and the low water content of the highly cross-linked hydrophobic particles. the nanoparticles dispersing timolol were encapsulated with linked nanoparticles enclosed within contact lenses, which increases the duration of drug release from 1 to 2 h to about four weeks. the drug-dispensing particles were dispersed in hydroxymethyl methacrylate (hema) gels that were commonly used for manufacturing contact lenses. xu and colleagues [58] developed micelles that could be loaded on the contact lenses for sustained release of timolol and latanoprost simultaneously for management of glaucoma. the micelles were synthesized by free radical polymerization of the hema monomer with timolol and latanoprost. the lenses released timolol and latanoprost in tear fluid for 144 and 120 h, respectively. the in vivo data showed sustained timolol and latanoprost release for 120 and 96 h in tear fluid, respectively. nanospheres /microspheresthe penetration of drugs loaded on the nanosphere depends on the size, charge, architecture, and surface of the carrier nanoparticle systems [59, 60]. the architecture of the nanospheres consists of a di-block copolymer that is a hydrophobic block [polycaprolactone (pcl)], a hydrophilic component [polyethylene glycol (peg)]. the unique structure of nanospheres allows a longer residence time on the surface of the cornea to provide the drug with a carrier followed by fusion with the corneal epithelial membrane, hence reducing the dosing frequency [61]. chiang et al. [62] initially reported 6 mmhg with brimonidine polylactic acid (pla) microspheres that results in reduced iop for one month in normotensive rabbit eyes. the in vitro analysis of the brimonidine microspheres showed a sustained release of the drug for 35 days. ohr1031it is a macromolecular drug for glaucoma management that incorporates into microparticles using a dissolvable hydrogel template technology [63]. the drug is dissolved into a plga polymer-solvent mixture, and the microparticles are formed using the dissolvable template technology. the median size of drug-loaded particles is 60 μm. the authors reported that the ohr1031 content in the microparticles was 57%-100% incorporation efficiency. the rai et al mindful-based stress reduction impact vol 1 | issue 2 | jul – sep 2022 indian j pharm drug studies | 43 drug release rate was nearly zero-order for over three months with virtually no initial burst. solidropsolidrop by otero therapeutics consists of a thermoresponsive hydrogel carrier and drug-loaded polymer microspheres. on administering a single brimonidine-loaded gel/microsphere drop, the ioplowering efficacy (reduced by 30% of baseline iop) was comparable to that of rabbits receiving twice-daily, standard brimonidine drops for 28 days. the gel drops were retained in the fornix during the entire period of the study [64]. env 515/travoprost xrenv515 pga/travoprost xr therapy is a particle replication in a nonwetting templatebased biodegradable polymer drug delivery system. the implant is rod-shaped and fits the anatomy of the iridocorneal angle in the anterior chamber, allowing its administration via an acceptably sized needle. the result from the phase ii clinical trial reported that a single low dose of env515 decreases the mean iop by 6.7 ± 3.7 mmhg over eleven months. the mean iop after a single low dose of env515 was 19.5 mmhg over the 11 months [68]. bimatoprost srbimatoprost sr is a biodegradable implant for the decrement of iop with a 4-month sustained release period [69]. in the first phase iii clinical study, bimatoprost sr reduced iop by 30% over the 12-week primary efficacy period. the results showed no requirement of supplementary treatment for iop lowering for one year after the last implant insertion. the magnitude of ioplowering efficacy with bimatoprost sr observed in this study is similar to that observed with daily topical prostaglandin analogs. bimatoprost sr was well tolerated in the majority of patients. graybuggraybug is a drug-encapsulated microparticle formulation to provide continuous iop lowering that is administrated by the treating physician every 3 –6 months using a subconjunctival injection. gb-203 is a preclinical stage dual mode of action, single molecular entity agent that can hydrolyze into an active agent that has the potential to lower iop and a second active agent that can provide longterm neuroprotection [70]. another pilot polymer depot formulation of gb-6249-103 developed on the graybug platform safely has been shown to deliver its payload in a sustained manner both in vitro and in vivo [71]. a significant reduction in iop was observed within the first week following injection of the formulation in rabbits. the results recorded a sustained maximum iop lowering of ~20% over two months. otx-tpthe otx-tp (ocular therapeutix) delivered travoprost to the ocular surface via an intracanalicular punctal plug for up to three months, resorbs, and drains through the nasolacrimal system [72]. it consists of pegbased hydrogel with embedded travoprost–loaded pla microspheres. these microspheres slowly degrade and show a sustained drug release over 30 days. perera et al. [73] in a study reported a 100% retention rate of the plugs, ten days post-implantation, and a reduction in iop by 5.4–7.5 mmhg. it is minimally invasive, contains fluorescein to monitor any retention, and clears from the body through absorption. the studies have shown an enhanced therapeutic benefit for 90 days with a consistent 90% retention rate. the phase ii trial did not find any serious adverse effects and showed only slightly less hypotensive effects as compared with timolol. latanoprost punctal plug delivery systemgoldberg and williams [74] used the latanoprost punctal plug delivery system (l-ppds) for lowering iop in oag patients. the data reported by the authors showed a reduction in mean iop by 5.7 mmhg. they also reported that 60% of subjects in the study showed at least 5 mmhg or higher iop reduction, and 47% of the subjects showed a reduction of at least 6 mmhg. a statistically significant 22.3% mean change in iop was recorded in the subjects with l-ppds when compared with controls. evoluteevolute, a punctal plug delivery system developed by mati therapeutics has been tested with latanoprost in patients with oag or ocular hypertension [75]. the plug consists of a drug core, which allows unidirectional sustained drug elution into the tear film. in phase ii clinical trial, an overall punctal plug retention rate of 96% was reported at 12 weeks. in the second phase, the retention rate of plugs was 92% in the 12th week. the punctal plugs loaded with travoprost reduced the pressure by 7 mm compared with a 5-mmhg decreased pressure with latanoprost. pentablock copolymer gelsthe pentablock copolymer gels were used as a vehicle for topical and intraocular delivery of glaucoma drugs like bimatoprost. the food and drug administration (fda) has approved five different pentablock copolymers for use in the eye. these include polyglycolic acid (pga), pcl, peg, pla, and plga [76]. the drug was introduced as an eye drop, then changes physical characteristics based on body temperature at contact. the change in viscosity gives the vehicle copolymer gel-like characteristics gets accumulated under the lower palpebra, releasing the drug over a longer period. microneedlesmicroneedles are drug delivery devices manufactured using metals or polymers with dimensions between 10 and 200 μm. the ultra-dimensions of these devices make the drug delivery less invasive and more targeted to the sites of drug action. jiang et al. used 500 to 750 μm longcoated stainless-steel microneedles delivering pilocarpine into the anterior chamber via the intrascleral route. the authors reported a 45-fold increase in drug absorption compared with conventional eye drops [77, 78]. rai et al mindful-based stress reduction impact vol 1 | issue 2 | jul – sep 2022 indian j pharm drug studies | 44 discussion in this review article, we described the development of welltolerated drug delivery systems that helps to overcome the factors limiting adequate drug delivery to the glaucomatous patients targeting infected tissues with traditional techniques. ocusert did not become a widely accepted method of drug delivery although the clinical studies showed positive outcomes the difficulty of device insertion, failure in satisfactorily controlling iop in many patients, ejection of the device from the eye, irritation during insertion, and the difficulty in device insertion [14]. the ease of availability and application of therapeutic contact lenses make them an ideal drug delivery system. the therapeutic contact lenses help in sustained and regulated ocular drug delivery due to their unique properties like extended wear and more than 50% bioavailability comparison with eye drop formulations [27, 28]. soft contact lenses are water-soluble polymeric hydrogels crosslinked to form networks. these hydrogel lenses are widely used for drug delivery, even though the delivery of watersoluble drugs, such as timolol and dorzolamide, elutes from the highly hydrated polymer networks rapidly [29]. in comparison, the soft contact lenses manufactured by polymerization n, n-diethyl acrylamide methacrylic acid deliver timolol over a prolonged period [30]. injectable systems are passive delivery systems capable of delivering medications to the target tissues for an extended period. the injectable systems are typically implanted at the site of drug release through a minimally invasive procedure, usually in an outpatient setting. the injectable systems use a polymer delivery vehicle to prolong delivery up to a few months around the surrounding tissue. both degradable and non-degradable polymers have been developed used as injectable systems for drug delivery in the eye [65]. degradable plgas are materials of choice for developing such a system. the non-degradable alternative such as the polymer of ethylene-co-vinyl acetate may lead to an immune response due to the prolonged presence of a foreign body [66]. the rate of dispersion of drugs from these systems is variable, with an initially more massive quantity release. the water solubility of the drug affects the efficacy because hydrophilic drugs interact poorly with degradable polymers as they are hydrophobic [67]. several methods of treatment of glaucoma in the patient’s eye were discussed in this review and also, we discussed the development of the well-tolerated drug delivery systems that helps to overcome the limiting factor of adequate drug delivery systems. in the past, many studies have emphasized the importance of adherence, compliance, and persistence for the management of glaucoma. we know the available drugs are efficacious in lowering iop and neuroprotection, the traditional methods of topical drug delivery have been not satisfactory due to poor target bioavailability, increased systemic absorption, and poor patient compliance. conclusion this review described the direction and ongoing innovation/research to address the challenges of safer and more effective drug delivery challenges associated with the previous one. the majority of the devices studied in this article are currently in various stages of development and are not commercially available. the impact of these devices on the patients can only be gauged once they are available for clinical use and extensive clinical data are available for scrutiny. notwithstanding the lack of data, the critical role of these devices in glaucoma management shortly needs to be emphasized. the potential of increasing patient compliance and persistence for optimum outcomes with the help of these devices is unprecedented. eye drop installation has always been a challenge, especially in the geriatric and pediatric age group patients. effective localized delivery will prevent drug loss due to systemic absorption and firstpass metabolism thereby, minimizing drug wastage. the prevalence of ocular surface disease in patients installing antiglaucoma drugs with added preservatives may be overcome with the newer devices. the 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department of pharmacology, hkes’s mtrips, kalaburagi. abstract aim and objective: to evaluate the role of zingiber officinale extract along with glibenclamide on lipid profile in alloxan & streptozotocin induced diabetes mellitus in rats. method and materials: the diabetes in rats was induced by the administration of intraperitoneal injection of alloxan (100mg/kg) and streptozotocin (65 mg/kg). after 2 days of injection, the hyperglycaemic rats (glucose level > 200 mg/dl) were separated and divided into five groups consisting of six rats in each group. the oral treatment was started from the same day for next three weeks. on 21st day the blood was collected for biochemical estimations by retro orbital puncture. the serum was obtained by centrifuging the blood samples at 3000 rpm for 10 m and used for estimation of sgpt, sgot, sod and cat. results: diabetic rats treated with glibenclamide and glibenclamide along with ginger extract showed significant (p<0.001) reduction in the elevated levels of total cholesterol, triglycerides and ldl cholesterol in comparison to diabetic control group. the effect was less significant in rats treated with only ginger extract (p<0.01). also, the hdl level was significantly (p<0.001) increased in glibenclamide and glibenclamide along with ginger treated group. serum biomarkers such as sgpt and sgot level were significantly elevated in diabetic control group. the animals treated with glibenclamide and glibenclamide in combination with ginger extract, the elevated sgpt and sgot levels were normalised significantly (p < 0.001, p < 0.01 respectively) as compared to the diabetic control. from antioxidant studies, it was found that alloxan and streptozotocin induced diabetic control animals showed a significant decrease in the levels of sod and cat as compared to normal control. the animals treated with glibenclamide and glibenclamide + ginger extract combination showed significant increase in cat and sod (p<0.01 and p<0.001 respectively) as compared to diabetic control. conclusions: the findings of the study suggest that, ginger shows complementary action with glibenclamide in the treatment of diabetes. so it can be considered as a safe supplementary in management of diabetes mellitus. further studies has to conducted, to check whether dosage of glibenclamide can be reduced when it is given along with ginger. key words: diabetes, bioenhancer, ginger, serum glutamic-oxaloacetic transaminase, separation of duty, common admission test. he major chronic complications associated with diabetes include retinopathy, neuropathy, nephropathy, atherosclerotic coronary artery disease and peripheral atherosclerotic vascular disease. besides hyperglycemia, several other factors like hyperlipidemia and enhanced oxidative stress play a major role in diabetic pathogenesis [1]. diabetes has been known to medical sciences longer than any other hereditary metabolic diseases. nevertheless, the existing methods of treatment for this age old illness are not completely satisfactory owing to low efficacy, associated adverse effects and compliance issues. among the therapies non pharmacologic therapy (e.g. diet, exercise and weight loss) remains to be critical component in access this article online received – 06th jan 2024 initial review – 11th feb 2024 accepted – 26th feb 2024 quick response code diabetes treatment. dietary management includes the use of traditional medicines mainly derived from plants. several herbal preparations are used to treat diabetes, but their reported hypoglycemic effects are complex or even paradoxical in some cases. several mechanisms have been proposed for the hypoglycemic effect of phytochemical, such as inhibition of carbohydrate metabolizing enzymes, manipulation of glucose transporters, -cell regeneration and enhancing insulin releasing activity. eugenia jambolana inhibits -amylase, -glucosidase, sucrase and increase glucose uptake by cells. also increase insulin secretion and inhibit insulinase activity. momordica charantia inhibits glucose-6-phosphatase, fructose-1, 6biphosphatase and stimulates of hepatic glucose-6-phosphate dehydrogenase activities [2]. __________________________________________________ correspondence to: ashoka shenoy m, associate professor, department of pharmacology, srinivas college of pharmacy, mangalore, india 574143. email: shenoyscp@gmail.com. t mailto:shenoyscp@gmail.com shenoy m & mahurkar complementary action of ginger as bioenhancers in the treatment of diabetes vol 3 | issue 1 | jan – mar 2024 indian journal pharm drug studies | 30 the development of new therapies that are able to improve glycemia management and even to cure diabetes is of great interest. use of plants for human health care is as ancient as human beings themselves india has one of the oldest, richest and diverse cultural traditions associated with the use of the plants and herbs for human, livestock and plant health [3]. due to change in the lifestyle, the number of people in the world with diabetes has increased dramatically over recent years. the world is facing an explosive increase in the incidence of diabetes mellitus. according to the world health organization (who) estimates, the number of adults with diabetes in the world will rise from 135 million in 1995 to 300 million in the year 2025, with one-third of affected individuals living in india and china alone [4]. herbs are staging a comeback and herbal ‘renaissance’ is happening all over the globe. the herbal products today symbolize safety in contrast to the synthetics that are regarded as unsafe to human. world health organization has recognized the potential of traditional and folk medicines in the management and self-reliance of health care system and currently it is encouraging and promoting the traditional systems in “national health care programmes” of various countries. the world health organization has estimated that 80% of the world's population use botanical medicine for their primary healthcare needs. herbal bioenhancers without possessing their own inherent pharmacological activity of their own but when coadministered with other drugs, enhances their bioavailability and hence efficacy. the interest for bioenhancers arises because of chemotherapeutic agents which are poorly bioavailable, administered for prolong periods, toxic and expensive. one of the unique ways to achieve reduction in drug dosage and therefore drug toxicity & cost is to increase drug bioavailability. the present pharmaceutical research is more concerned with different aspects of exploring new chemical molecules having new modes of action. new drug development technologies were developed from the economics of treatment. there is a revolutionary shift in the way medicines are administered due to recent developments for enhancing the bioavailability. the present global focus is on methods aimed at reducing drug treatment period leading to decrease in drug treatment cost. the reduction in cost of therapy will make more affordable for financially challenged wide sections of society [5]. materials & methods experimental animals: wistar rats (180 to 200 g) of either sex were used for this study. they were maintained under standard conditions (temperature 22±2oc, relative humidity 60±5% and 12 h light/dark cycle).the animals were housed in sanitized polypropylene cages containing sterile paddy husk as bedding. they had free access to standard pellet diet and water ad libitum. all the animals received humane care according to the criteria outlined in the “guide for the care and use of laboratory animals” prepared by the “national academy of sciences” and published by the “national institute of health”. all the procedures were performed in accordance with institutional animal ethics committee constituted as per the direction of the committee for the purpose of control and supervision of experiments on animals (cpcsea), under ministry of animal welfare division, government of india, new delhi, india. chemicals: all the chemicals and reagents used were of analytical grade and were purchased from yarrow chem, loba chem, himedia and agappe diagnostics. methodology preparation of aqueous extract of gingiber officinale rhizomes: aqueous ginger extract was prepared from locally available ginger rhizomes. ginger rhizomes (500g) were peeled on crushed ice and were cut into small pieces and homogenized in750 ml cold, 0.9% nacl solution and 250 ml ice cold water to make the volume up to 1000 ml. the homogenization was carried out in a blender for 12 m. the homogenized mixture was filtered three times through cheese cloth. the filtrate was centrifuged at 2000 rpm for 10 m and the clear supernatant fraction was separated and volume made up to 1000 ml with normal saline. the concentration of this ginger preparation was considered to have 500 mg / ml on the basis of the weight of the starting material. the extract was stored in sample tubes at -4°c until fed to animals [6]. routes of drug admistration: the vehicle, standard drug and test drugs were administered orally with the help of an oral feeding needle. pharmacological screening alloxan induced anti-diabetic activity: fasting blood glucose was determined after depriving food for 16 h with free access to drinking water. hyperglycemia was induced by single i.p injection of 100 mg/kg of alloxan monohydrate in normal saline. after 2 days of alloxan injection, the hyperglycemic rats (glucose level > 200 mg/dl) were separated and divided into five groups consisting of six rats in each group. the oral treatment was started from the same day except diabetic control groups for three weeks. the animals had free access to feed with water ad libitum. experimental design: animals were randomly divided into 5 groups of 6 each. the different groups were assigned as follows: group i vehicle control (normal saline), group ii diabetic control (alloxan100mg/kg), group iii diabetic rats + ginger extract (500mg/kg), group iv diabetic rats + glibenclamide (5mg/kg), group v diabetic rats + glibenclamide (5 mg/kg) + ginger extract (500mg/kg) shenoy m & mahurkar complementary action of ginger as bioenhancers in the treatment of diabetes vol 3 | issue 1 | jan – mar 2024 indian journal pharm drug studies | 31 streptozotocin induced anti-diabetic activity: fasting blood glucose was determined after depriving food for 16 h with free access to drinking water. hyperglycemia was induced by single i.p injection of 65 mg/kg of stz in citrate buffer, freshly prepared and injected immediately to prevent degradation. after 2 days of streptozotocin injection, the hyperglycemic rats (glucose level > 200 mg/dl) were separated and divided into five groups consisting of six rats in each group. the oral treatment was started from the same day except diabetic control groups for three weeks. the animals had free access to feed with water ad libitum. experimental design: animals were randomly divided into 5 groups of 6 each. the different groups were assigned as follows: group i: vehicle control (normal saline), group ii: diabetic control (streptozotocin 65mg/kg), group iii: diabetic rats + ginger extract (500mg/kg), group iv: diabetic rats + glibenclamide (5mg/kg), group v: diabetic rats + glibenclamide (5 mg/kg) + ginger extract (500mg/kg). collection of blood and serum samples: the above treatment was carried out in each group of animals for 21days. on 21st day the blood was collected for biochemical estimations by retro orbital puncture. the serum was obtained by centrifuging the blood samples at 3000 rpm for 10 m and they were used for estimation of sgpt, sgot by using a corresponding kit from agappe diagnostics pvt. ltd. the intensity of the colored complex formed after treating with these reagents was estimated in semi-auto analyzer. the parameters studied were as follows:  biochemical parameters such as a. serum lipid profile b. serum glutamic pyruvate transamase (sgpt) c. serum glutamic oxaloacetate transamase (sgot)  endogenous antioxidant parameters include a. superoxide dismutase (sod) b. catalase (cat) antioxidant parameters tissue preparation  animals were sacrificed by cervical dislocation.  the whole liver was perfused in situ with ice-cold saline, dissected out, blotted dry and immediately weighed.  a liver homogenate was prepared with ice-cold salineedta.  the homogenate was centrifuged at 10,000 rpm for 10 m and the pellet discarded. the supernatant was again centrifuged at 20,000 rpm for 1 hour. statistical analysis: results of biochemical estimation were reported as mean ± s.e.m. the total variation present in a data was analyzed by one way analysis of variance (anova). results diabetic rats treated with glibenclamide and glibenclamide along with ginger extract showed significant reduction in the elevated levels of total cholesterol, triglycerides and ldl cholesterol in comparison to diabetic control group. the effect was less significant in rats treated with only ginger extract. also, the hdl level was significantly increased in glibenclamide and glibenclamide along with ginger treated group as summarized in (table 1 & table 2). serum biomarkers: after 21days of experiment, serum biomarkers such as sgpt and sgot level were significantly elevated in diabetic control group. in animals treated with glibenclamide and glibenclamide in combination with ginger extract, sgpt and sgot levels were decreased significantly as compared to the diabetic control as shown in (table 3). antioxidant parameters: from antioxidant studies, it was found that both alloxan and stz induced diabetic control animals showed a significant decrease in the levels of sod and cat as compared to normal control. animals treated with glibenclamide and glibenclamide + ginger extract combination showed significant increase in cat and sod as compared to diabetic control as shown in (table 4). table 1: effect of ginger, glibenclamide and their combination on serum lipid profile in alloxan induced diabetic rats group cholesterol (mg/dl) triglyceride (mg/dl) hdl (mg/dl) ldl (mg/dl) normal control 62.12±0.84 62.02±1.25 47.32±1.30 42.20±0.14 diabetic control 6.13±1.57 89.45±0.35 20.18±0.34 97.24±0.37 ginger extract (500 mg/kg) 82.15±0.34* 83.3±0.17* 27.56±0.56* 71.35±0.25* glibenclamide (5 mg/kg) 60.34±0.47** 74.19±0.78** 33.24±0.25** 66.16±1.65** glibenclamide (5 mg/kg) + ginger extract (500 mg/ kg) 56.28±0.67** 64.33±1.26** 39.33±0.58** 57.46±1.07** values are expressed as mean± s.e.m (n=6). one way anova followed by dunette’s test. *p<0.01, **p<0.001 when compared with diabetic control group. shenoy m & mahurkar complementary action of ginger as bioenhancers in the treatment of diabetes vol 3 | issue 1 | jan – mar 2024 indian journal pharm drug studies | 32 table 2: effect of ginger, glibenclamide and their combination on serum lipid profile in streptozotocin induced diabetic rats group cholesterol triglycerides hdl ldl normal control 66.23±2.15 60.58±0.05 48.51±1.54 43.19±3.21 diabetic control 151.5±0.12 110.26±1.69 16.86±8.12 104.31±1.58 ginger extract (500 mg/kg) 118.3±0.25* 98.0±6.18* 31.57±3.51* 87.68±1.95* glibenclamide (5 mg/kg) 105.61±6.12** 82.84±2.89** 36.85±0.24** 75.21±2.68** glibenclamide (5 mg/kg) + ginger extract (500mg/ kg) 89.88±1.59** 64.42±3.25** 42.19±4.25** 61.32±2.18** values are expressed as mean ± s.e.m (n=6). one way anova followed by dunette’s test. *p<0.01, **p<0.001 when compared diabetic control group. table 3: effect of ginger, glibenclamide and their combination on serum sgpt and sgot in alloxan and streptozotocin induced diabetic rats group alloxan stz sgpt sgot sgpt sgot normal control 56.43±1.28 64.52±1.45 58.61±2.65 62.63±3.75 diabetic control 142.51±1.95 150.38±1.65 139.48±2.38 148.72±1.05 ginger extract (500 mg/kg) 116.21±3.24 * 123.51±2.43 * 113.45±2.09 * 121.34±1.34 * glibenclamide (5 mg/kg) 102.81±2.65 ** 98.91±2.85 ** 92.80±2.11 ** 84.64±1.69 ** glibenclamide (5 mg/kg) + ginger extract (500 mg/ kg) 89.38±1.65 ** 87.45±2.25 ** 79.52±3.54 ** 76.57±1.95 ** values are expressed as mean ± s.e.m (n=6). one way anova followed by dunette’s test. *p<0.01, **p<0.001 when compared diabetic control group table 4: effect of ginger, glibenclamide and their combination on serum sod and cat in alloxan and streptozotocin induced diabetic rats group alloxan stz sod cat sod cat normal control 12.65±1.18 8.25±1.65 11.36±3.25 6.45±2.57 diabetic control 4.56±1.75 4.14±2.65 3.28±1.78 2.35±1.15 ginger extract (500 mg/kg) 6.16±1.34* 4.76±2.12* 5.56±1.76* 3.25±1.43* glibenclamide (5 mg/kg) 7.55±2.76** 5.92±1.85** 8.46±1.32** 4.05±2.96** glibenclamide (5 mg/kg) + ginger extract (500 mg/ kg) 8.21±2.65** 6.85±1.25** 9.52±2.54** 4.46±2.75** values are expressed as mean ± sem (n=6). one way anova followed by dunette’s test. *p<0.01, **p<0.001 when compared diabetic control group discussion the present study was undertaken to evaluate the antidiabetic activity of a standard synthetic antidiabetic drug glibenclamide in combination with an herbal bioenhancer drug ginger in diabetic rats. diabetes is a global disease with a huge adverse impact on health and mortality, particularly from cardiovascular disorders. it occurs at any time of life from infancy to old age. type-2 diabetes is primarily a lifestyle disorder, which accounts for around 90% of diabetes cases and increasing at an astonishing rate, particularly in developing countries like india [7]. diabetes mellitus is a group of disorders characterized by increased blood sugar, polyhydria, polyuria and weight loss. it is treated either by allopathic or with traditional system of medicines which utilizes herbs for cure. two national surveys examined the prevalence and pattern of use of complementary and alternative medicine (cam) among individuals with diabetes. one study by medical expenditure panel survey data in 2016 reported that individuals with diabetes were 1.6 times more likely to use cam than persons without diabetes. data from a national representative survey conducted from 2017 to 2022 reported that 35% of respondents with diabetes used cam to treat their condition [8]. herbs are often administered in combination with therapeutic drugs, raising the potential of herb–drug interactions. there is very little information published on herb–drug interactions while the use of herbs is progressively growing across the world. certain herbal supplements can cause potentially dangerous side effects when taken with prescription drugs and the number of cases reported for the emerging herb–drug interactions are already on the rise [9]. in the present study, diabetes was induced using alloxan and streptozotocin (stz). alloxan is a cyclic urea derivative, is reported as a potent diabetogenic agent and has widely been used for the induction of experimental diabetes in animal species by damaging the insulin secreting pancreatic β-cells, resulting in a decrease in endogenous insulin release. alloxan shenoy m & mahurkar complementary action of ginger as bioenhancers in the treatment of diabetes vol 3 | issue 1 | jan – mar 2024 indian journal pharm drug studies | 33 produces oxygen radicals which causes pancreatic injury and could be responsible for increased blood glucose seen in the animals [10]. over production (excessive hepatic glycogenolysis and gluconeogenesis) and decrease utilization of glucose by the tissues are the fundamental basis of hyperglycemia in diabetes mellitus. streptozotocin is a broad spectrum antibiotic, induces diabetes in a wide variety of animal species by damaging the insulin-secreting cells of the pancreas [11]. streptozotocin (stz) induced diabetes is a valuable model for induction of diabetes mellitus. diabetes mellitus induced by stz may be due to pancreatic β-cells destruction resulting in a decrease in endogenous insulin release. iddm can be induced by injecting stz to adult rats whereas niddm is induced by administration of stz to neonatal rats [12]. in this study the animals survived without insulin treatment and showed improvement by glibenclamide which act by stimulating residual beta cells of the pancreas indicate incomplete destruction of pancreatic beta cells of the diabetic rats in the present study. the model can therefore be considered as type ii diabetic model showing symptoms like hyperglycemia, glycosuria, polyuria, loss of body weight in spite of polyphagia [13]. non insulin dependent diabetes mellitus (niddm) also called as type 2 diabetes is a complex metabolic disorder that involves abnormalities in both insulin secretion and action at peripheral tissues. it is a more prevalent form of diabetes and responsible for 90% of the disease. in niddm, the kinetics of insulin release in response to meal or glucose is altered. so, postprandial blood glucose remains high and leads to glucose intolerance. postprandial hyperglycemia plays an important role in the development of diabetic complications. poor glycogen content in insulin dependent tissues such as liver, skeletal muscle and adipose tissues were observed in niddm due to insulin resistance. in niddm, partial or total deficiency of insulin causes derangement in carbohydrate metabolism [14]. most employed oral hypoglycemic agents are sulfonylureas and biguanides. these drugs, however, have disadvantages such as primary and secondary failure of efficacy as well as the potential for induction of severe hypoglycemia. there is a need, therefore, for new candidate molecules that may effectively reduce insulin resistance or potentiate insulin action in genetically diabetic or obese individuals. new drugs that reverse insulin resistance without stimulating insulin release from β -cells also fulfill a major medical need in the treatment of non-insulin-dependent diabetes mellitus (niddm). the search for such drugs with a potential to reduce long-term complications of niddm is, therefore, of current interest [15]. glibenclamide is a second generation sulphonylurea derivative, oral hypoglycemic agent and found to be effective in diabetic rats that retain functioning of islet β-cells. hence the principle mechanism of action is to stimulate the production and secretion of insulin by the β-cells of pancreas. this drug may lower down the output of glucose from the liver by insulin independent mechanism [16]. ginger is the rhizome of the plant zingiber officinale which is a proven herbal bioenhancer. aqueous extract of ginger of concentration 500mg/ml was prepared and used for the study. in the present study, the blood lipid levels in both alloxan and stz treated rats were significantly increased as compared to normal rats. whereas the group treated with standard drug glibenclamide and those treated with glibenclamide + ginger extract combination showed significant reduction in lipid profile. the results indicate that the combination of glibenclamide and ginger extract could lead to increase in the effect of glibenclamide that may be helpful to reduce the dose of glibenclamide. also, to minimize the adverse effects. insulin deficiency leads to various metabolic aberrations in the rats; the rise in ldl level is accompanied by increase in sgpt and sgot level. in this present study, glibenclamide and glibenclamide + ginger extract combination treated animals serum showed significant reduction in sgpt and sgot level [17]. oxidative stress plays a major role in the pathogenesis of both types of diabetes mellitus. free radicals are formed in diabetes by glucose oxidation, protein glycation and the subsequent degradation of glycated proteins. high levels of free radicals and the simultaneously declined antioxidant enzyme levels lead to cell damage, inactivation of enzymes and lipid peroxidation. superoxide dismutase and catalase play an important role in the detoxification of super oxide anion and h2o2 respectively. in present study, catalase and sod which are most important antioxidant enzymes were found to be decreased in diabetic control group. treatment with glibenclamide and glibenclamide + ginger extract combination restores the level of both enzymes [18]. conclusions intraperitoneal administration of alloxan and streptozotocin produced elevated levels of lipid triglycerides, ldlcholesterol, hyperglycemia, loss of body weight, increase in serum biomarkers such as sgpt and sgot (liver damage), increased oxidative stress due to decrease in antioxidants such as sod and cat. the animal groups treated with glibenclamide and glibenclamide in combination with ginger extract showed antidiabetic effect by restoring the above markers. the antidiabetic effects are better in animals treated with combination of glibenclamide and ginger extract in comparison to glibenclamide alone. the findings of the study suggest that, ginger shows complementary action with glibenclamide in the treatment of diabetes. so it can be considered as a safe supplementary in management of diabetes shenoy m & mahurkar complementary action of ginger as bioenhancers in the treatment of diabetes vol 3 | issue 1 | jan – mar 2024 indian journal pharm drug studies | 34 mellitus. further studies has to conducted, to check whether dosage of glibenclamide can be reduced when it is given along with ginger. acknowledgements: the authors are grateful to management of hkes’ matoshree taradevi rampure institute of pharmaceutical sciences, kalaburagi and srinivas college of pharmacy, mangalore for providing necessary facilities to carry out the experiments. references 1. patil um, singh a, chakraborty ak. role of piperine as a bioavailability enhancer. inter j recent adva in pharmac res. 2017; 1(4):16-23. 2. drabu s, khatri s, babu s, et al. use of herbal bioenhancers to increase the bioavailability of drugs. res j pharmac, bio & chem sci. 2018; 2(4):108-119. 3. vendruscolo a. antiinflammatory and antinociceptive activities of zingiber officinale roscoe essential oil in experimental animal models. indian j pharmac. 2017; 38(1):58. 4. 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diabetes. indian j pharm drug studies. 2024; 3(1):29-34. funding: none; conflicts of interest: none stated http://www.ijp-online.com/searchresult.asp?search=&author=a+vendruscolo&journal=y&but_search=search&entries=10&pg=1&s=0 http://scialert.net/asci/author.php?author=r.c.r.&last=latha http://scialert.net/asci/author.php?author=p.&last=daisy http://www.ijp-online.com/searchresult.asp?search=&author=guy+bertrand+sabas+nya+njomen&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijp-online.com/searchresult.asp?search=&author=f+gy+mbongue&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijp-online.com/searchresult.asp?search=&author=p+kamtchouing&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijp-online.com/searchresult.asp?search=&author=o+jl+essame&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijp-online.com/searchresult.asp?search=&author=agung+endro+nugroho&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijp-online.com/searchresult.asp?search=&author=agung+endro+nugroho&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijp-online.com/searchresult.asp?search=&author=ni+kadek+warditiani&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijp-online.com/searchresult.asp?search=&author=rakesh+barik&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijp-online.com/searchresult.asp?search=&author=rakesh+barik&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijp-online.com/searchresult.asp?search=&author=deep+qwatra&journal=y&but_search=search&entries=10&pg=1&s=0 das k et al. heparininitiated thrombocytopenia: hemodialysis’s 1-month study vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 183 original article incidence of thrombocytopenia one month after initiation of heparin during hemodialysis: an observational study krisnendu das1, utpal bhui2, arghya majumdar3, sanmoy karmakar4 from, 1assistant professor, school of pharmaceutical sciences, the neotia university, diamond harbour, west bengal, 2pg student, department of pharmacology, school of pharmaceutical sciences, lovely professional university, phagwara, punjab, 3director & head of nephrology, department of medicine, amri hospitals, 4professor, department of pharmacology, jadavpur university, kolkata, west bengal, india abstract thrombocytopenia, a platelet count below 150 × 109/l, may be acquired or congenital. in patients with chronic kidney disease on hemodialysis, thrombocytopenia may be multi-factorial. however, the most worrisome of these is heparin-induced thrombocytopenia (hit), as it causes thrombosis in various vital organs in addition to bleeding. heparin is the mainstay of anticoagulation on hemodialysis to prevent clotting in the extra-corporeal circuit. hit may be either type 1 (non-immune) or type 2 (immune). the protocol of testing for the presence of thrombocytopenia 1 month after starting heparin on hemodialysis is seldom followed and the exact incidence of hit in eastern india is unknown. we studied hemodialysis patients in a tertiary care hospital in kolkata over a period of 9 months. all of them had platelet counts checked after starting hemodialysis as per protocol. no patients had symptoms or signs of hit. a drop in platelet count was noticed in 3 patients, but it was transient and attributable to other causes. it did not necessitate stopping of heparin and the platelet count spontaneously improved. so, it is reassuring that hit is rare in the eastern part of india. key words: contaminated heparin, oversulfated chondroitin sulphate, heparin-induced thrombocytopenia, end-stage renal disease, anti heparin platelet factor 4 antibodies hrombocytopenia, or low platelet count, defined as a platelet count below 150 × 109/l, can be broadly classified as congenital and acquired. acquired thrombocytopenia could be immune or non-immune [1]. thrombocytopenia could be a result of decreased marrow production, increased destruction or sequestration/ consumption in the periphery, or a combination of decreased production and sequestration. initial steps in the evaluation of thrombocytopenia include a review of the peripheral blood smear to exclude pseudothrombocytopenia due to platelet clumping [2]. the peripheral blood smear may also provide clues toward other causes of thrombocytopenia when combined with the complete blood count and a good patient history access this article online received – 06th august 2023 initial review – 16th august 2023 accepted – 22nd august 2023 quick response code and physical examination. platelet size and the presence of schistocytes, polychromasia, or spherocytes are some of the other features on the peripheral blood smear that help in diagnosing the etiology of thrombocytopenia [3]. thrombocytopenia could be a serious medical condition such as thrombotic thrombocytopenic purpura (ttp) or heparin-induced thrombocytopenia (hit). therefore, it is always important for the treating physician to evaluate thrombocytopenia in a timely fashion so that the treatment for some of the serious conditions is not delayed [4]. the relevance of thrombocytopenia in the individual patient is variable and depends on the clinical presentation. because platelets play an essential role in preserving vessel wall integrity [5]. thrombocytopenia is associated with a defect of primary hemostasis. clinically significant spontaneous _______________________________________ correspondence to: krishnenedu das, 36a, mahanirban road, kolkata 700029, west bengal, india. email: krishnendas96@gmail.com tel.: +91 9874377408 t mailto:krishnendas96@gmail.com das k et al. heparininitiated thrombocytopenia: hemodialysis’s 1-month study vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 184 bleeding does not usually occur until the platelet count is less than 10-20 109/l [6]. however, the presence of thrombocytopenia can aggravate surgical or traumatic bleeding or prevent the administration of effective treatment for several conditions (eg, antiviral therapy for chronic hepatitis c virus infection or cancer chemotherapy) [7]. in other situations, a low platelet count is the only initial manifestation of an underlying disorder that poses greater risks than thrombocytopenia itself (eg, hiv infection or myelodysplastic syndromes) or is an important marker of disease activity (eg, thrombotic microangiopathies) [8]. establishing the cause of thrombocytopenia has obvious clinical repercussions, but is sometimes quite challenging. this is particularly the case for hospitalized patients, in whom thrombocytopenia appears frequently in the background of a multisystem disorder and may be determined by multiple mechanisms [9]. conversely, in the outpatient setting, thrombocytopenia is often isolated and asymptomatic, and the diagnosis of the specific cause is usually straightforward. thrombocytopenia in pregnancy deserves special consideration because of the possible consequences on the fetus [10]. a structured approach to the diagnosis of thrombocytopenia involves the integration of clinical findings and appropriate support from the laboratory and other medical disciplines [11]. material and methods the study was conducted in a prospective mode and was based on patients who were observed during the study period from 21st september 2020 to 4th may 2021. the study was carried out in the dialysis department of amri group of hospitals, kolkata, dhakuria. formal permission for the study has been obtained from the respective iec. result statistical analysis shows that in comparison of platelet count according to age and sex before and after heparin, 61-70 age group patients have more changes in platelet count than other age groups. females have more changes in platelet count than males. no patients had symptoms or signs of hit. two patients dropped platelet count below 1l and one patient below 1.5l but did not. develop thrombotic events. so heparin was continued. the thrombocytopenia was attributed to transient episodes of sepsis. the platelet count spontaneously improved again to >1l of two patients and one patient’s platelet count improved again to >1.5l (table 1). table 1 – patient details age sex platelet count before heparin platelet count after heparin 58 f 2.8 2.2 73 m 1.7 1.5 70 m 3.68 2 69 m 1.7 1.5 70 f 1.7 1.5 59 f 1.5 1.51 66 m 1.5 1.6 78 m 1.5 1.6 65 m 1.1 0.75 65 m 1.6 1.6 68 m 1.6 0.9 58 m 1.5 1.3 53 m 1.6 1.6 55 m 2.1 2.2 83 f 2.2 2 64 f 3.68 2 56 m 3 2 66 m 1.9 1.6 51 m 2.27 2.2 the pie chart (figure 1) illustrates the platelet count before and after heparin according to age, whereas it reveals that the high platelet count before and after heparin is bigger at 61 to 70 age compared to other age groups. according to figure 2, which shows the platelet count before and after heparin according to sex, male patients have higher platelet counts both before and after heparin than female patients. figure 3 shows based on age and sex, it compares platelet counts before and after heparin, with the rate of platelet counts before heparin being higher than the rate of platelet counts after heparin. this shows that once heparin was started during hemodialysis, the amount of platelet count decreased. figure 4 shows the differences in platelet count before and after heparin based on age, clearly demonstrating that the platelet count before heparin is higher than the platelet count after heparin at various age groups. the changes in platelet count are also significant, with a 0.56 change from 61 to 70 years old. the graph charts (figure 5) illustrate how the platelet count has changed according to sex, with the platelet count before heparin being higher than the platelet count following heparin. additionally, compared to men, women have greater variations in platelet count, which is 0.53. das k et al. heparininitiated thrombocytopenia: hemodialysis’s 1-month study vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 185 figure 1 – platelet count before and after heparin at different age groups. figure 2 -platelet count before and after initiation of heparin at different sex groups. figure 3 drop of platelet count before and after heparin based on age and sex. figure 4 – changes in platelet count before and after heparin based on age. figure 5 changes in platelet count before and after heparin based on sex. 36.8 47.4 10.5 5.3 age 51-60 61-70 71-80 81-90 26.3 73.7 sex female male 2.03 1.66 0.00 0.50 1.00 1.50 2.00 2.50 before heparin after heparin platelet count 0.00 0.50 1.00 1.50 2.00 2.50 51-60 61-70 71-80 81-90 2.11 2.05 1.60 2.20 1.86 1.49 1.55 2.00 0.25 0.56 0.05 0.20 platelet count & age platelet count before heparin platelet count after heparin change in platelet count 0.00 1.00 2.00 3.00 female male 2.38 1.911.84 1.60 0.53 0.31 platelet count & sex platelet count before heparin platelet count after heparin change in platelet count das k et al. heparininitiated thrombocytopenia: hemodialysis’s 1-month study vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 186 discussion takfumi matsuo et al. conducted a study on heparin induced thrombocytopenia and haemodialysis in dialysis patients with an unexpected fall in the platelet count, and/or unexplained thrombotic events, particularly visible clotting in the circuit under an adequate heparin dose, that begins between 5 and 10 days ( between 7 and 30 days, mostly by the third to fifth session) after heparin initiation elisa test result came positive, so in this case alternative non-heparin anticoagulants like a direct thrombin inhibitor argatroban was used as an alternative to heparin. argatroban contributed to the rapid recovery of the platelet count and the disappearance of visible circuit clotting [12]. robert e.cronin et al., is conducted a study on unfractionated heparin for haemodialysisin the 10,564 maintenance hemodialysis patients in the united kingdom, the prevalence of hit was 0.26per 100 patients and only 17% of these had complications related to the disorder. the disorder was typically discovered 5–10 days following exposure to unfractionated heparin and was characterized by a drop in platelet count and variable occurrence of a clotting event. arterial disorders. the drop in platelet count was typically 30–50% below baseline and rarely reduced to the low levels seen with other drug-induced thrombocytopenias. when heparin was withdrawn, platelet counts typically recover to baseline within 2 weeks [13]. jenny i. shen, md, ms and wolfgang c. winkelmayer et al. conducted a study on the use and safety of unfractionated heparin for anticoagulation during maintenance hemodialysis in a 50-year-old man with a history of diabetes mellitus and end-stage renal disease (esrd) became hypotensive 15 minutes into his dialysis session. he had been receiving maintenance hemodialysis through a left arteriovenous fistula 3 times a week for the past 5years without complication. he does not have a history of a bleeding disorder but takes 81 mg of aspirin daily. however, his abdominal pain worsened, so the hemodialysis treatment was discontinued and he was sent to the emergency department for further evaluation, later he had been transfused a total of 8 units of packed red blood cells. his hemoglobin level had stabilized at 9.5 g/dl. his platelet levels never decreased. the patient underwent anticoagulant-free hemodialysis acutely. after discharge, he was instructed to avoid taking aspirin [14]. c. wu et al., is conducted a study on rivaroxaban for the treatment of suspected or confirmed heparin-induced thrombocytopenia studying twenty-two consecutive adults with suspected or confirmed hit received rivaroxaban 15 mg bid until a local hit assay result was available. participants with a positive local assay result continued rivaroxaban 15 mg bid until platelet recovery (or until day 21 if they had acute thrombosis at study entry), then stepped down to rivaroxaban 20 mg daily until day 30. the primary outcome measure, incidence of new symptomatic, objectively-confirmed venous and arterial thromboembolism at 30 days, occurred in one hitpositive participant (4.5%; 95% confidence interval) and one hit-positive participant required limb amputation despite platelet recovery. platelet recovery was achieved in nine out of 10 hit-positive patients with thrombocytopenia [15]. timothy k. liem, md et al., conducted a study on lepirudin as a safe and effective anticoagulant for patients with heparin-associated antiplatelet antibodiesineighteen haab-positive patients received lepirudin. lepirudin use was analyzed for indication, duration, and effectiveness of anticoagulation, and adverse events. haab presence was determined by platelet aggregation .9 had previous documentation of haab, 6 had thrombocytopenia while receiving heparin, and 3 had haab after a thrombotic event. the indications for lepirudin anticoagulation included thromboembolism prophylaxis arterial thrombosis pulmonary embolus or deep venous thrombosis and one each for atrial fibrillation, myocardial infarction, artificial heart valves, and hemodialysis access. the average duration of therapy was 4.04 days. fifteen patients achieved adequate anticoagulation (activated partial thromboplastin time [aptt] ratio > 2.0) with lepirudin. seven patients had aptts that were sometimes supra therapeutic (aptt> 100 seconds) but did not bleed. in all patients who had heparin-induced thrombocytopenia, platelet counts were normalized while they received lepirudin. there were two complications: one patient fell and had a calf hematoma (aptt ratio 3.24) [16]. joana gameiro et al. conducted a study on haemodialysis-related-heparin-induced thrombocytopenia case series and literature review in 5 patients between the ages 71 to 85 and had a history of hypertension, had chronic kidney disease, between this patient some had multiple myeloma and some had a bacterial infection, atrial fibrilization, and anemia. these 5 patients came with thrombocytopenia with low levels of platelet count in the hospital, warfarin and sodium citrate gave good results in platelet recovery and thrombocytopenia [17]. makoto harada et al. conducted a study on a case of heparin-induced thrombocytopenia that developed in the therapeutic course of anti-neutrophil cytoplasmic antibody-associated vasculitis in an 87-year-old woman who presented with appetite loss and leg edema was das k et al. heparininitiated thrombocytopenia: hemodialysis’s 1-month study vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 187 admitted for evaluation. blood examination revealed an inflammatory response (c-reactive protein level was 7.85 mg/dl), kidney dysfunction (blood urea nitrogen was 37.4 mg/dl, and the serum creatinine level was 2.25 mg/dl), and hypoalbuminemia. heparin calcium therapy was also administered. reduction of 50% or more of the platelet count and a platelet decrease between 5 and 10 days after using heparin are consistent. additionally, because the fdp-d-dimer was high, she might have had thrombosis. heparin was discontinued 29 days after hospitalization, and argatroban therapy was administered. after starting argatroban therapy, her platelet count gradually increased. fifty-two days after her hospitalization, the platelet count improved to 200,000/μl. however, because kidney function was not recovered, she was administered maintenance hemodialysis [18]. in this study, 19 patients experienced hit after taking heparin recently (within 14 days). through the analysis of sociodemographic data, it was found that the percentage of male patients (73.7%) was higher than the percentage of female patients (26.3%) (figure 2). male preponderance is seen in gender distribution in our study, which is similar to reports from other studies in countries of asia. in india, less number of females may be due to higher illiteracy, social stigma, and the need for the male relative to be concerned and accompany the female for hospital visits. maximum patients in this study were of age group 61-70 years (47.4 %) followed by 51-60years (36.8 %), 71-80 years (10.5 %), and 8190 years (5.3%) (figure 1). a bimodal distribution is seen with the incidence of hit. with a peak incidence in elderly patients and then in the first decade. hit has been recorded in individuals receiving hemodialysis while utilizing heparin anticoagulation, though heparin exposure for other purposes could not be ruled out. this study revealed 19 patients who experienced hit after taking heparin recently (within 14 days) just to keep their blood vessels healthy while receiving renal replacement therapy. none of the patients showed an abrupt (within hours) development of hit after heparin exposure, indicating a longer period possibly up to four months without prior heparin exposure. the immediate discontinuation of heparin and the initiation of alternative anticoagulation are recommended for treating patients with hit. this approach decreases the 38–76% risk of thrombosis that persists for days to weeks (figure 3). platelet counts typically recover more rapidly when patients receive alternative anticoagulant therapy, compared with historical control therapy. in the patients reported here, partial recovery of platelet counts occurred before argatroban discontinuation, the rate of new thrombosis was similar to that reported in previous studies of argatroban therapy in hit, no one died of thromboembolic complications, and the bleeding risk was acceptably low (figure 4, 5). conclusion in 9-month research conducted in a kolkata tertiary care hemodialysis facility, no incidences of hit were found. a small number of patients developed thrombocytopenia without developing thrombotic events. so heparin kept going. the platelet counts spontaneously improved. as a result, we can conclude that hit is probably fairly uncommon in the eastern part of india and the direct thrombin inhibitors lepirudin and bivalirudin, which are primarily cleared by renal and renal/enzymatic processes, respectively, have also been used for hit therapy. however, argatroban, which is metabolized by the liver, is generally considered to be a better choice for patients with renal failure. the present findings emphasize the importance of heightened awareness of hit in heparintreated patients undergoing hemodialysis and of suspecting. reference 1. i ahmed, a majeed, r powell. heparin induced thrombocytopenia: diagnosis and management update postgrad med j 2007;83:575–582. 2. makiko morita: acute non‐heparin‐induced thrombocytopenia during haemodiafiltration in a patient with multiple myeloma , john wiley & sons ltd 2019;7:699–702. 3. kd tripathi, essentials of medical pharmacology, 7th edition; new delhi; jaypee brothers medical publishers (p) ltd ; 2013 ;613-633. 4. gowthami m. arepally, heparin-induced thrombocytopenia, blood 2017;129(21):2864-2872. 5. robert e. cronin, robert f. reilly, unfractionated heparin for hemodialysis: still the best option,semin dial. 2010; 23(5): 510–515. 6. michael haase,use of fondaparinux (arixtra) in a dialysis patient with symptomatic heparin-induced thrombocytepenia type ii, nephrol dial transplant 2005(20): 444–446 7. theodore e. warkentin, heparin-induced thrombocytopenia in critically ill patients, thieme med pub. 2015(1):49-60. 8. john g. kelton, donald m. arnold, shannon m. bates. c.m non-heparin anticoagulants for heparin-induced thrombocytopenia, n engl j med 2013;368:737-44. 9. jack hirsh, frcpc, fracp, frsc, dsc; nancy heddle, msc; john g. kelton, treatment of heparin-induced thrombocytopenia, arch intern med. 2004;164:361-369. 10. giuseppe coluccil, michael nagler, nicole klaus, et al. practical guidelines for argatroban and bivalirudin in patients with heparin-induced thrombocytopenia j transl sci, 2015;1(2): 37-42. 11. chieh tsai, laura quinn marcus, priya patel, et al. use in hemodialysis patients with atrial fibrillation: a systematic das k et al. heparininitiated thrombocytopenia: hemodialysis’s 1-month study vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 188 review of stroke and bleeding outcomes, canadian journal of kidney health and disease. 2017(4): 1–13. 12. steven a baroletti, samuel z goldhaber, heparin-induced thrombocytopenia, american heart association 2006;8:355356. 13. maciej dryjsk p, hanna dryjski. heparin induced thrombocytopenia, eur j vasc endovasc surg 11 1996;260269. 14. faraz kerendi, vinod h thourani, john d puskas, et al. impact of heparin-induced thrombocytopenia on postoperative outcomes after cardiac surgery, ann thorac surg 2007;84:1548 –55. 15. joana gameiro, sofia jorge, josé antonio lopes, haemodialysis-related-heparin-induced thrombocytopenia: case series and literature review, nefrologia 2018;3 8(5):551–557. 16. junaid h mudaliar, timothy k liem, walter k nichols,et al. lepirudin is a safe and effective anticoagulant for patients with heparin-associated antiplatelet antibodies. j of vascular surgery. 2001(1):17-20. 17. guillermo delgado-garcia, roberto monreal-robles, daniel gallegos-arguijo et al. apixaban as a therapeutic option in chronic kidney disease patients with heparin-induced thrombocytopenia (hit). gac med mex. 2015;151:742-5. 18. theodore e warkentin, andreas greinacher, an dreas koster, bivalirudin, thrombhaemost 2008; 99: 830–839. how to cite this article: krisnendu das, utpal bhui, arghya majumdar, sanmoy karmakar. incidence of thrombocytopenia one month after initiation of heparin during hemodialysis: an observational study. indian j pharm drug studies. 2023; 2(4):183-188. funding: none conflict of interest: none stated mhamane & kadam use of citrullus lanatus in cosmetic face pack formulation vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 73 original article herbal face pack formulation from citrullus lanatus supriya mhamane1, revati kadam2 from, 1sahyadri college of pharmacy, methwade, 2assistant professor, department of pharmacognosy, sahyadri college of pharmacy, methwade, sangola, maharashtra, india. abstract watermelon (citrullus lanatus) belongs to the family cucurbitaceae. even though watermelon is commonly classified as a vegetable, it is botanically considered as a fruit and used primarily as a dessert. the main aim of this study is, to evaluate and formulate the citrullus lanatus face pack. watermelon rind powder, turmeric powder and sandal wood powder were used for this formulation. the dried powders were mixed and passed from sieve no. 120. particle size of the powder was found to be 125 micron. the organoleptic evaluations, irritancy test, rheological evaluations, ph value, ash value determination, moisture content evaluation were performed. the herbal face pack is used to stimulate blood circulation, recreate the muscle, regulate the elasticity of skin and cleansing the skin. the herbal formulations has the less side effects. so, we decided to work on the herbal face pack. keywords: citrullus lanatus, herbal face pack, rind powder, humectant, anti-acne. watermelon contains vitamins (a, c and b6), amino acid (citrulline), anti-oxidant (lycopene), bioactives such as curcurbitacin, triterpenes, sterols and alkaloids. it also contains dietary fibres in 16%, minerals such as potassium in 4%, and anti-oxidant like phenol in 1415ppm and flavonoids in 732ppm [1]. the major part of the body and which is act as a mirror which reflects health of that person is skin. to maintain the health of the skin, the diet should contain amino acids, lipids and carbohydrates. from ancient times, the womens take care of their skin because they are very conscious about it [2]. as mentioned in ayurveda, face packs can help to reduce wrinkles, dark circles, pimples and acne. fairness and smoothness of skin can be increased by using herbal face packs. from many years, the face packs are used for removing the dirt from the skin pores [3]. materials & methods the rind of citrullus lanatus were collected and cut into small pieces. then it is dried under the sunlight for 2-3 days. then it is powered by using mixer grinder and passed from sieve no. 120 for uniform particle size i.e. 125 micron. access this article online received – 03rd jul 2023 initial review – 30th jan 2024 accepted – 08th mar 2024 quick response code then this rind powder of citrullus lanatus was authenticated by dr. tembhurne r. r. from dept. of botany, sangola college, sangola. then collected the turmeric powder and sandal wood powder. then mixed all these three powder in a given proportion. then crush it by using mortar and pestle. then evaluate the formulation by using different parameters such as organoleptic evaluations, irritancy test, rheological evaluations, ph value, ash value determination, moisture content evaluation [3]. formulation table 1: formulation of face pack ingredient quantity (gm) uses watermelon rind powder 30 humectant, antiacne turmeric powder 5 anti-oxidant sandal wood powder 15 antiinflammatory evaluation of formulation 1) organoleptic evaluation: it is used to determine the various parameters of the powder such as nature/ appearance, color, odor, taste, texture and smoothness by using sense organs like eyes, nose, teeth and skin [4]. __________________________________________________ correspondence to: supriya mhamane, korwali, tal. mohol, dist. solapur, maharashtra, india. email: supriyamhamane99@gmail.com tel.: +91 8080244609. mailto:supriyamhamane99@gmail.com mhamane & kadam use of citrullus lanatus in cosmetic face pack formulation vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 74 2) irritancy test: in a pinch of face pack, added 2-3 drops of rose water. mixed it well and then it is applied on the skin for 10 min. then it is observed [5]. 3) rheological evaluation: in this, bulk density, tapped density and angle of repose were determined. a) bulk density: weighed 20gm formulation and added in a clean glass measuring cylinder. then the volume is measured as bulk volume [3]. it is calculated by using following formula: bulk density = mass of powder bulk volume b) tapped density: weighed 20gm formulation and added in a clean glass measuring cylinder. then tapped it for 10 times and measured the tapped volume [3]. it is calculated by using following formula: tapped density = (mass of powder)/(tapped volume) c) angle of repose: weighed 20gm formulation. on the surface place white paper. pour the formulation on a clean funnel which has adjusted height at 10cm from the surface by using burette stand. then measure the height of pile and draw a circle around it. then measure its radius three times [3]. calculate the average radius by using following formula: average radius (r) = (r1+r2+r3+⋯+ rn)/(no.of radius) by using height of pile and its average radius we can calculate angle of repose by using following formula: angle of repose (ɵ) = tan−1( ℎ 𝑟 ) 4) ph value: adjust the 10ml clean distilled water at a neutral ph by using dil. hcl and dil. naoh. in that, added the 10mg formulation. stir it well. then take ph meter and deep it in a solution and compare its color with standard [4]. 5) proximate analysisw i. moisture content: weighed 1.5gm of formulation and empty crucible as w1. then weighed the formulation and crucible together as w2. then it is transferred in a hot air oven for 15 minutes. then place it at room temperature for few minutes, then transfer it in a desiccator for cooling. then weighed it as a w3 [4]. then by using following formula it is calculated: moisture content = w2−w3 w2−w1 × 100 ii. ash value: it is used to calculate ash of the formulation. a) total ash: weighed 2gm of formulation and empty crucible. then formulation added in that crucible, it is placed in a muffle furnace for 15 minutes. then it is kept at room temp. for few minutes and then it is cooled in a desiccator. then weighed it and calculated total ash by using following formula: total ash = weight of ash drug taken × 100 b) acid-insoluble ash value: weighed 2gm of formulation and empty crucible. then formulation added in that crucible, it is placed in a muffle furnace for 15 minutes. then it is kept at room temp. for few minutes and then it is cooled in a desiccator. then in a clean beaker add 25ml dil. hcl then it is placed on a hot plate along with some water in another beaker for boiling. then it is cooled at room temperature and then filtered by using ashless filter paper and washed with hot water. then wrap it and place in a crucible and again place it in a muffle furnace for 15 min. then cool it at room temperature and then in a desiccator [4]. then weighed it and calculated the acidinsoluble ash by using following formula: acid-insoluble ash value = crusible with ash wt.−empty crusible wt. wt.of drug taken × 100 c) water-soluble ash value: weighed 2gm of formulation and empty crucible. then formulation added in that crucible, it is placed in a muffle furnace for 15 minutes. then it is kept at room temp. for few minutes and then it is cooled in a desiccator. then in a clean beaker add 25ml distilled water then it is placed on a hot plate along with some water in another beaker for boiling. then it is cooled at room temperature and then filtered by using ashless filter paper and washed with hot water. then wrap it and place in a crucible and again place it in a muffle furnace for 15 min. then cool it at room temperature and then in a desiccator [4]. then weighed it and calculated the acidinsoluble ash by using following formula: water-soluble ash value = crusible with ash wt.−empty crusible wt. wt.of drug taken × 100 figure 3: powder of citrullus lanatus figure 4: powder of turmeric figure 5: powder of sandalwood mhamane & kadam use of citrullus lanatus in cosmetic face pack formulation vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 75 figure 8: irritancy test fig 9: bulk density, fig 10: tapped density, fig 11: angle of repose, figure 12: ph value figure 13: moisture content figure 14: acid-insoluble ash value figure 15: water-soluble ash value results 1. organoleptic evaluation: organoleptic evaluation are the external appearance of the citrullus lanatus. table 2: organoleptic evaluation parameter observation nature/appearance powder colour butterscotch (yellow) odor slight taste characteristic texture fine smoothness slightly rough 2. irritancy test: this test includes the traumatic effect of the citrullus lanatus after its use on face which are depicted in following table table 3: irritancy test parameter observation irritation no irritation erythema no erythema edema no edema itching no itching swelling no swelling 3. rheological evaluation: rheological evaluation consist of evaluation of flow properties. table 4: rheological evaluation parameter observation bulk density 0.34gm/ml tapped density 0.43gm/ml angle of repose 22.210 4. ph value: the observed ph value is 6-7 5. proximate analysis: these analysis consists of laboratory investigations of the specimen. table 5: proximate analysis parameter observation (%) moisture content 2 total ash value 19 acid-insoluble ash value 10.50 water-soluble ash value 5.5 graph 1: formulation of face pack mhamane & kadam use of citrullus lanatus in cosmetic face pack formulation vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 76 discussion the rind of citrullus lanatus contains plant constituents such as vitamin (a, c and b6), amino acid (citrulline), antioxidants, curcurbitacin, triterpenes, sterols, alkaloids. it also contain dietary fibres (16%), minerals (4%). this constituents shows anti-acne, humectant property. herbal face pack from watermelon (citrullus lanatus) rind were firstly formulated. because of its properties and less side effects of herbal drugs [6]. the various evaluation tests were performed to check the property of the powder. organoleptic evaluation is to confirm its morphological characteristics. then the irritancy test to check allergic reaction on body. the rheological evaluation determines the bulk density, tapped density and angle of repose. proximate analysis were performed to check presence of moisture, ash value in acid, water. ph value is also determined [8]. conclusion the results of the evaluation test show that, the formulation is good to use. organoleptic evaluation gives knowledge about its morphological characteristics like nature, color, odor, taste, texture, smoothness. then irritancy test shows that this formulation does not caused any irritation, erythema, edema or itching. rheological evaluation shows calculation of bulk density i.e. 0.34gm/ml, tapped density i.e. 0.43gm/ml and angle of repose i.e. 22.210. proximate analysis include moisture content i.e. 2%, total cash value i.e. 19%, acidinsoluble ash value i.e. 10.5% and water-soluble ash value i.e. 5.5%. the observed ph value is 6-7. these all observation shows that, this formulation has anti-acne and humectants property without allergic reactions. references 1. https://www.vedantu.com/evs/uses-of-watermelon-rind 2. avinash o. maske. formulation and evaluation of herbal face pack for glowing skin. inter j adv in pharmaceutics. 2019; 8(1):e5184 3. yadav n, yadav r. preparation and evaluation of herbal face pack. inter j rec sci res. 2015; 6(5):4334-4337. 4. ramakrishna s et al. formulation and evaluation of herbal face pack. jetir. 2021; 8(12):179-186. 5. bhawana bhatt et al. preparation and evaluation of herbal face pack. indo american j pharmac res. 2018: 8(5):1253-1259. 6. sachin b somwanshi et al. formulation and evaluation of cosmetic herbal face pack for glowing skin. int j res ayurveda pharm. 2017; 8(3):199-203. 7. priti r. neware et al. formulation and evaluation of herbal face pack for acne-prone skin and dull skin. ijfans. 2022; 31-39. 8. miss. telange-patil p.v et al. formulation and evaluation of face pack using by orange peel. ijcrt. 2022; 10(10):566-577. 9. sachin bhagwat aglawe et al. formulation and evaluation of herbal face pack. inter j pharm biologi sci. 2018; 8(4):49-52. 10. swati siddheshwar londhe et al. formulation and evaluation of polyherbal face pack. wjpmr. 2020; 6(7):159-165. how to cite this article: supriya mhamane, revati kadam. herbal face pack formulation from citrullus lanatus. indian j pharm drug studies. 2024; 3(2):73-76. funding: none; conflicts of interest: none stated https://www.vedantu.com/evs/uses-of-watermelon-rind sanjay s s a sectorial overview on the synthesis of zoledronic acid vol 2 | issue 3 | jul – apr 2023 indian j pharm drug studies | 107 review article an overview of solvent-free and solvent/s-involved phosphorylation to synthesize zoledronic acid sanjay sukumar saralaya from, assistant professor, department of chemistry, sri dharmasthala manjunatheshwara institute of technology (sdm it), [affiliated to visvesvaraya technological university (vtu), belagavi], ujire, dakshina kannada, karnataka, india. abstract this work provides a complete overview of solvent-free and solvent-involved phosphorylation strategies employed to synthe size the renowned biphosphonate drug, zoledronic acid. in this regard, all the disclosed patents and journal publications were considered and reviewed as per the yearly chronology towards the use of solvent/s or in the absence of it for the phosphorylation. interestingly, a prolonged reaction time, sticky lump formation, exothermicity, enormous hcl release, tedious workup, etc had allowed the researchers to venture various synthetic and isolation approaches to overcome the process-specific setbacks. in line to this context, various solvent/s were used alone or in combination with another solvent to synthesize zole dronic acid in varied yields. a few attempts were also reported under solvent-free conditions in reasonably good yields. more over, along with the above variations, a few different p-reagent/s are also reported towards the synthesis of zoledronic acid. key words: biphosphonates, zoledronic acid, phosphorylation, p-reagents, hydrolysis, green-solvent. ynthesis of zoledronic acid z involves the reaction of an acid derivative (2) or its salt (3) with selective p-reagent/s {phosphoric acid (h3po4), phosphorous acid (h3po3), phosphorous trichloride (pcl3), phosphorous oxychloride (pocl3), triphosgene, mesyl chloride, etc} in the presence of suitable solvent/s or in the absence of solvent at a suitable temperature. after the phosphorylation, the reaction mixture was hydrolyzed by refluxing in water or hcl solution and further workup processes will lead to the isolation of z (scheme 1). numerous synthetic strategies were adopted by various researchers and are reported in various publications (patents/journals). the present review attempt will provide a distinct sectorial overview of the solvent-free or solvent/s-involved phosphorylation approaches disclosed in patents and journal publications to synthesize z. access this article online received – 13th june 2023 initial review – 17th june 2023 accepted – 26th june 2023 quick response code n n oh o oh po3h2 po3h2 n n z n n h (1) (2) n n oh o (3) (or) 1. solvent/s or no solvent, p-reagent/s, heating 2. hydrolysis by water or hcl solution, heating 3. anti-solvent addition, cooling, filtration, drying hcl optional recrystallization optional scheme 1. a renowned pathway to synthesize zoledronic acid via phosphorylation and hydrolysis of (2) or (3). details gathered from patent publications numerous patents were published/filed comprising a lot of information on the synthesis and biological activities of z. _______________________________________ correspondence to: dr. sanjay sukumar saralaya, assistant professor, department of chemistry, sdm it, opposite to siddhavana gurukula, dharmasthala road, ujire, belthangady tq, dk, karnataka, india574 240 email: sanjayss@sdmit.in s mailto:sanjayss@sdmit.in sanjay s s a sectorial overview on the synthesis of zoledronic acid vol 2 | issue 3 | jul – apr 2023 indian j pharm drug studies | 108 to the context, a critical examination of disclosed processes was done to identify the solvent-free and solvent/s-based strategies employed to isolate z. jaeggi ka & widler l., in 1989, had reported the phosphorylation of key starting material 1-h-imidazol-1ylacetic acid hydrochloride (3) by the renowned p-reagents like h3po4 (85%) and pcl3 in the presence of chlorobenzene to isolate z (yield: 41.0%) [1]. hu w., et al, in 2002, had illustrated the condensation of key starting material 1-h-imidazole (1) with ethyl-chloroacetate (4) and then hydrolyzed to isolate (3). phosphorylation of (3) using the p-reagents like h3po3 and pcl3 in chlorobenzene to get z (yield: 48.1%) [2]. de fl., et al, in 2002, had reported the use of tributyl ammonium chloride (tbac) as the solvent instead of hydrocarbon-based solvents. phosphorylation of 1-h-imidazol-1-ylacetic acid (2) was done by using h3po3 and pcl3 in the tbac medium to get z (yield: 25.95%) [3]. lidor hr., et al, in 2003, had illustrated the phosphorylation of key starting material (2)/(3) in silicon oil alone or with toluene using the preagents h3po3 and phosphorous oxychloride (pocl3) to isolate z (yield: 38.0-79.0%) [4]. aronhime j & lifshitz lr., in 2004, had disclosed the phosphorylation of (2)/(3) in solvents like silicon oil, chlorobenzene, toluene, and peg-400 in distinct experiments using h3po3 and pocl3 to get z (yield: 13.4100%). interestingly, the use of chlorobenzene or silicon oil for phosphorylation gave a better yield compared to other solvents [5]. patel vm., et al, in 2004, had reported the phosphorylation of (2) in sulfolane using h3po3 and pcl3 to isolate z (yield: 70.7%). a similar attempt was done using 1, 2-dimethoxyethane as the solvent to isolate z (yield: not mentioned) [6]. patel vm., et al, in 2004, had disclosed the condensation of (1) with chloroacetyl chloride (5) and benzyl alcohol (6) to isolate the intermediate benzyl-1-h-imidazol-1-ylacetate (7). it was then reduced by pd/c or hydrolyzed by 10% hcl to isolate (2). it was subjected to phosphorylation using h3po3 and pcl3 in sulfolane to get z (yield: 70.7%) [7]. pulla rm., et al, in 2004, had illustrated the condensation of (1) with methyl chloroacetate (8) to get (2), it was then converted to (3) by the treatment with isopropanol-hcl. phosphorylation of (3) using h3po4 and pcl3 in the presence of various solvents like ethylene dichloride, cyclohexane, and chlorobenzene gave z (crude yield: 79.0-85.0%). a better yield was obtained in an experiment performed using ethylene dichloride as the diluent for phosphorylation [8]. grassi s & volante a,. et al, in 2004, had reported the phosphorylation of (3) using h3po3 and pocl3 in the absence of solvent to isolate z (yield: 62.0%) [9]. cai wz., in 2005, had illustrated a onestep process to condense (1) with (4) in 1, 4-dioxane using 60% sodium hydride (nah), and an in situ phosphorylation was done using h3po4 and pcl3 to get z (yield: 32.0%). similarly, (1) was reacted with chloroacetonitrile (9) in 1, 4-dioxane using potassium carbonate (k2co3) and phosphorylation was done using h3po4 and pcl3 to isolate z (yield: 29.9%) [10]. pandey sc., et al, in 2005, had disclosed the phosphorylation of (2) in n-octane using h3po3 and pcl3 to get z (yield: 64.89%). a similar attempt of phosphorylation in 1, 4-dioxane for (2) gave z (yield: 51.91%) [11]. vecchioli a., et al, in 2006, had illustrated the phosphorylation of (2) in methanesulfonic acid (msa) using pcl3 to isolate z (crude yield: 83.0%). the process efficiently avoids the use of h3po4 or h3po3 for the reaction [12]. deshpande pb & luthra pk., in 2006, had reported an efficient phosphorylation of (2) in diphenyl ether (dpe) using h3po3 and pcl3 to isolate z (crude yield: 75.0%) [13]. yadav rp., et al, in 2006, had disclosed the condensation of (1) with (8) to isolate the intermediate methyl 1-h-imidazol-1-ylacetate (10). it was hydrolyzed to (2) and then subjected to phosphorylation in the absence of solvent using h3po3 and pcl3 or pocl3 to isolate z (crude yield: 74.62-78.79%) [14]. samsel eg & wu tc., in 2007, had illustrated the condensation of (1) with t-butyl chloroacetate (11) to get (2). it was then phosphorylated in the presence of diglyme using h3po4 and pcl3 to isolate z (crude yield: 28.0%). an experiment was also conducted in peg-400 instead of diglyme to isolate z (yield: 7.0%) [15]. baptista j & mendes z., in 2007, had reported the phosphorylation of (2) in the presence of an aprotic polar solvent n, n’-dimethylethyleneurea (dmeu) using h3po3 and pcl3 to obtain z (crude yield: 85.6%) [16]. liu y & delaup aj., in 2008, illustrated the phosphorylation of (2) in sulfolane using h3po3 and the pcl3 to isolate z (crude yield: 53.0-64.0%). the experiments were done by the modulated mode of addition of p-reagents (coaddition/alternate addition etc) [17]. nazarenko ab & fedorov ve., in 2009, had demonstrated the phosphorylation of 1-h-imidazol-1ylacetonitrile (12) in msa using alone pcl3 to isolate z (yield: 85.0-92.0%). a few experiments were conducted using different equivalents (1.25, 2.5, and 3.0) of msa for phosphorylation [18]. dembkowski l., et al, in 2009, disclosed the conversion of (2) to (3) by the addition of hcl solution and immediate phosphorylation in the absence of solvents/diluents using pcl3 alone to isolate z (crude yield: 41.0-49.0%). initially added water itself will act as the diluent for the process. moreover, it avoids the sanjay s s a sectorial overview on the synthesis of zoledronic acid vol 2 | issue 3 | jul – apr 2023 indian j pharm drug studies | 109 use of h3po4 or h3po3 for phosphorylation [19]. kas m., et al, in 2009, had reported a few pathways to convert (1) to (2) by the condensation with (8). moreover, (2) was phosphorylated in a peg-400 medium using h3po3, pcl3, or pocl3 to isolate z (yield: 31.3-32.9%). instead of peg400, diethyl carbonate (dec) was used to convert (2) to z (yield: 59.0%). interestingly, the combination of diluents like peg-400 and dec for phosphorylation gave a better atom economy of z (crude yield: 75.0-84.0%). furthermore, propylene carbonate (pc) was used along with peg-400 / peg-600 / peg-1000 as the solvent combination in distinct experiments to isolate z (crude yield: 97.0-99.0%) [20]. hu y., et al, in 2010, had disclosed a one-pot synthetic strategy to condense (1) with (4) using 1, 4-dioxane or tetrahydrofuran (thf) and the in situ phosphorylation using h3po3 and pcl3 to isolate z (crude yield: 55.058.3%) [21]. lanxiang s., et al, in 2011, had disclosed the phase transfer reagent mediated condensation of (1) with (4) and its further hydrolysis to isolate (3). it was phosphorylated in trifluoroacetic acid (tfa) or sulfuric acid (h2so4) using h3po4 and pcl3 to get z (crude yield: 57.0-58.8%) [22]. yinchuan z., et al. in 2011, had reported the phosphorylation of (2) or (3) in liquid paraffin medium using h3po4 and pcl3 to get z (crude yield: 56.6-81.1%) [23]. keglevich g., et al, in 2012, had reported the phosphorylation of (2) in an msa medium using a different set of p-reagents like triphosgene, mesyl chloride, pcl3, etc to get z (crude yield: 59.0-74.0%). the process avoids the use of routine p-reagents like h3po4 or h3po3 for phosphorylation [24]. kai s., et al, in 2012, had illustrated the phosphorylation of (2) in commercially affordable aliphatic hydrocarbon-based solvents like nhexane, n-decane, n-tetradecane along with water using pcl3 alone to isolate z (crude yield: 81.0-91.0%) [25]. n n oh o n n po3h2 oh po3h2 z n n h n n oh o hcl (1) (2) (3) o o cl (4) cl o cl (5) oh (6) n n o o (7) o o cl (8) cl n (9) n n o o (10) o o cl (11) n n n (12) n n o o (13) figure 1. list of various key reactants and intermediates featuring in different synthetic strategies of zoledronic acid hao e., et al, in 2015, had illustrated the condensation of (1) with (3) in the presence of ionic liquid ([bmim]bf4) to get (3). phosphorylation of (3) in the presence of ionic liquid ([bmim]bf4) using h3po4 (85%) and pcl3 gave the sodium salt of z monohydrate (yield: 60.0%). numerous experiments were done to optimize the process using different ionic liquids, variations in reaction temperature, and changes in pcl3 addition duration. different ionic liquid {n-ethylpyridine tetrafluoroborate [epy][bf4], [bmim][pf6], loh, lcn, looh} was used in distinct experiments to obtain z (yield: 90.0-92.0%). meanwhile, the ionic liquid facilitated the phosphorylation as an effective reaction mixture diluent [26]. wu y., et al, in 2016, had disclosed the phosphorylation of (2) in chlorobenzene and also in sulfolane using h3po3 along with pcl3 in high scale to isolate z (crude yield: 78.1% and 65.0% respectively). a few experiments were also done by conducting the phosphorylation of (2) in the absence of solvent using h3po3 along with pcl3 or pocl3 to isolate z (crude yield: 83.7-87.3%) [27]. details gathered from journal publications many researchers had reported their work on z in numerous national/international journals.. in all those, the synthetic method part was examined critically to tabulate the disclosures about solvent-free and solvent/s-based strategies to isolate z. widler l., et al, in 2002, had reported the phosphorylation of (2) in chlorobenzene using h3po4 (85%) and pcl3 to isolate z (yield: 67.0%) [28]. srinivasa rdvn., et al, in sanjay s s a sectorial overview on the synthesis of zoledronic acid vol 2 | issue 3 | jul – apr 2023 indian j pharm drug studies | 110 2007, had reported the phosphorylation of (2) in p-cresol using h3po3 along with pcl3 to get z (crude yield: 80.0%) [29]. keglevich g., et al, in 2011 and 2012, had illustrated and explained the mechanistic aspects behind the phosphorylation of (2) in msa using pcl3 and with/without h3po3 to obtain z (crude yield: 0-71.0%). the work proved that, in the presence of msa as the medium for reaction, alone pcl3 can induce the phosphorylation and hence there is no requirement of h3po3 to obtain z (crude yield: 23.0-71.0%) [30, 31]. mustafa da., et al, in 2011, had disclosed the phosphorylation of (2) in sulfolane medium using h3po3 along with pcl3 under the assistance of microwave irradiation (3-4 min) to isolate z (yield: 70.0%). interestingly, the same experiment when conducted in a conventional pathway resulted in the formation of z (yield: 67.0%) with not much deviation in outcome. but the conventional process takes more reaction time than the microwave irradiation pathway [32]. lenin r., et al, in 2013, had reported the phosphorylation of (2) using h3po3 and pcl3 in the presence of silica gel under microwave irradiation (3-4 min) to isolate sodium salt of z (yield: 80.0%) [33]. kovács r., et al, in 2014, had reported a review article regarding the use of greener solvent msa for the phosphorylation of (2) requiring alone pcl3 [34]. ratrout ss., et al, in 2015, had reported the phosphorylation of t-butyl-imidazol-1-yl acetate (13) in the presence of msa and chlorobenzene using h3po4 and pocl3 to isolate sodium salt of z (yield: 85.0%). it was later converted to z (yield: 79.0%) by the treatment with concentrated hcl (37.0%). the work also disclosed the route of synthesis of (13) by condensation of (1) with (11) in acetonitrile using nah [35]. keglevich g., et al, in 2015, had disclosed the phosphorylation of (2) in msa using pcl3 to isolate sodium salt of z and then to z (yield: 49.0%) using 1 n hcl solution [36]. nagy di., et al, in 2016, had reported a review article regarding the use of different solvents for the synthesis of hydroxymethylenebisphosphonic acids. the work covers the synthesis of z (yield: 31.0-53.0%) using msa to phosphorylate (2) using pcl3 and with/without h3po3. similarly, the use of chlorobenzene in various disclosures using h3po4 or h3po3 and pcl3 gave z (yield: 41.0-100%). furthermore, the use of sulfolane for phosphorylation gave z (yield: 67.0-71.0%). attempts of solvent-free conditions for phosphorylation of (2) using h3po3 and pcl3 / pocl3 gave z (yield: 61.0-81.0%). use of different ionic liquids as the diluent also gave z or its sodium salt (yield: 26.0-92.0%). similarly, the use of pcresol as the solvent gave z (yield: 80.0%). additionally, phosphorylation of (2) in n-octane gave z (yield: 65.0%). the work covers the use of other solvents like cyclohexane, 1, 4dioxane, diphenyl ether, propylene carbonate, a mixture of propylene carbonate and peg 600, peg 400, dimethoxymethane, dimethoxyethane, diglyme, 1, 2-dichloroethane, n, n-dimethylurea, and silicon oil [37]. nagy di., et al, in 2017, had reported a review article covering the vital role of pcl3 and h3po3 in specific molar equivalents for the formation of hydroxymethy lenebisp hosphonic acids from the corresponding carboxylic acids. the work disclosed the impact of p-reagent/s for the phosphorylation of (2) in msa or sulfolane medium to isolate z [38]. nagy di., et al, in 2018, had illustrated the phosphorylation of (2) in sulfolane using h3po3 and pcl3 to isolate z dihydrate (yield: 74.0%). the use of ionic liquid [bmim][bf4] as the reaction medium gave z dihydrate (yield: 75.0%). similarly, the use of sulfolane and [bmim][bf4] for phosphorylation resulted in the formation of z dihydrate (yield: 93.0%) [39]. nagy di., et al, in 2018, had emphasized the phosphorylation of (2) in msa using the p-reagent pcl3 alone to isolate z (yield: 53.0%) [40]. grün a., et al, in 2019, had reported the phosphorylation of (2) in sulfolane, or the presence of an ionic liquid, or both together as the medium for the reaction using pcl3 and h3po3 to obtain z (yield: 74.093.0%). the combination of solvent sulfolane and the ionic liquid [bmim][bf4] gave a promising output of z (yield: 93.0%) [41]. ábrányi bp., et al, in 2021, had reported a review article covering the phosphorylation of (2) using only pcl3 using msa to isolate z (yield: 46.0-53.0%). similarly, the use of h3po3 and pcl3 in sulfolane gave z (yield: 63.074.0%) [42]. grün a., et al, in 2021, had disclosed the phosphorylation of (2) using different equivalents of h3po3 and pcl3 in diethyl carbonate (dec) as a green solvent medium to isolate z (0-61.0%). the work extends to cover the use of msa and dec, alone or in different combination ratios to synthesize z (yield: 0-53.0%). this approach was observed to be less efficient as compared to the use of sulfolane for phosphorylation. but, found reasonably better as compared to the use of msa for phosphorylation to isolate z. [43]. sanjay ss., in 2023, had reported a review article regarding the synthesis and purification of z. it covers the disclosures provided in various patents regarding the till date adopted synthetic strategies in detail to isolate z and its few forms [44]. summary as per the prior arts, phosphorylation in msa medium requires only pcl3. if the same was performed in solvents other than msa, then both pcl3 and h3po3 are required in sanjay s s a sectorial overview on the synthesis of zoledronic acid vol 2 | issue 3 | jul – apr 2023 indian j pharm drug studies | 111 optimum equivalents. numerous solvents are being used for the phosphorylation to isolate z and its salt, all those were tabulated in table 1. table 1 list of solvent-free and solvent-based strategies employed for the phosphorylation to synthesize z. solvent/s based synthetic strategies solvent/s for phosphorylation references chlorobenzene [1], [2], [5], [8], [27], [28], [35] tributylammoniumchloride [3] silicon oil [4], [5] toluene [5] peg-400 [5], [15], [20] sulfolane [6], [7], [17], [27], [32], [39], [41] 1, 2-dimethoxyethane [6] cyclohexane [8] ethylene dichloride [8] 1, 4-dioxane [10], [21] n-octane [11] methanesulfonic acid [12], [18], [24], [30], [31], [34], [35], [36], [40], [43] diphenyl ether [13] diglyme [15] n, n’-dimethylethyleneurea [16] diethyl carbonate [20], [43] diethyl carbonate & peg-400 [20] propylene carbonate & peg-400 [20] propylene carbonate & peg-600 [20] propylene carbonate & peg-1000 [20] tetrahydrofuran [21] trifluoroacetic acid [22] sulfuric acid [22] liquid paraffin [23] n-hexane [25] n-decane [25] n-tetradecane [25] ionic liquid/s [26], [39], [41] p-cresol [29] silica gel [33] sulfolane & ionic liquid [39], [41] methanesulfonic acid & diethyl carbonate [43] solvent-free synthetic strategies phosphorylation in the absence of solvent/s [9], [14], [19], [27] conclusion an exceptionally complex phosphorylation forms a crucial step to synthesize z. to ensure the scalability and the industrial adaptability of phosphorylation, numerous reaction optimization experiments were reported by many researchers. as a part of it, solvent-free and different solvent/s involved phosphorylation reactions were demonstrated with varied purity and yields of z. this work primarily focused to give an overview of all the solvents used and the solvent-free synthetic strategies adopted for the phosphorylation to prepare z. the supportive details were extracted separately from the filed patents and the journal publications, comprising the specific illustrations towards the synthesis of z. references 1. jaeggi ka, widler l. substituted alkanediphosphonic acids and pharmaceutical use. us31596289a, 1989. 2. hu w, zhang y, zhang g. synthesis of product prepared from imidazole acted with halogenated acetate ethyl ester. cn02138852a, 2002. 3. de fl, turchetta s, massardo p et al. preparation of bisphosphonic acids and salts thereof. ib0204941w, 2002. 4. lidor hr, harel z, lifshitz lr et 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2018. p. 199–213. 41. grün a, rádai z, sőregi-nagy di, et al. rational synthesis of α-hydroxyphosphonic derivatives including dronic acids. phosphorus sulfur silicon relat elem. 2019;194(4–6):386–7. available from: http://dx.doi.org/10.1080/10426507.2018.1555537 42. ábrányi bp, greiner i, keglevich g. a mechanistic study on the formation of dronic acids. molecules. 2021;26(24):7587. available from: http://dx.doi.org/10.3390/molecules26247587 43. grün a, szalai z, keglevich g. “greener” synthesis of zoledronic acid from imidazol-1-yl-acetic acid and p-reagents using diethyl carbonate as the solvent component. lett org chem. 2021;18(1):8–12. available from: http://dx.doi.org/10.2174/1570178617999200730203738 44. sanjay ss. an exhaustive methodological review of patents on the synthesis and purification of zoledronic acid. world j. pharm. res. 2023;12(9):2731-2777. available from: http://dx.doi.org/10.20959/wjpr20239-28454 how to cite this article: sanjay sukumar saralaya. an overview of solvent-free and solvent/s-involved phosphorylation to synthesize zoledronic acid. indian j pharm drug studies. 2023; 2(3) 107-112. funding: none conflict of interest: none stated http://dx.doi.org/10.1021/jm020819i http://dx.doi.org/10.1080/00397910701578545 http://dx.doi.org/10.1016/j.tetlet.2011.03.093 http://dx.doi.org/10.2174/138955712799829285 http://dx.doi.org/10.1016/j.tetlet.2011.02.058 http://dx.doi.org/10.1007/s00044-012-0153-4 http://dx.doi.org/10.1515/gps-2013-0107 http://dx.doi.org/10.1007/s11094-015-1205-0 http://dx.doi.org/10.1080/10426507.2015.1072194 http://dx.doi.org/10.3390/molecules21081046 http://dx.doi.org/10.2174/1385272821666170417122441 http://dx.doi.org/10.1080/00397911.2017.1410894 http://dx.doi.org/10.1080/10426507.2018.1555537 http://dx.doi.org/10.3390/molecules26247587 http://dx.doi.org/10.2174/1570178617999200730203738 http://dx.doi.org/10.20959/wjpr20239-28454 d kolekar et al. chemometric assisted qbd method for estimation of bilastine and montelukast sodium vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 16 original article chemo metric assisted spectrophotometric method development through quality by design approach for the estimation of bilastine and montelukast sodium in combined solid dosage form bhagyashri d kolekar1, namrata n gawade1, g k dyade1, nilesh y jadhav2 from, 1dept of post graduate studies in pharmaceutical quality assurance, svpm’s college of pharmacy, malegaon (bkii), baramati, pune, 2sspm’s dr n. j. paulbudhe college of pharmacy, survey no 45/1b, vasant tekadi, savedi, ahmednagar, maharashtra, india abstract objective: quality by design (qbd) is a systematic process for pharmaceutical development recommended by regulatory agencies like usfda. development of various pharmaceutical processes including analytical methods by applying quality by design aids in ensuring the robustness of the method. qbd approached chemo metric assisted uv-vis spectrophotometric analytical method was developed for the estimation of bilastine (bse) and montelukast sodium (mks) from their combined dosage forms. materials and method: simultaneous equation method was selected from the nature of spectra, solvent 50 % alcohol was utilised; and for method 274.5 nm and 351.5 nm was the wavelength for measurement of absorbance of bilastine and montelukast sodium respectively. effect of input variables on spectrum characteristics were studied for selection of critical parameters and developed method was validated as per ich q 2 r1 regulatory guidelines. linearity of the drugs was ascertained over the conc range 1-32 mcg/ml (microgram/ml) for bse and 1-20 mcg/ml for mks. results and discussion: the percentage purity of assay was found 98.09 % for bse and 103.62 % for mks; and the accuracy study data were varied from 0.2523 to 0.5221 for bse and 0.2512 to 1.2515 for mks. precision study was shown acceptable data as sd data varied from 0.1902 to 0.5773 for bse and from 0.2828 to 0.5458 for mks. conclusion: the developed method is rigid, robust and efficient for the estimation of bse and mks from the composition of dosage form. qbd was applied to build rigid robust method through risk assessment at early stage and defining the design space at the later stage. key words: bilastine, montelukast sodium, qbd, ich, simultaneous equation method ilastine (bse) chemically 4-[2-[4-[1-(2-ethoxy ethyl)-1h-benzimidazole-2-yl]-1-piperidinyl] ethyl]α, α-dimethyl benzene acetic acid [1] is an antihistamine, non-sedative histamine h1 receptor antagonist; by binding and preventing activation of h1 receptor bilastine reduces development of allergic symptoms due to release of histamine from mast cell [2]. literature survey revealed that various analytical methods have been reported for estimation of bse such as uv spectrophotometric methods lonely [3-6], uv spectrophotometric method with mks or other drug [7], for estimation of bse with mks or other by rp-hplc [8-14], stability indicating rp-hplc [15-17], green qbd hplc [18], lc-ms/ms [19], stability indicating uplc [20] and rpuflc [21] alone or in combination with other drugs. access this article online received – 21th dec 2023 initial review – 30th dec 2023 accepted – 08th jan 2024 quick response code montelukast sodium (mks) chemically sodium [1-[[[(1 r)-1-(3-[(e) 2-(7-chloroquinoline-2-yl) ethenyl] phenyl)-3-[2(1-hydroxy-1-methylethyl) phenyl] propyl] sulfanyl] methyl] cyclopropyl] acetate [1] is an anti-asthmatic, leukotriene receptor antagonist. it can completely block the binding of cyslt’s to receptor that they can inhibit the binding of inflammation mediator ltd4 [2]. literature survey revealed that various analytical methods have been reported for estimation of mks includes uv spectrophotometric method lonely [22-28], uv spectrophotometric method with bse or other drug [29-33], for estimation of mks qbd technique [34], rp-hplc methods [35-40], stability indicating hplc with other [41-43] and hptlc [44] alone or in combination with other drugs. montelukast sodium is official in indian and british pharmacopoeia [45, 46]. chemical structure of both these drugs is shown in (figure 1). _________________________________________ correspondence to: g k dyade, department of pg studies in pharm quality assurance, svpm’s college of pharmacy, malegaon (bkii), baramati, pune, maharashtra, india. email: pharmacyresearchsvpmcop@gmail.com b mailto:pharmacyresearchsvpmcop@gmail.com d kolekar et al. chemometric assisted qbd method for estimation of bilastine and montelukast sodium vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 17 figure 1: chemical structure of drug molecule quality by design concept is applied for the development of pharmaceutical processes to assure a predefined product quality. qbd concepts are mentioned in ich guidelines q8 (r2) (pharmaceutical development), q9 (quality risk management), and q10 (pharmaceutical quality system) [4749] shown in (figure 2). ich guidelines q8 (r2) defines qbd as a “a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management”[50]. qbd approach in analytical method summarizes a complete understanding of how the analytical technique attributes and operating conditions affect the analytical performance. factors to study in analytical quality by design (aqbd) approach may include the type of analytical technique chosen, reagents used and instrument parameters. fig no 2: analytical qbd approach there are similar advantages of applying qbd principles to analytical methods as to manufacturing processes and product [51]. a qbd approach can be beneficial in the development of suitable, robust, low cost and eco-friendly (eco-friendly solvent, chemicals) method which is applicable at any stage of the lifecycle of the product. also some regulatory guidelines have mentioned flexibility of changing analytical method without revalidation if the aqbd approach has been implemented during analytical method development. the first stage of aqbd approach is to fix an analytical target profile (atp) for the method. atp defines the goal of the analytical method development process and it is the sign of method performance [52, 53]. for analytical method validation ich q2 (r1) has given various method performance characteristics for an analytical method. in development of uv-vis spectrophotometric method, qbd approach was implemented with the study of the effect of method input variables on spectral shape, intensity of absorbance, and absorbance maxima λmax and critical parameters were selected for the proposed method and method was validated as per ich guidelines q2 (r1). materials and methods instrumentation: analysis was performed with a shimadzu double beam uv-visible spectrophotometer (shimadzu, kyoto, japan) with spectral bandwidth of 2 nm and wavelength accuracy of ± 1 nm with 10 mm matched quartz cells was used. electronic balance afcoset balance (the bombay burmah trading corpo ltd) with accuracy ±0.1 mg model no. er 200a was utilised for weighing and for degassing the solution digital ultrasonic cleaner 1.8 ltr (labman scientific instruments chennai) was used. reagents and chemicals: pharmaceutically pure samples of bse and mks from glenmark pharmaceuticals, nashik, maharashtra, india were procured as a gift sample and the commercial formulation bilafav-m tablet containing bilastine 20 mg and montelukast sodium 10 mg was procured from the local market. aqbd approach application in method development: aqbd approach was applied to study the influence of input variable parameters on spectrophotometric analytical method performance shown in (figure 3). figure 3: diagram showing the relationship between input variable parameters and the spectrophotometric method performance characteristics d kolekar et al. chemometric assisted qbd method for estimation of bilastine and montelukast sodium vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 18 solvent selection: bse is freely soluble in 10% acetic acid glacial, chloroform and in ethanol, slightly soluble in 1 n hcl and soluble in 1n naoh and sparingly soluble in water; whereas mks is very soluble in water, methylene chloride and ethanol. although the solubility of the procured drugs were studied in alcohol 90%, 0.1 n hcl and 0.1 n naoh separately; and found that bse is soluble in ethanol, 0.1 n naoh, slightly soluble in 0.1 hcl however mks soluble in ethanol and insoluble in naoh and hcl. both drugs solubilises in 50% alcohol, hence selected as a common solvent. each drugs solution with known conc was scanned in uv range of 400 nm to 200 nm. it was found that alcohol 50% is suitable with respect to stable, robust and precise in producing result. preparation of stock solutions and standard solutions: 10 mg each of drug bse and mks were separately and accurately weighed; and transferred into separate 25 ml volumetric flask. dissolved into solvent alcohol 50% and volume was made to 25 ml with this solvent. working standard solution was prepared by diluting 5 ml to 10 ml with 50 % alcohol. subsequent standard solution of each drug with conc 16μg/ml was prepared by diluting aliquot of stock solution to 10 ml with 50% alcohol into 10 ml capacity volumetric flask. selection of wavelength and conc range: from uv spectra it was found that bse has measurable absorbance at 274.5 nm and 281.5 nm (figure 4) and less interference was observed by mks; similarly mks has maximum absorbance at 351.5 nm and negligible interference by bse was accounted. chemometric method using simultaneous equation was applied and which was reasonable remedy to overcome interference at each other’s absorbance. to study linearity, working conc range 1 to 32μg/ml for bse and 1 to 20μg/ml for mks was selected. also combined drug solution was prepared simulated to marketed formulation. selected critical parameters based upon above discussion, observations were listed in (table 1) and by using these; method was validated as per ich guidelines and by analysing marketed preparations. experimental method for estimation: from the overlain spectra simultaneous equation method was applied for estimation of both the analytes from their combined dosage form. figure 4: overlaid spectra of bse and mks table no 1: selected critical parameter for uv-vis analytical method of bse and mks parameter selected variables for simultaneous equation method bse mks wavelength 274.5 351.5 solvent 50% alcohol 50% alcohol scan speed fast fast sampling interval 0.2 nm 0.2 nm simultaneous equation method for estimation of bilastine and montelukast sodium: bse was shown maximum absorbance i.e. λmax at 274.5 nm where moderate interference by mks found and mks has maximum absorbance i.e. λmax at 351.5 nm where negligible interference by bse observed. at 274.5 nm the λmax of bse, the mks was shown consistency in the absorptivity; hence two wavelengths 274.5 and 351.5 nm were considered as 1 and 2 respectively for the said method to estimate bse and mks. the equation a= abc was applied for x (bse) and y (mks) determination. working standard solutions of bse and mks containing 12μg/ml conc were separately prepared and used for the method. where cx = conc of bse in sample solution a1 and a2 = absorbance of sample solution at 1 and 2 wavelength ay1 and ay2 = absorptivity of mks at 1 and 2 wavelength of standard solution ax1 and ax2 = absorptivity of bse at 1 and 2 wavelength of standard solution cy = conc of mks in sample solution as = absorbance of sample solution at 2 wavelength validation of the method selected critical parameters should meet the performance characteristics of the analytical method so as to attain analytical target profile of the method. an ich guideline q2 r1 was applied to study methods performance with critical parameters in order to implement aqbd approach. the method was validated as per ich guidelines system suitability: system suitability is studied to demonstrate the suitability of the developed procedure under consideration for the analytical method. six replicates of working standard solutions with conc 20μg/ml and 16μg/ml of bse and mks respectively were prepared separately and absorbance was recorded, calculated sd and % rsd of the response. d kolekar et al. chemometric assisted qbd method for estimation of bilastine and montelukast sodium vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 19 linearity: the linearity of an analytical method is its ability to obtain response i.e. absorbance which is directly proportional to the conc of analyte. series of working standard solutions were prepared in conc. range of 1-32 μg/ml for bse and 1-20 μg/ml for mks and scanned in 400 to 200 nm range in spectrum mode of the spectrophotometer, absorbance of the standard solutions were recorded at their respective wavelength; i.e. 274.5 for bse and 351.5 nm for mks in spectrum order. microsoft office excel software tool was used to obtain the standard regression curve and its analysis as slope, intercept, and correlation coefficient. assay of formulation: assay was carried out by proposed methods and assay was validated by statistical parameters. estimation of formulations by simultaneous equation method: tablet powder equivalent to 10 mg bse and 5 mg mks was weighed and transferred into 25 ml volumetric flask. dissolved into 50% alcohol, mixed well for 10 mins and volume was made to 25 ml with the solvent. solution was filtered through what man filter paper and aliquots of solution were further diluted with the 50% alcohol to obtain tablet sample solution. solution was scanned in the range of 400 to 200 nm to obtain absorbance of tablet solution at 274.5 nm and 351.5 nm in spectrum order. obtained absorbance were utilised to estimate unknown conc of formulation; and results were statistically validated to obtain % of nominal conc, standard deviation and % of rsd. accuracy and precision: the accuracy of an analytical method expresses the closeness of an agreement between test result and true result. accuracy study was performed by recovery study i.e. standard addition method; diluted standard solutions of bse and mks were prepared and standard solutions added in 80,100 and 120% proportionate to the tablet solution. three replicates at each of these three levels were prepared, measured and % of conc, sd and rsd were calculated. the precision study was carried out by performing assay of tablet six times; also the reproducibility in result was studied by inter day and intraday precision. limit of detection (lod) and limit of quantitation (loq): the lod and loq of bse and mks by the proposed method were determined using calibration graph method and calculated as 3.3σ/s and 10 σ/s for lod and loq respectively; σ is the standard deviation of calibration curve and s is the slope of regression line. robustness and ruggedness: it is measure of capacity of analytical procedure to remain unaffected by small but deliberate variations in method parameter. results and discussion method development comprises numerous steps, and of which solvent selection, selection of method for measurement are significant one. uses of aqueous solvents, eco-friendly solvents like hydrotropic have got remarkable weightage due to low cost, readily available and environmentally sound. drugs underlying analysis must have appreciable solubility in the selected solvent. chemical structure of the drug and physico-chemical properties available in the literature guides about use of appropriate solvent in the method. from uv spectra two wavelengths were selected as 274.5 nm (λmax of bse) and 351.5 nm (λmax of mks) for calculation of both drugs in combined solution shown in (fig no 4). system suitability: the absorbances of six replicates of standard solutions of respective drugs conc are reported in (table no 2). the sd and % rsd was found for bse and mks and meets the system suitability requirements indicate method was suitable for analysis. table no 2: system suitability study of bse and mks conc in μg /ml absorbance of bse conc in μg /ml absorbance of mks 20 μg /ml 0.3124 16 μg /ml 0.5269 20 μg /ml 0.3110 16 μg /ml 0.5443 20 μg /ml 0.3281 16 μg /ml 0.5146 20 μg /ml 0.3115 16 μg /ml 0.5156 20 μg /ml 0.3395 16 μg /ml 0.5332 20 μg /ml 0.3292 16 μg /ml 0.5581 sd rsd 0.006662 0.41972 sd rsd 0.01281 0.29142 linearity: the calibration curve of both drugs was found to be linear shown in (figure 5) in the conc range of 1-32 μg/ml for bse and 1-20 μg/ml for mks as shown in (figure 6). the regression equation of line and parameters slope, r2 value and intercept (figure 7) are tabulated in (table 3), which proved the linear relationship between conc and obtained response. assay: the assay was carried out by the proposed method. the spectrum of formulation by method was shown in (fig no 8). the assay of formulation was carried out by proposed method and calculated % of nominal conc and rsd was found within acceptable limits are summarized in (table no 4). the results indicated applicability of the method for estimation of formulation. accuracy and precision: the accuracy study was carried out at 3 levels; and the results of accuracy are summarised in (table 5), the obtained results were within acceptable limit; and methods accuracy was justified by calculating % drug content. the precision study was carried out by performing assay of solutions; further the reproducibility in result was studied by interday and intraday precision. the values obtained sd and % rsd was shown methods precision and are summarised in (table 5). d kolekar et al. chemometric assisted qbd method for estimation of bilastine and montelukast sodium vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 20 limit of detection (lod) and limit of quantitation (loq): the lod and loq of bse and mks by the proposed method were calculated and shown in (table 6). robustness and ruggedness: robustness was studied and capacity of analytical procedure to measure analyte was remain unaffected by small but deliberate variations in method parameter. the analytical method was found rugged during development; similarly the result was produced shown in (table 6) by performing the analysis by different analyst. figure 5: overlaid spectra of bilastine obtained in linearity study figure 6: overlaid spectra of montelukast obtained in linearity study fig no 7: calibration curve of bse and mks table 3: parameters of regression equation obtained in microsoft excel parameters bse mks detection wavelength 274.5 351.5 beer’s law limit (μg/ml) 1–32 μg/ml 1–20 μg/ml correlation coefficient (r2) 0.9967 0.9998 regression equation (y = mx + c) y = 0.0203x 0.0037 y = 0.0302x 0.0013 fig no 8: spectra of formulation obtained in the assay table 4: results of assay of formulation by proposed method name of the formulation bilafav m (bilastine 20 mg, montelukast sodium 10 mg) b no -23s2gtb843, mfg datejul 2023, exp date–jun 2025 formulation drug label claim (mg/tablet; n=6) amount found/mg drug content % std deviation % rsd method bse 20 19.618 98.092 0.15702 0.16092 mks 10 10.362 103.62 0.21932 0.24942 table no 5: results of accuracy and precision parameter level of study drug name s.d. % rsd precision intraday precision bse 0.19025 3.9635 mks 0.28281 0.2828 inter day precision bse 0.57732 0.83165 mks 0.54582 0.64584 accuracy study of bse and mks 80% bse 0.25236 0.21612 100% 0.28842 0.24982 120% 0.52218 0.44192 80% mks 0.25123 0.34451 d kolekar et al. chemometric assisted qbd method for estimation of bilastine and montelukast sodium vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 21 100% 0.68602 0.88492 120% 1.25152 1.81023 table no.6: results of lod and loq, robustness parameters bse mks lod μg/ml 0.7253 0.6726 loq μg/ml 1.1761 0.8458 robustness (conc 20 bse and 16 μg/ml mks) 0.3724 -to0.3973 (± 2 nm) 0.5308 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development and its validation, int journal of pharma science. 2011; (3):179-187. how to cite this article: bhagyashri d kolekar, namrata n gawade, g k dyade, nilesh y jadhav. chemo metric assisted spectrophotometric method development through qbd approach for the estimation of bilastine and montelukast sodium in combined solid dosage form. indian j pharm drug studies. 2024; 3(1):16-23. funding: none conflict of interest: none stated key words: bilastine, montelukast sodium, qbd, ich, simultaneous equation method osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 41 review article some members of genus cassia (senna): their ethnobotany, potency and prospects of drug discovery salome osunga1, omari amuka2, alex k. machocho3, albert getabu4, martin o. onani5 from 1 department of chemistry, kisii university, p.o box 408-40200, kisii, kenya. 2 depatment of applied plant sciences, maseno university, private bag, maseno, kenya. 3 department of chemistry, kenyatta university, p.o. box43844-00200, nairobi, kenya. 4 department of aquatic and fisheries sciences, kisii university, p.o box 408-40200, kisii-kenya. 5 department of chemistry, university of the western cape, bellville, south africa. corresponding to: salome osunga, department of chemistry, kisii university, p.o box 408-40200, kisii, kenya. email: sosunga@kisiiuniversity.ac.ke abstract plants from cassia species have been used traditionally all over the world in the treatment of various diseases. various compounds with diverse bioactivities have been isolated from the cassia species. however, this has not been done exhaustively in all the species. moreover, only a few drugs are associated with cassia species as their origin despite the knowledge that plants are a good source of drugs. further investigations are required to isolate more compounds from these species, verify their bioactivities, conduct clinical and toxicological studies and eventually come up with lead drugs. this review relates the ethnobotanical uses of various cassia species with the isolated bioactive compounds. it pinpoints the limited compounds that have been isolated compared to their traditional benefits justifying the potential and potency of the species as a drug source. key words: cassia, senna, ethnopharmacology, phytochemicals, bioactivity edicinal plants are critical to developing novel medications [1-3]. 70-90% of the population in asia, africa, latin america and the middle east rely on traditional medicine for primary healthcare [3]. the global acceptability of popular and efficient species from europe, north america, africa and asia is expanding due to increased demand for medicinal plants in many countries [4]. the percentage of individuals using herbal plants has increased: to 40–50% in germany, 42% in the usa, 48% in australia, and 49% in france [3,5]. there is also an ever increase in microbial resistance to antibiotics. the trend is causing concern when it involves the continuous evolution of new strains that use resistance strategies against antimicrobial drugs. it could be possible that plant extracts may offer a solution to the puzzle.such disease management methods rely on ethnobotany and ethnopharmacology methods used in identifying the plants of interest for pharmacological and phytochemical studies [6]. ethnobotany has played and will continue to play a significant role in drug discovery [7]. in addition, traditional medical practices involving plant or plant parts are embedded in virtually all community cultures [8].during the literature review of chamaecrista nigricans syn. cassia nigricans, it became clear that there was significant documentation; however, there was still room to add more knowledge to this already exciting genus senna (cassia). cassia genus belongs to the family fabaceae and comprises about 600 species of herbs, shrubs, and trees distributed in tropical and subtropical countries, mainly africa, asia, and south america [9,10]. the species have been used, anciently in vogue, to treat various ailments that include: skin diseases (such as scabies, eczema, and ringworm), helminthiasis, impetigo, ulcers, pesticide, laxatives, rheumatic diseases, headache, and fever [2]. most of these species are used as anti-inflammatory, antimicrobial, antivirals, antimutagenic antioxidants, antiplasmodial anticancer, laxative hypoglycaemic, and hyperglycaemic [11-13]. several secondary metabolites have been isolated and identified from the cassia species. they include peridine alkaloids, anthraquinones, anthracenes, tannins, phenylpropanoids, pentacyclic triterpenoids, essential oils, polyphenols, flavonoids, fatty acids, γ-naphthopyrones, sterols and polysaccharides [13-16]. such compounds have been proven to be bioactive and found in different parts of plants, such as flowers, seeds, fruits, leaves, roots, and bark [11]. this article has attempted to give an overview of the ethnopharmacology and bioactivity of the phytochemical compounds isolated from various species of the genus cassia and their potency and potential as new drug sources. it summarises advances in bioactive isolates from this genus and the discovery of new therapeutic agents. it is expected to emphasize the importance and traditional utilization of the genus for novel drug discovery. this effort will incentivize more research to isolate bioactive constituents and develop m mailto:sosunga@kisiiuniversity.ac.ke osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 42 drugs from the already bioactive compounds from the genus. virtually all the members of this genus are of economic importance as fodder for foraging bees and nitrogen fixers, thus increasing soil fertility in essential nutrients [14]. the relevant information on the botanical description, ethnopharmacological uses, phytochemicals, and the bioactivities of the isolated compounds were collected from various search engines, including google scholar, google, springer, elsevier, pubmed, science direct, and researchgate. cassia or senna and its associated plant names were employed as keywords to find the pertinent information. the chemical structures of the compounds were drawn using chemdraw ultra 8.0 software. pubchem and chemspider databases were used to verify the iupac names of the isolated phytochemicals. the data includes species name, habitat distribution, extracted phytochemical compounds, and the bioactivities performed on the phytochemical compounds. cassia auriculata linn synonyms are senna auriculata and c.densistipulata (l.) roxb. it is a fast-growing shrub to a small tree used in green manuring, ornamental, soil reclamation, and tannin. the plant possesses some cardiac glycosides [15]. it is commonly found in asia [16]. the whole plant treats liver ailments [17]. the leaves have been used for ulcers, skin diseases, anthelmintics, and leprosy [18]. the bark is an astringent [19], while the roots have been used to treat skin conditions such as leprosy, tumours, urethroea, and asthma [20]. the roots are also used in managing ailments in the urinogenital system, fever, constipation, and diabetes [21,22]. the flowers cure nocturnal emissions, urinary discharges, throat irritation, and diabetes [23]. flowers are also used as a body coolant, treating yellow fever and in blood and liver purification [24]. the seeds are used in chylous urine, diabetes, ophthalmic, and aphrodisiac complaints [25]. the leaves have also been used as hair cleaner and to cure common cold, whereas roots also cure diarrhea, abdominal pains, and vomiting [26]. the plant is also used generally for intestinal problems, female infertility, worms, leprosy, conjunctivitis, rheumatism, and diarrhea [27]. some compounds that have been isolated from the species include 4-(4-chlorobenzyl)-2,3,4,5,6,7-hexahydro-7-(2ethoxyphenyl)benzo[h][1,4,7]triazecin-8(1h)-one (1), an anticancer compound, isolated from ethanolic leaves extracts of the plant. this compound has been confirmed to inhibit the growth of human colon cancer cells [28]. oleanolic acid. (2) isolated from methanolic leaf extracts has antimicrobial activity against klebsiella pneumonia, proteus mirabilis, escherichia coli, and salmonella typhi [29]. methanol and chloroform crude extracts showed potent inhibitory activity against the above microbes [30]. ayurvedic hydro-alcoholic seed extracts of c. auriculata have been reported to possess antidiabetic activity [31]. antidiabetic compounds: 1,3,8-trihydroxyanthraquinone (emodin) (3) and quercetin (4) have been isolated from nbutanol seeds extracts, while gallic acid (5), quercetin-3-orutinoside (6), caffeic acid (7), ferulic acid (8), and ellagic acid (9) were isolated from methanol: water (1:1)seeds extract [32]. 5-o-methylquercetin-7-o-glucoside (10) has been isolated from the plant's 50% acetone flower extracts and has antiinflammatory activity [33]. furthermore, α-tocopherol-β-dmannoside (11) is also an anti-inflammatory compound isolated from the plant's methanolic leaf extracts [34]. refluxed dried powdered leaves concentrates in 1n naoh solution contain luteolin (12), quercetin (4), kaempferol (13), and kaempferol-3-o-β-d-rutionoside (14). these compounds inhibit the aluminium corrosion activity without harming living organisms, unlike chemical inhibitors, which are quite toxic and expensive, harmful to bio-organisms and nonbiodegradable [35,36]. cassia glauca (lam) synonym is senna sulfurea (collad.) h.s.irwin & barneby a shrub usually with yellow flowers and is used as an ornamental. it is found in tropical asia, india, australia, south america, malaysia, pakistan, and china [37,38]. the leaves have been used to manage blennorrhagia [39,40]. the seeds treat skin diseases and leucoderma, whereas the bark and leaves treat gonorrhea and diabetes [37]. the plant has been used for common cold, as an antimalarial, central depressant, purgative, and diuretic [41]. a compound of biological interest that has been isolated is: kaempferol 3-o-βd-rutinoside (14) from the methanolic leaves extracts and has been reported to harbour in vitro cytotoxic effects against human liver carcinoma (hepg-2) and human breast adenocarcinoma (mcf-7) cell lines [42]. moreover, it can be combined with other chemotherapeutic drugs to boost their cytotoxic activity and guard against their side effects, suggesting that kaempferol 3-o-β-d-rutinoside can be a potent anticancer agent [38]. cassia angustifolia vahl synonyms are senna alexandrina mill, c.acutifolia delile, c.lanceolata forssk, c.senna l., senna acutifolia (delile) batka and .s.angustifolia (saheed s.a & illoh h.c). the plant is also referred to as indian senna. it is found in india, saudi arabia, pakistan, egypt, somalia, arabia, and yemen [43,44]. the leaves have been used to manage hepatomegaly, anemia, constipation, malaria, loss of appetite, indigestion, jaundice, ringworm, splenomegaly, and to increase peristaltic movement of the colon [9,43]. the leaves and pods are used for splenic enlargements, cholera, antipyretic in typhoid, anthelminthic, and laxative [44]. dry tubers have been used as an aphrodisiac, general debility tonic, and rheumatism [45]. various isolates from parts of the plant, including quercimeritrin (15), scutellarein (16), and rutin (17), have been isolated from methanol, ethyl acetate, and ethanol seed powdered extracts. all these extracts possess anticancer and antioxidant activities. in addition, the mentioned three compounds have antimicrobial activity; they inhibit the microbial growth of e. cloacae, p. aeruginosa, s. mercescens, and s. typhi [44]. the leaves and the pods of this species have been reported to contain dianthone glucosides, sennosides a (18) and b (19), commonly used as laxatives [46-49]. http://www.theplantlist.org/1.1/about/#synonym http://www.theplantlist.org/tpl1.1/record/ild-29621 http://www.theplantlist.org/tpl1.1/record/ild-29621 osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 43 n n o n h cl o 1 h3c ch3 coohh3cch3 ho h3c ch3 2 oh ho oho o o o ho oh oh oh oh ho oh oh ho o 3 4 5 o o ho oh o oh oh o ho oh oh o o h3c ho oh oh o ho oh o o o o o ho ho oh oh 6 9 ho ho oh o 7 8 o ho ho o ho ho o oh oo o oh oh o oh oh ho o ho 10 11 ooh ho o oh oh oh ooh ho o oh 12 13 osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 44 o ooh ho o oh o oh o o oh oh oh oh oh ch3 14 other compounds also isolated from methanolic leaves extracts of c.angustifolia and known to display various activities, including antimicrobial compounds such as 2, 5dimethyl-4-hydroxy-3(2h)furanone (20), 4a-acetoxy-5,5,8a,trimethyloctahydrobenzo[b] pyran (21) and 1-ethynyl-4-fluorobenzene (22); anti-inflammatory compounds such as caryophyllene(23), estragole(24) and 1-(1,5-dimethyl-4 hexenyl)-4-methylbenzene (25); antihyperglycemic compound such as anethole (26); antioxidant compounds such as 2methylene-(3ß,5α)-5-cholestan-3-ol (27), 2-methoxy-4vinylphenol (28); anticancer compounds such as 5hydroxymethylfurfural (29), ß-curcumene (30) as well as antiviral compound such as 2-[6-(2,6,6-trimethylcyclohex-1enyl)-4-methyhexa-1,3,5-trienyl]cyclohexanal (31) [46]. cassia fistula l synonyms are: bactyrilobium fistula (l.) willd; c.bonplandiana dc; c. excels kunth; c.fistuloides collad; c.rhombifolia roxb; cathartocarpus excelsus g. don; cathartocarpus fistula (l.) pers; cathartocarpus fistuloides (collad) g. don; and cathartocarpusr hombifolius (roxb) g. don.the medicinal use of the species date from ancient times and has been the main factor in its spread. it is called "aragvadha," a word that can be translated as "elimination of diseases" in sanskrit. the plant must have originated from the indian subcontinent. it is widespread in east africa and several of the indian ocean islands [48]. the roots, bark, leaves, flowers, and seeds are all used for therapeutic purposes. the leaves have been used as a purgative against ringworms [49]. the whole plant treats anorexia, skin diseases, rheumatism, jaundice, and inflammatory diseases [50]. the roots also cure heart diseases, dysentery, joint pain, retained excretions, chest pain, fever, and migraine [51]. ayurvedic medicines recognize the use of the plant for skin diseases, tubercular glands, adenopathy, burning sensations, syphilis, and leprosy [52]. the fruit, seeds, flowers, and pulps are all used for skin diseases; the pulp is also used to treat gout and rheumatism, while the leaves have been used as a laxative [53]. in addition, flowers, leaves, bark, root, and pulp have been used in wound healing, liver protection, and as an antimycotic [54]. the whole plant also treats ulcers, purgative, impetigo, and helminthiasis, although the leaves and the seeds act as a liver tonic, cardiotonic, laxative, antihelmintic, ophthalmic, antiperiodic expectorant as well as in treating constipation and bronchitis [55]. the species' seeds have been used in treating swollen throats, oral sores, jaundice, and biliousness [56]. the plant is also used as a hair cleanser and in the treatment of venereal diseases, diarrhea, toothache, muscle pain, cold, inflammation, reducing body heat, vomiting as well as diabetes [26,48]. phytochemical elucidation of cassia fistula has led to isolating compounds with therapeutic values.:4-hydroxy benzoic acid hydrate (32) (from the ethyl acetate extracts of the flower) was confirmed to possess antifungal activity against trichophyton mentagrophytes and epidermophyton floccosum [57]. rhein (1,8-dihydroxyanthraquinone-3carboxylic acid) (33) has also been isolated from ethyl acetate extract of c. fistula flower and showed inhibition against fungi such as trichophyton mentagrophytes, trichophyton simii, trichophyton rubrum, and epidermophyton floccosum [58]. benzyl 2-hydroxy-3,6-dimethoxybenzoate (34) and dibenzyl-׳tetramethoxy-biphenyl-1,1-״6,״dihydroxy-3,6,3-׳2,2 dicarboxylate (35) isolated from methanol extracts of seed showed antifungal activity against cladosporium cladosporioides and cladosporium sphaerospermum [59]. phytol (36), lutein (37), and di-lineolylgalactopyranosylglycerol (dlgg) (38) are antiplasmodial compounds that have been isolated from chloroform leaf extract of the plant. dilineolylgalactopyranosyl-glycerol portrayed weak toxicity against cytotoxicity test carried out using the chinese hamster ovarian (cho) cell line, while phytol and lutein were nontoxic [60]. rhein (33), 2(3h)-furanone (39), thymol (40), and oleic acid (41) have been isolated from ethyl acetate extract of seeds and pulp of c. fistula [61]. butanol extracts of the seed yielded inositol (42) and palmitic acid (43), whereas the butanol extract of the pulp yielded inositol (42) and 2-pyrrolidone (44). all these compounds were reported to inhibit breast cancer (mcf7) and human cervical cancer (siha) cell growth, and they also induced cell death [61]. cassia alata l synonyms are senna alata l and herpetica alata. it is distributed in africa, south and north america, and india [62,63]. the plant leaves treat ulcers, scabies, ringworm, and skin diseases such as eczema, itching, and pruritis [62]. the leaves and stem bark are used to treat burns, diarrhea, jaundice, gastroenteritis, and hepatitis, whereas the immature leaves are effective against food poisoning and constipation [64]. this plant is good against insect bites, worms, goiter, fever, blemishes, sexually transmitted diseases, and fungal skin infections [65-67]. the leaves also cure asthma and bronchitis [67]. the roots are used for uterus disorder [68,69]. some compounds of interest have been isolated from this species. cannabinoid alkaloids, 4-butylamine-10methyl-6hydroxy cannabinoid dronabinol (45), have been isolated from the ethanolic seed extracts of the plant and is known to possess bioactivities against microbes such as pseudomonas aeruginosa, klebsiella pneumonia, escherichia coli, osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 45 staphylococcus aureus, candida albicans and aspergillus niger [70]. luteolin (12), kaempferol (13), and aloe-emodin (46) have been isolated from methanolic leaf extracts, and they all showed antibacterial activity against vibrio cholerae and shigellaflexneri., with aloe-emodin showing the highest activity [71]. kaempferol (13) and ω-hydroxyemodin (47) have been isolated from the acetone leaves extracts ωhydroxyemodin showed antibacterial activity against methicillin-resistant staphylococcus. aureus (mrsa) and kaempferol posses antioxidant activity [72]. oo oh oh oh oh o oho oh o oh oh o o ho ho oh ho oho oh oh o ho 15 16 o o o ho oh oh ohho oh 17 o o o oh cooh cooh oho o o oh ho ho oh ho ho oh oh h h o o o oh cooh cooh oho o o oh ho ho oh ho ho oh oh h h 18 19 o o ho 20 o o o 21 f 22 23 o 24 25 o 26 ho 27 o oh 28 oo oh 29 30 o 31 osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 46 cassia tora l synonyms are senna obtusifolia (l.) h.s.irwin & barneby,cassia numilis collad, c. obtusifolia (l.), c.toroides raf., diallobus uniflorus raf and senna toroides roxb. it is found in africa, fiji, southeast asia, northern australia, and latin america [73,74]. the species has been used in folklore medicine in that the leaves of the plant are used for the treatment of jaundice, eczema, ringworm, intestinal impetigo, and helminthiasis; the decoction of leaves and flowers are used against asthma and bronchitis; and the seeds are used to treat leprosy, psoriasis, earache, itching, eye diseases, liver problems while pods are used against dysentery and in eye diseases [75]. its fermented leaves have purgative properties [72]. traditionally, the plant has been widely employed in managing hemorrhoids, vitiated tridosha, hepatitis, skin diseases, dandruff, cough constipation, and fever [74]. the leaves and seeds possess cardiotonic, ophthalmic, antimicrobial, and liver tonic properties [76]. emodin(3), aurantio-obtusin (48), chryso-obtusin-2-o-β-dglucoside (49), and obtusifolin (50) have been isolated from ethyl acetate soluble extracts of the seeds of c. tora. they are known to inhibit diabetic complications (advanced glycation end products (ages)) and cataract formation (rat lens aldose reductase (rlar)) [77]. ononitol monohydrate (6methoxycyclohexane-1,2,3,4,5-pentaol hydrate) (51) occurred in the leaves of ethyl acetate extracts and was found to exhibit in vivo hepatoprotective activity [78] aurantio-obtusin (48), chrysophanol (52), and chryso-obtusin (53) have been isolated from dichloromethane fraction of methanolic seed extracts, and the compounds showed significant antimugenic activity [79]. additionally, friedelin (54), a triterpene, is a compound that was isolated from ethanolic leaves extract of the plant and found to possess anticancer activity against human cancer cell lines; hela (cervical carcinoma) and hsc-1 (squamous carcinoma) [73]. two phenolic triglucosides: toralactone 9-o[β-d-glucopyranosyl-(1→3)-o-β-d-glucopyranosyl-(1→6)o-β-dglucopyranoside] (55) and torachrysone 8-o-[β-dglucopyranosyl(1→3)-o-β-d-glucopyranosyl(1→6)o-β-dglucopyranoside] (56) occurred in 70% ethanol extracts of the seeds and was found to possess estrogenic activity [80]. o o o oh oh oh o ho oh h o 32 33 h 34o o o o oh o oh meo o o o meo oh ome ome 35 ho ho oh o oh oh ho oh o o o o 36 37 38 o o ho o oh 39 40 41 ho ho oh oh ohho o ho n h o 42 43 44 osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 47 o ch3 oh n h ch3 h o o oh oh oh45 46 o o oh oh 47 ho o o o o o o oh h3co ho och3 oh ch3 och3 ho h3co och3 o ch3 o oh oh oh oh och3 oh ch3 ho oh oh ohho o h oh 48 49 50 51 hhh o o ooh oh ch3 o ooch3 h3co h3co och3 oh ch3 52 53 54 ch3 ch3 oh h3co o o o o o oh ho ho o ho o oh oh ho ho oh oh oh h3co o o o o oh ho ho o ho o oh oh ho ho oh oh o ch3 o 56 55 advanced chemical studies and elucidation of ethyl acetate soluble seed extract yielded emodin (3), rhein (33), aloeemodin (46), torachrysone (57) and toralactone (58), which possess significant antibacterial activity against methicillinresistant staphylococcus aureus [81]. cassia abbreviata oliv synonyms are cassia afrofistula brenan, cassia beareana holmes and cassia kassneri bak. it is a medium-sized tree widely distributed in the tropics of asia and other tropical worlds [82] and used as medicine. the roots treat malaria, fever, impotence, abdominal pain, wounds, osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 48 dysentery, syphilis, snake bite, body weakness, and hernia [83[84]85]. the plant is used to treat cancer [86]. its root bark manages vaginal candidiasis [87]. the leaves, roots, and bark have been used to treat cough, epilepsy, diarrhea, fever, convulsion, vomiting, abortion, infertility, earache bilharzia, syphilis, hemorrhoids, gonorrhea, jaundice hernia, and stomach ache [88]. the bark and the roots cure dysentery, bloody vomiting, and menstrual cycle problems [89]. fruits are used against eye infections and malaria [82]. the bark is also used in treating toothache and sexually transmitted diseases [90]. the roots are also used as an aphrodisiac [91]. further, advanced studies in chemistry identified compounds that could enhance drug discovery. such compounds are trimmericproanthocyanidins; 3,7,4'trihydroxyflavan-(4β→8)-3,5,7,4'-tetrahydroxyflavan-(3'→6)3,5,7,2',4'-pentahydroxyflavan (cassinidin a) (59) and 3,7,2',4'tetrahydroxyflavan-(4α→8)-3,5,7,4'-tetra-hydroxyflavan(4α→6)-3,5,7,2',4'-pentahydroxyflavan (cassinidin b) (60) which were isolated from methanol root bark extracts and they exhibited antibacterial activities on escherichia coli, bacillus subtilis, staphylococcus aureus and candida mycoderma [92]. 2,3-dihydro-5-hydroxy-8-methoxy2-(4methoxyphenyl)chromen-4-one (61) and 3,4-dihydro-2-(4 hydroxy-phenyl)-4-methoxy-2h-chromen-7-ol (62) have been isolated from methanol root extracts and found to have antiplasmodial activities on both chloroquine-resistant and chloroquine-sensitive strains of plasmodium falciparum [93]. cassiabrevone (63), guibourtinidol-(4α→8)epiafzelechin (64), taxifolin(65), oleanolic acid (66), piceatannol(67), and palmitic acid (43) were isolated from ethanolic bark and root extracts and posses anti-hiv-1 activity [94,95]. cassia nigricans vahl synonym is chamaecrista nigricans vahl the roots and leaves are vermifuge and antiperiodic [80]. the leaves also treat fever, sore throat, rheumatoid pains, gastrointestinal disorders, and family planning [12, 96,97]. the plant treats skin diseases, ulcers, diarrhea, and gastrointestinal disorders [98,99]. various compounds have been isolated from this species, justifying its continued use in folklore medicine. it was reported that 1,3,8 trihydroxy-6-methyl-9,10-anthracenedione (emodin) (3) isolated from methanol extract of the whole plant showed in vitro antiplasmodial activities against plasmodium falciparum[100]. emodin has also been isolated from the ethyl acetate leaf extract and found to be highly cytotoxic despite having antimicrobial activity [101]. emodin (3), luteolin(12|), citreorosein (68), and emodic acid (69) have been isolated from n-hexane, ethyl acetate, and methanol combined extracts. emodin, citreorosein, and emodic acid showed larvicidal activity on anopheles gambiae larvae [102]. steroidal ester and hydroxyestranic acid ethyl ester (70) have been isolated from methanol leaves extracts and showed antimicrobial activities on staphylococcus aureus, streptococcus pyogenes, corynebacterium pyogenes, bacillus subtilis, salmonella typhi, escherichia coli, pseudomonas aeruginosa, candida albicans, neisseria gonorrhea and klebsiella pneumoniae [103]. cassia sieberiana d.c. this species is a medium-sized tree widely distributed in sub-saharan africa, from senegal to east africa [104]. the root bark and rootlets treat toothache, abdominal pains, skin diseases, hemorrhoids, helminths, and leprosy [105]. the roots also treat hemorrhoids and skin irritation and manage indigestion, stomach pains, and gastric ulcer [106,107]. the seeds have been used in weight reduction, clearing of acne, detoxification, and driving out internal heat [108]. the folklore uses of c. sieberiana have led to the isolation of such compounds as quercetin (4), cassiberianol a (71), and piceatannol (72) from methanol extracts of the roots. they all had inhibitory activity on 15-lipoxygenase, while piceatannol and quercetin also showed acetylcholinesterase inhibitory activity [109]. flavanoids, epiafzelechin (73), have been isolated from ethanol extracts of the plant's stem bark and possess antioxidant activity [110]. moreover, cassiphenol (74), spectaline (75), and iso-6-cassine (76) are also compounds that have been isolated from ethanol extracts of the stem bark. cassiphenol has weak antibacterial activity against pseudomonas aeruginosa, providencia stuartii, enterobacter aerogenes, and klebsiella pneumoniae. spectaline and iso-6-cassine were the only compounds that portrayed antioxidant activity [111,112]. cassia absus l. synonym is chamaecrista absus h.s.irwin & barneby it is a small hairy herb, a native of africa; however, a common monsoon weed in south east asia [113]. in folklore medicine, the seeds have been used to treat syphilitic ulcers, skin infections, leukoderma, ophthalmia and as a cathartic [114]. the leaves are used for nasal diseases, cough, and as an astringent to the bowel [115]. the leaves are used in curing tumours, while the roots are used against constipation [116]. ch3 ch3 ohoh h3co o o ohoh h3co o ch3 57 58 osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 49 oho oh oh oh ho oh o ho oh oh oho oh oh oho oh oh oh oh o ho oh oh oho oh oh ho ho 59 60 o och3 och3 oh o o oh och3 ho 61 62 o o o o ho oh ho oh oh h h h o o o oh oh oh ho ho oh oh o oh oh ho oh o oh h ho h cooh h ho oh oh oh 63 64 65 66 67 o o h h h h 70ch3h3c oh o ooh ho oh oh o ooh ho oh oh o 68 69 osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 50 ho oh ho oh h3co oh och3 oh oh 71 72 ho o o h3c n ho h3c h ch3 n ho h3c h ch3 o o 74 75 76 ho oh o oh oh 73 from this ethnobotanical information, guided chemical profiling has been done, and the following noble compounds like aloe-emodin (46), chrysophanol (52), and chaksine (77) have been isolated from the ethanol root extracts [117, 118]. the ethnomedicinal use of the roots is associated with the presence of chrysophanol and aloe-emodin and has also been verified by the bioactivity of these compounds from this genus. it has been reported that chaksine has antidepressant activity, anesthetic activity, and antibacterial activity against streptococcus hemolyticus [113]. 5,7,4′-trihydroxy-8,3′dimethoxyflavone-5-o-α-l-rhamnopyranosyl-7-o-β-dxylopyranosyl-(1→4)-o-β-d-galacto-pyranoside (78) is a compound that has been isolated from methanolic seeds of the plant. reports indicate that the isolates possess antimicrobial activity on staphylococcus aureus, pseudomonas aeruginosa, penicillium digitatum, and fusarium oxysporum [119]. senna septemtrionalis h.s.irwin & barneby synonyms are cassia aurata roxb., c. elegans kunth, c. floribunda kunth c. laevigata willd, c. quadrangularis, c. septemtrionalis viv and c. vernicosa clos. a shrub to a sub-shrub native to america now spreads in the tropic and subtropics up to india and south africa [120]. the roots treat malaria, gonorrhea, syphilis, intestinal worms, and blood cleansing [120,90]. the leaves are used as a vermifuge [121,122]. it is also used in treating stomach aches, wounds, fever, gastroenteritis, snakebites, burns, epilepsy, hemorrhoids, anxiety, and as a laxative, fungicide agent, antidiuretic as well as expectorant [123]. this plant has also been used as a medication for rabies, cold, alopecia, earache, bile diseases, pain, inflammation, and cholera [124,125]. its chemical elucidation yielded two compounds. physcion (79) has been isolated from this plant and possesses bioactivities against chlorella fusa and bacillus megaterium [126]. rutin (80) and kaempferol 3-o-rutinoside (14) hexoside are also compounds isolated from this plant, posing both anti-inflammatory and antinociceptive activities [125]. senna petersiana (bolle) lock synonym is cassia petersiana (bolle) lock this small tree is generally found in tropical and sub-tropical africa and can grow to 12 m [127]. the root extracts treat infertility, malaria and are also used as inhalers and for deworming [128-130]. the leaves cure skin diseases, backaches, stomach aches, and febrifuge [127,130]. the plant is used to treat arthritis and management of diabetes [131]. the roots also treat a loss of appetite [132]. the plant also cures cholera, cold, and fever [133]. the plant's roots, leaves, and stems have been used for convulsion and relieving pain in the bones [134]. as a result of its use in traditional medicine, several natural compounds have been isolated from its various parts. luteolin (12) isolated from ethanol extracts of the seed possesses bioactivities against bacillus cereus, bacillus pumilus, serratia marcescence, pseudomonas aeruginosa, and staphylococcus aureus [135]. four compounds: 7-acetonyl-5-hydroxy-2methylchromone (petersinone a) (81), 5-hydroxyl-2-methyl-7(propan-2βol)-chromone (petersinone b) (82), glyceryl-1hexacosanoate (83) and stigmastosterol-3-o-β-d-glucoside (84) were isolated from ch2cl2-meoh (1:1) leaves extract of this plant. stigmastosterol-3-o-β-d-glucoside showed the highest anti-inflammatory antitumor, antioxidant, macrophage osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 51 proliferation, and immune proliferative activities [127]. senna singueana del lock synonyms are s. goratensis fresen, cassia zanzibarensis vatke, c. singueana delile, and c. tettensis bolle. the species is a medium-sized tree used as ornamental and, like most family members, can fix gaseous nitrogen into the soil, thus enriching it with an essential nutrient. it is widely distributed in east and west africa [136]. various parts of the plant have been used in traditional medicine in different parts of the world [137]. the leaves and bark have been used to treat skin cancer [138]. the stem, bark, leaves, and roots treat a sprain, stomach pain, and tooth infection [139]. the leaves cure hepatitis, vomiting, and loss of appetite, and the stem bark prevents stillbirth [140]. the roots treat epilepsy and agitation [141]. the flowers are used as an anti-inflammatory, anti-ulcer, antispasmodic agent and in treating respiratory tract infections, malaria, and typhoid [142] ). the root bark is also used for abdominal pain, bilharziosis, mental disorder, women's infertility, constipation, convulsion, painful uterus, swollen breast, fever, menstruation, gonorrhea, anti-emetic, hernia, and in managing snakebites [143,144]. some beneficial compounds that have been isolated from the plant through phytochemistry include 3β-o-acetyl betulinic acid (85) isolated from the ethyl acetate bark of this plant. it inhibited α-glucosidase and αamylase activity, thus delaying carbohydrates digestion and glucose assimilation, eventually restraining postprandial hyperglycemia, thus treating type 2 diabetes (t2d) [145]. lupeol (lup-20(29)-entriterpene) (86), eugenol (4-allyl-2methoxyphenol) (87) and 8-11-octadecadienoic acid methyl ester (methyl-8, 11-octadecadienoate) (88) are compounds which have been isolated from its methanolic root extracts. eugenol showed antioxidant activity and a broad spectrum of antimicrobial activity against candida albicans, staphylococcus aureus, escherichia coli, streptococcus pneumoniae, and pseudomonas aeruginosa. lupeol and methyl8,11-octadecadienoate also possessed certain antioxidant activity and antimicrobial activity levels against s. aureus [146]. senna didymobotyra (fresen.) h.s.irwin and barneby synonyms are cassia didymobotrya fresen, c. nairobiensis h. bailey, c.verdickii de wild and chamaesenna didymobotrya sunarno. it is an invasive shrub that forms a dense growth that hinders the development of other plant species in a given area [147]. it is distributed in north, west, east, southern africa, and madagascar [148]. this plant's leaves, stem, and roots have been used in treating sickle cell anaemia, backache, fibroids, inflammation of fallopian tubes, and hemorrhoids [149]. the bark is an antihaemorrhagic [150]. the plant has been used in treating skin disease, jaundice, purgative, malaria, sexually transmitted diseases, intestinal worms, and as an appetizer and antibiotic [148,151,152]. the leaves are used in curing dysentery, diarrhoea, and as an emetic and diuretic, while the root is used to treat ringworm, malaria, intestinal worm, fever, and jaundice [151]. through natural product chemistry, some compounds have been isolated from this species; they include: chrysophanol (52), physcion (76), 3β-sitosterol {17-(4-ethyl1,5-dimethyl-hexyl)-10,13,dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1h cyclopenta[a]phenanthren-3-ol} (89) and stigmasterol {(3s,9s,10r,13r,14s,17r)-17-[e,2r,5s)-5-ethyl-6methylhept-3en-2-yl]-10,13-dimethyl-2,3,4,7,8,911,12,14,15,17-dodecahydro-1-h-cylopentanal[a]-3-ol (90) were compounds isolated from the hexane and dcm root bark extracts of this plant. the specific activity of the individual compounds was not reported; however, the crude extracts showed antimicrobial activities on staphylococcus aureus, escherichia coli, bacillus subtilis and candida albicans[153]. hn n o nh nh2 n o o o nh2 h3c h3c h h 77 o och3 oh och3 o o o o ho o ho ho ho ho oh o o h3c ho ho oh 78 o o o oh oh 79 o o ho oh o o o ho oh oh o h3c ho ho oh 80 osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 52 o o oh o o o oh oh o ooh oh h hh h oo ho oh oh ho 81 82 83 84 o h cooh o h h h 85 ho h h 86 h hh h hho och3 87 o o 88 ho ho 89 90 senna bicapsularis (l) roxb synonyms are cassia bicapsularis l., c.emarginata l, c. berterii colla, adipera bicapsularis (l.), adipera spiciflora pittier, cathartocarpus bicapsularis (l.) ham isandrina emarginata (l.) britt. & rose ex britt. & wilson, chamaefistula inflate g.don and isandrina arborescens raf. it is distributed in south america and tropical countries [154] the species is cultivated in many parts of the world; however, it has escaped being naturalized and has an unpleasant odor [155]. roots and leaves are used for stomach aches [156]. the leaves have been used in treating skin ailments [157]. the flowers have been used as an abortifacient and contraception agent for both males and females [158]. the plant has been used to treat pain as well as a muscle relaxant and purgative [159]. due to its aromatic nature, several compounds osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 53 like emodin (3), rhein (33), chrysophanol (52), physcion (76), and stigmasterol (90) have been isolated from it with biological activities including sedative effect, anti-inflammatory, analgesic effect and muscle relaxing effect [159]. cassia spectabilis (d.c.) h.s. irwin & barneby synonyms: cathartocarpus humboldtianus loudon; cathartocarpus speciosus (d.c.) g.don; cathartocarpus trinitatis (d.c.) g.don; cassia trinitatis rchb.ex d.c. cassia carnaval speg, pseudocassia spectabilis (dc.) britton & rose, senna speciosa roxb, senna surattensis (burm. f.) irwin & barneby. cassia amazonica ducke and senna spectabilis (d.c.) irwin & barneby. the species is a native of south america, brazil, and coastal ecuador and has migrated throughout central america, the west indies, the tropics, and sub-tropic parts of the world [160-162]. the leaves are used in throat inflammation and diarrhoea [162]. the plant is used to cure headaches, malaria, and dysentery, while the leaves treat anxiety, epilepsy, insomnia, constipation, and anxiety [163]. the species has also been used in treating ringworm, skin disease, flu, cold, whooping cough, menstrual cramps, and diabetes [160,164,165]. natural products that have been isolated from this plant are: (-)-spectaline (72), (+)-2-methyl-3-feruloyl-6(dodecyl-11′-one) piperidine ((+)-3-o-feruloylcassine) (91) and (-)-3o-acetylspectaline (92), isolated from methanol extract of green fruits of this plant and they showed moderate antioxidant activities [162]. iso-6-spectaline (14-[(2r, 3r, 6r) 3-hydroxy-2-methylpiperidine]-tetradecan-13-one) (93) was isolated from ethanol leaf extracts and showed central nervous system (cns) depressant and anticonvulsant activities [166]. piperidine alkaloids; (–)-3-o-acetylspectaline (92) and iso-6 spectaline (93), have also been isolated from the flowers and fruits of the plant, and they possessed dna damaging activities in saccharomyces cerevisiae [162,167]. (−)-cassine (94) has been isolated from the leaves, flowers, and fruits of this plant and possesses anti-inflammatory and antinociceptive activities [160]. additionally, alkaloids; (−)-spectaline (75) and (−)-cassine (94) were isolated from ethanol extracts of the flower of senna spectabilis, and they showed antiproliferative activity on hepg2 cells and antitumor activity against hepatocellular carcinoma cells [168]. (−)-spectaline (75) and (−)-cassine ((+)3-o-feruloylcassine) (91) have also been reported to possess schistosomicidal activity against adult worms and cercaricidal activity [169]. several anthraquinones have been isolated from this plant, including physcion (76) and 1,3,8-trihydroxy-2methylanthraquinone (95), which were isolated from methanol leaf extracts of senna spectabilis and showed antimicrobial activity against mycobacterium tuberculosis, bacillus subtilis, sarcina lutea, staphylococcus albus, and staphylococcus aureus. chrysophanol (52) and physcion (76) isolated from the plant possessed hepatoprotective, antimicrobial, and anticancer activities [161]. n h 94 ho ch3 o 92n o o 93n ho o n h o ch3 o o ho h3co 91 h h o o oh ho oh 95 o o o oh o o o ho 98 n o ho 96 n o o o o 97 osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 54 cassia siamea lam synonyms are senna siamea lam.; senna sumatrana roxb, cassia florida vahl, cassia arayatensis naves, cassia sumatrana roxb, cassia arborea macfad, cassia gigantea dc., and sciacassia siamea lam. it is widely spread in south africa, east africa, brazil, mexico, china, west indies, and asia [170]. the fruits prevent convulsion and expel intestinal worms [171,172]. the plant treats asthma and microbial infections [170]. leaves treat stomach pains, malaria, constipation, sleeplessness, liver disorder, hypertension, cough, and toothache; the roots are used for diabetes mellitus, malaria, and snake bite; flowers and seeds are used to cure convulsion, typhoid fever, snake and scorpion bites while the stem is used against herpes, scabies, rhinitis, urogenital diseases, diabetes and as a laxative [173,174]. the plant is also used to reduce blood sugar levels and to treat abdominal pain, fever, typhoid, menstrual pain, and jaundice [175]. compounds isolated from this plant include: cassiarin a (96) and cassiarin b (97), isolated from the methanol leaf extracts, and these compounds showed moderate in vitro antiplasmodial activity against plasmodium falciparum [176]. chrobisiamone a (98) has also been isolated from methanolic leaf extracts and possesses antiplasmodial activity [177]. cassiarin a (96) and barakol (99) also showed vasorelaxant activity [178,179]. barakol (99), isolated from this plant's ethanol flower extracts, also showed antioxidant activity [180,181]. chrysophanol (52), physcion (76), betulinic acid (85), lupeol (86), and lupenone (100) are some of the compounds that have been elucidated from the methanol stem bark and showed anti-polio virus activity with lupeol possessing the most significant activity [182]. cassia mimosoides l synonymns are chamaecrista mimosoides l., chamaecrista nictitans (l.) moench, cassia aeschynomene d.c,cassia aspera muhl. ex ell., cassia multipinnata pollard, cassia procumbens stickman, and cassia nictitans sickmann. it is native to china but has spread to different countries [183]. the leaves cure swelling of the legs during pregnancy; [184], facilitate urination, and act as an anti-inflammatory agent [183]. roots treat colic pain, stomach spasms, and diarrhoea [185]. the plant also cures chronic hepatitis and other liver diseases, including liver cirrhosis and fibrosis [186]. phytochemicals such as emodin (3), luteolin (12), oleanolic acid (66), βsitosterol (101), α-l-rhamnose (102), and carotene (103) have been isolated from this species, and showed anti-hmg-coa reductase activity, with emodin and luteolin showing the highest activity; an indication that the plant has potential in treating liver diseases. chrysophanol(52), an essential bioactive component of the plant, possesses blood lipid regulation, antidiabetic, antiinflammatory, and anticancer activities, as well as alleviating metabolic disorders and obesity properties [187]. cassia occidentalis l synonyms are senna occidentalis (l.) roxb, cassia caroliniana walter, cassia obliquifolia schrank, cassia ciliata raf, cassia planisiliqua l, cassia falcate l, cassia macradenia, cassia torosa cav, and ditrimexa occidentalis (l.) britt & rose. it is distributed in asia, south america, australia, and africa [188]. the plant treats diarrhoea and dysentery [189]. the leaves are used for throat infections, itching, and bone fractures. it also manages fever, anaemia, leprosy, tuberculosis, menstrual, and liver problems [190]. moreover, the leaves and seeds are used for skin disorders such as eczemas and mycoses [191]. additionally, the plant is used to treat cancer, eye inflammation, and venereal diseases [192]. the roots cure diabetes, elephantiasis, epilepsy, and convulsion [193,194]. the c. occidentalis isolates include 1, 3,8-trihydroxy-6methyl-anthraquinone (emodin) (3) and 4, 5-dihydroxy-9, 10dioxo-4a, 9, 9a, 10tetrahydro-anthracene-2-carboxylic acid ( rhein) (33); isolated from alcoholic extracts (r-spirit) and found to possess antimicrobial activity on β-lactum resistant strains of aspergillus niger, pseudomonas aeruginosa, aspergillus clavatus, candida albicans, escherichia coli, streptococcus pyogenus, and staphylococcus aureus [195]. chrysophanol (52) was isolated from the leaf extracts and showed wound healing activities [192]. cassia italica mill synonyms are senna italica mill, cassia obovata collad, and cassia aschrek fors., its origin is in the equatorial region and surrounding areas [196]. the plant treats venereal diseases [197]. the roots are used for dysmenorrhoea, nausea, and liver problems, whereas the pods and the leaves are used for burns, skin diseases, and ulcers [198]. leaves are used as hair conditioners, while roots are used to cure diarrhoea [199]. the whole plant is used as a urinary tract purifier and laxative, while its leaves, seeds, and pods are used as a purgative and also to treat elephantiasis and eye diseases [200]. compounds isolated from the species is 2methoxy-emodin-6-o-β-d-glucopyranoside (104), which was isolated from dcm/meoh (1:1) extract of aerial parts of the plant and possessed mild anticancer activity against hepatocellular carcinoma (hepg-2) and mammary gland breast cancer (mcf-7) [201]. cassia afrofistula brenan synonyms are cassia beareana holmes and cassia kassneri bak. f. it is distributed in madagascar, mozambique, tanzania, and kenya [202]. the roots treat hernia and body weaknesses, while the stem is used for kidney diseases and liver pains [83]. the bark is used as an aphrodisiac and laxative. the bark also cures pneumonia, fever, stomach aches, backache, and blood pressure [203]. it is also used to manage uterine complaints, fever, malaria, syphilis, gonorrhea, pneumonia, and snakebites [203]. no bioactive compounds have been reported from this species. cassia falcinella oliv synonym is chamaecrista afalcinella oliv. it is found in kenya, tanzania, uganda, rwanda, namibia, zambia, zimbabwe, mozambique, botswana, and dr congo [202]. the roots are used as an aphrodisiac and in treating gonorrhea [204]. its leaves are used to cure broken bones and rheumatism, while the roots are also used to treat diarrhoea [202]. bioactive compounds have not cassia kirkii oliv synonym is chamaecrista kirkii oliv. it is widely spread in tropical and https://www.gbif.org/species/2949769 https://www.gbif.org/species/2949769 osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 55 subtropical africa [205]. leaves are used for upsetting pains [206]. the plant is used for skin diseases and fertility [207]. bioactive compounds have not been isolated from this species. cassia leptocarpa benth. synonyms are senna hirsuta l, cassia caracasana jacq, cassia hirsuta l, cassia tomentosa l, cassia. longisiliqua blanco, cassia. venenifer rodsch. ex g.mey, cassia neglect vogel var. acuminata benth, cassia pubescens jacq, and cassia gooddingii a. nelson. it is distributed in north, central, south america, and tropical regions [208,209]. it is used to treat liver diseases, malaria, high blood pressure, diarrhoea, typhoid fever, and skin rashes and to reduce cholesterol levels [9,210]. various compounds have been isolated from this plant; however, there is no literature report on their bioactivities. ho h hh h 101 o ho ohho oh 102 103 o o h3c ch3 oh 99 oh ho 100 o o oh ho oh ho o ooh oh och3 ch3 104 conclusion the genus cassia has been widely used in chinese, ayurveda, african, and south american folklore medicines to manage various ailments. various studies have been done on their crude extracts, and isolated phytochemicals from different plant parts of individual species of this genus have been accomplished. it has been established that several species have a wide range of bioactivities: antimalarial, larvicidal, antimicrobial, wound healing, laxative, antiasthmatic, hepatoprotective, antidiabetic, antiparasitic, antioxidant, analgesic, anti-inflammatory and anticancer. this makes the genus a hive under pharmacological research. despite the genus portraying a lot of bioactivities, most of the compounds responsible for these bioactivities have not been entirely isolated and their activities verified. additionally, studies on synergistic effects, toxicology, in vivo activities, and clinical trials on isolated compounds with biological potency have not been done effectively. unfortunately, this has been a setback in discovering new drugs that could have solved drug resistance issues and side effects. regardless of all these challenges, some countries use dosage forms of cassia, including senna, under different brand names [12]. some compounds isolated from cassia species, such as sennosides, are currently being used as allopathic laxative medicine with a prescribed dosage, while others, such as ononitol monohydrate and chrysophanol, are still under clinical assessment as hepatoprotective agents. this indicates osunga et al. their ethnobotany, potency and prospects of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 56 the potential of the isolated compounds as leads for new drug discovery as most conventional drugs are currently unaffordable; hence, the genus cassia species continues to serve communities as a cheap drug source. they are assumed to be safe even though many side effects have been reported from herbs, including interactions with allopathic medicines and herbs, preparations, contaminations, allergic reactions, transformed food consumption, distorted body and organ weights [211,212]. hence, further toxicological studies, dosage determination, and regimes are imperative to avoid side effects and complicated conditions in these herbal preparations. it is expected that this review will stimulate more research for the elucidation and isolation of bioactive compounds from the genus leading to more new drug discoveries for the sake of humanity. references 1. busia k. medical provision in africa past and present. phyther res. 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of drug discovery vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 62 207. sikolia sf. medicinal plants of kakamega forests and their consistency applications : opportunities and challenges to-date . iosr j pharm biol sci (iosr-jpbs. 2018, 13:48–54. 208. queensland department of agriculture and fisheries. invasive plant risk assessment: hairy sicklepod senna hirsuta. 2016. 209. hidayati e, wardani i, susyanti d, et al. research journal of pharmaceutical , biological and chemical sciences antimicrobial assay and gc-ms analysis of leaves extracts medicinal plant senna hirsuta ( l .). res j pharm biol chem sci. 2020, 11:215–9. 210. agbafor kn, nwaka ac, dasofunjo k, et al. glucose-6phosphate dehydrogenase activity in albino rats treated with aqueous extract of fresh leaves of morinda lucida. ©idosr publ int digit organ sci res. 2017, 2:10–7. 211. ogunbanjo o, onawumi o, gbadamosi m, et al. chemical speciation of some heavy metals and human health risk assessment in soil around two municipal dumpsites in sagamu, ogun state, nigeria. chem speciat bioavailab. 2016, 28:142–51. 212. zeng x, xu x, boezen hm, et al. children with health impairments by heavy metals in an e-waste recycling area. chemosphere. 2016, 148:408–15. 10.1016/j.chemosphere.2015.10.078 how to cite this article: salome osunga, omari amuka, alex k. machocho, albert getabu, martin o. onani. some members of genus cassia (senna): their ethnobotany, potency and prospects of drug discovery. indian j pharm drug studies. 2023: 2(2) 41-62. funding: none conflict of interest: none stated gaonkar s et al. pharmacology review on brassica oleracea var.italica sprouts vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 103 review article a brief review on pharmacological potential of brassica oleracea sprouts sonia gaonkar1, ashoka shenoy m2, a r shabaraya3 from 1pg scholar,, 2assistant professor, department of pharmacology, 3principle and head of the department of pharmaceutics, srinivas college of pharmacy, valachil, post farangipete, mangalore, karnataka, india-574143. abstract broccoli (brassica oleracea) is an important vegetable crop of the family are essential to maintain quality. glucosinolate levels mirror visual quality in broccoli as they generally decrease during post-harvest handling, with low temperatures (<4 °c) slowing the loss of both quality and glucosinolates. glucosinolates content in broccoli florets declined by 82% after 5 days at 20 °c, but by only 31% at 4 °c. a high relative humidity (rh) of 98–100% is recommended to maintain post-harvest quality in broccoli. controlled atmosphere (ca) storage is very effective in maintaining broccoli quality and can double post-harvest life. ideal atmospheres to maintain quality were 1–2% o2; 5–10% co2 when temperatures were kept between 0 and 5 °c. care needs to be taken that o2 does not drop below 1% as this can cause the development of off-order. broccoli is a vegetable with a high nutrient content in brassica oleracea. it possesses a wide range of nutrients, including vitamins a and k, antioxidants, βcarotene, calcium, riboflavin, and iron, as well as phytochemicals such as phenols, flavonoids, glucosinolates, minerals, and selenium. the biological abilities of these defense-related secondary metabolites, such as their anti-diabetic, anti-carcinogenic, anti-inflammatory, and antioxidant activities, have been favorably connected. the anti-cancer and antioxidant properties of broccoli sprouts have drawn the most interest and have been the subject of substantial research in recent years. key words: botanical description, biological abilities, controlled atmosphere storage, chemical constituents, phytochemical, relative humidity. bout cauliflower, the history, and evolution of broccoli are examined, as well as how it has changed over time to include both annual and biennial varieties [1]. china and india produced the majority (19.7 million tonnes) of this vegetable in 2019, accounting for 73% of global output, while the united states, spain, and mexico each contributed about 1 million tonnes [2]. the bioactive chemicals found in broccoli are thought to be responsible for many of the vegetable's functional qualities. these include functions that regulate enzymes, cell cycles, apoptosis, antioxidant activity, and other processes [3]. isothiocyanate, an organic phytochemical substance generated after glucosinolates were hydrolyzed by an enzyme, is one of broccoli's components that promotes health. in addition, broccoli contains minerals like iron, access this article online received – 09th june 2023 initial review – 17th june 2023 accepted – 21st june 2023 quick response code zinc, and selenium, vitamins like ascorbic acid and tocopherol, and polyphenols like kaempferol and quercetin. these substances probably play a role in lowering the risk of degenerative illnesses including cancer and cardiovascular conditions [4, 5]. in addition to being a great source of fiber, broccoli also has a lot of other nutrients, including a lot of water (89%), carbs (6.27%), protein (2.57%), lipids (0.34%), ash (0.83%), and the aforementioned vitamins and minerals (1%). despite the nutritional benefits of broccoli, people with developed palates prefer to consume it [6]. the sulfur-containing molecules are thought to produce a strong odor and unpleasant taste. however, these substances have demonstrated antitumor action [4]. table 1 depicts the vernacular names of brassica oleracea and table 2 elaborates its taxonomical classification. ________________________________________ correspondence to: sonia gaonkar, pg scholar, department of pharmacology, srinivas college of pharmacy, valachil, post farangipete, mangalore, karnataka, india. email: soniangaonkar@gmail.com a mailto:soniangaonkar@gmail.com gaonkar s et al. pharmacology review on brassica oleracea var.italica sprouts vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 104 table 1: vernacular names of brassica oleracea [7] languages name english broccoli sanskrit kapishank kannada kosugadde malayalam breakkeali hindi brokolee table 2: taxonomical classification of brassica oleracea [7, 8] kingdom plantae order brassicales family brassicaceae genus brassica species brassica oleracea habitat brassica oleracea, or broccoli, is a highly prized hardy biennial crop produced as a cool-season annual. although broccoli may be grown in a variety of soil types, choosing the right soil unintentionally has an impact on how the plant grows. as porous and well-drained soil encourages higher root penetration, impacting broccoli development, sandy and silt loam soils with a ph range of 5.5–6.5 are often favoured for the growth of broccoli. a cool-weather crop, broccoli is ready for harvest before daily highs reach over 75°f [8]. the head is chopped with shears around 25 mm (1 in) from the tip. broccoli should be pluked before the blossoms on the head bloom brilliant yellow [9]. distribution beginning in the northern mediterranean region in the sixth century bc, land-race brassica crops were crossed to produce broccoli. the roman empire produced early varieties of broccoli, which were later enhanced by artificial selection in sicily or the southern italian peninsula. by the 18th century, broccoli had reached northern europe, and italian immigrants had brought it to north america in the previous century. the most widely used cultivars since then are "premium crop," "pack-man," and "marathon," which were created via the breeding of american and japanese f1 hybrids during world war ii to boost yields, quality, growth speed, and geographical adaptability [10, 11]. botanical description: the cabbage family's (family brassicaceae, genus brassica) enormous blooming head, stalk, and little accompanying leaves are all consumed as vegetables in the form of broccoli. large, typically darkgreen flower heads on a thick, typically light-green stalk are the distinguishing feature of broccoli. these flower heads are arranged in a tree-like structure. leafy bushes around the multitude of flower heads [12]. broccoli buds: the vegetable broccoli is a flower. a crown of broccoli has several small flower buds that have not yet blossomed or opened out. a bud is each of the little, green pieces of the crown. before it blooms, broccoli is picked so that the little buds can be eaten. the adorable buds will eventually bloom into little yellow flowers [13]. broccoli crown: broccoli develops on a sturdy, mediumheight stem. it is cut at the base of the stalk when taken from fields or gardens. the portion that is left over is known as the broccoli's head or crown. the tops (heads) of the broccoli stem, which are made up of the florets, steam, and other components, are known as broccoli crowns. in essence, it is the entire stalk, excluding the actual stalk [13]. broccoli florets: it is a short segment of broccoli buds that have been cut off from the crown [13]. broccoli leaf: compared to a typical head of broccoli, broccolini or broccoli rabe often have more leaves. broccoli leaves are the plant's most delicate and brittle component [13]. broccoli stalk: the stem of a plant is called a stalk. also, the stem of a broccoli plant is referred to as the stalk. the area between the stem and the florets with broccoli bud-filled florets is called the stalk [13]. broccoli stem: the component of the broccoli plant that sprouts from the soil is the stem. it is substantial and ultimately divides into distinct stalks that emerge from the stem's base. as the stem is rough, most people chop it off and don't eat it [13]. chemical constituents: the typical broccoli phytochemical is sulfur-containing compounds, including glucosinolates, hydroxycinnamic acids (sinapic and caffeoyl-quinic\acid derivatives), minerals (calcium, phosphorus, potassium, sodium), vitamins (b, c, e, k), \fibers, indoles, flavonoids (kaempferol), glucoraphanin, smethyl cysteine, sulfoxide \isothiocyanates, and indole-3carbinol, etc [14]. pharmacological activities anti-amnesic activity: researchers tested amyloid beta [a]-induced neurotoxicity in vitro and in vivo to determine the anti-amnesic properties of broccoli [brassica oleracea var. italica] leaves. a notable neuronal cell-protective effect and acetylcholinesterase [ache] inhibition were demonstrated by the broccoli leaf chloroform fraction (cbl). the y-maze, https://en.wikipedia.org/wiki/brassica_oleracea gaonkar s et al. pharmacology review on brassica oleracea var.italica sprouts vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 105 passive avoidance, and morris water maze tests were used to assess the ameliorating impact of cbl on a1-42-induced learning and memory deficits. the findings showed that the cbl group's cognitive performance was improving. as a result, our research suggests that cbl may be exploited as a natural resource to treat learning and memory problems brought on by a1-42 [15]. anti-oxidant activity: samples made from fresh broccoli sprouts using freeze-drying or water distillation were tested for antioxidant activity. antioxidant activity that was dosage dependent was present in all samples. the maximum dpph radical scavenging activity was found in the broccoli methanol extract. both samples of dichloromethane extract from broccoli sprout water distillate and freeze-dried broccoli sprouts had a strong antioxidant activity that was equivalent to bht. it is well known that these isothiocyanates have antioxidant qualities. the compounds 4-(1-methyl propyl) phenol (0.012 g/g), 4-methyl phenol (0.159 g/g), and 2-methoxy-4-vinylphenol (0.009 g/g) may be phenolic antioxidants. the current study shows that broccoli sprouts are an excellent source of natural antioxidants [16]. anti-diabetic activity: after 28 days of therapy, type 2 diabetic hfd-stz rats with the plant's ethanolic extracts of brassica oleracea var. italica showed clear antihyperglyce mic capabilities. these effects were tested in stz-induced type 2 diabetic sd rats at dosages of 800 mg/kg [17]. anti-cancer activity: in addition to being effective in treating other diseases, broccoli plays a significant and advantageous role in treating cancer in its various forms that can occur in various parts of the body. uncontrolled cell division is the end consequence of a multi-step process called cancer. the primary active components of many cruciferous vegetables, isothiocyanates, inhibit the development of tumors by producing reactive oxygen species or by causing cycle arrest that results in cell apoptosis. broccoli sprouts with added selenium are superior to regular broccoli sprouts in that they cause prostate cancer cells to die, restrict cell growth, and secrete less prostate-specific antigens. phytoch emicals offer significant promise for preventing cancer given their degree of safety, cost, and oral absorption [18]. anti-obesity activity: obesity has emerged as a major global health issue and is a precursor to harmful metabolic illnesses including type 2 diabetes and cardiovascular disease. according to a few studies, broccoli sprouts reduce obesity by modulating lipid metabolism. in double-blind clinical research, the use of broccoli sprout powder as adjunctive therapy in type 2 diabetes patients may have positive effects on lipid profiles and the ox-ldl/ldl ratio, risk factors for obesity, and cardiovascular disease. based on a recent study, glucoraphanin from broccoli sprouts can boost nrf2 activation and reduce lipid build-up. it results in the expression of several genes involved in gluconeogenesis and lipogenesis being down regulated, which reduces obesity [19]. anti-microbial activity: according to a study, broccoli sprouts high in gallic acid, esculetin, ferulic acid, and myri cetin have antibacterial activity against foodborne pathogens like salmonella typhimurium and escherichia coli as well as gram-positive and gram-negative bacteria with minimum inhibition concentrations (mic) ranging from 390 to 1560 g/ml. similar to previously reported findings from other plant extracts, this study found that gram-positive bacteria were more susceptible to broccoli sprout extract than gramnegative bacteria. these findings indicate that broccoli sprout extracts might serve as a potential source of antimi crobial agents in the food and pharmaceutical industry [20]. anti-inflammatory activity: several studies proved that broccoli sprouts and their bioactive components possess antiinflammatory activity. using broccoli sprouts and microgreens might help prevent and cure inflammatory bowel conditions including crohn's disease and ulcerative colitis. the main anti-inflammatory effects of broccoli sprouts have been demonstrated in both in vitro and in vivo studies as well as clinical trials. these studies found that broccoli sprouts had high concentrations of sulforaphane and a group of phenolic compounds, such as anthocyanins, isoquercitrin, chlorogenic, and cinnamic acids, which inhibited inflammatory mediators like nitric oxide (no), decreased levels of proinflammatory cytokines. moreover, type 2 diabetes individuals' blood levels of high-sensitive c reactive protein (hs-crp), il-6, and tnfwere shown to be lower after eating broccoli sprouts rich in sulforaphane [21]. hepatoprotective activity: in-depth research has been done on the preventative and therapeutic benefits of broccoli extracts on paracetamol-induced liver injury in rats. according to histological research and the measurement of biochemical indicators, broccoli extracts exhibit protective effects against liver injury. three chemicals, obtucarbamate, n-(4-hydroxy phenyl) acetamide, and p-hydroxy benzoic acid, were isolated by phytochemical analysis of an 80% ethanolic extract and are suitable candidates for the prevention of liver damage brought on by paracetamol [22]. conclusion brassica oleracea belonging to the family brassicaceae is widely accepted for its traditional medicinal uses and pharmacological potential. various parts of brassica oleracea have reported the presence of several gaonkar s et al. pharmacology review on brassica oleracea var.italica sprouts vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 106 phytochemicals such as glucosinolates, dithiolthiones, indoles, glucoraphanin, s-methyl cysteine sulfoxide, isothiocyanates, indole-3-carbinol, and other constituents. brassica oleracea revealed various pharmacological activities such as anti-microbial, anti-inflammatory, antidiabetic, anti-oxidant, anti-cancer, anti-obesity, and hepatoprotective activity. every part of the broccoli plant possesses medicinal and nutritional value, with numerous other applications. further scientific investigation on different parts of brassica oleracea must be undertaken to unveil additional pharmacological properties. reference 1. gray ar. taxonomy and evolution of broccoli (brassica oleracea var. italica). 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el-shabrawy ar, et al. hep atoprotective activity of brassica oleracea l. var. italica. egyptian pharmaceutical journal. 2013 jul 1;12(2):177. how to cite this article: sonia gaonkar, ashoka shenoy m, a r shabaraya. a brief review on pharmacological potential of brassica oleracea sprouts. indian j pharm drug studies. 2023: 2(3) 103-106 funding: none conflict of interest: none stated ghuman et al. alternative medicine in dentistry vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 156 review article application of alternative medicine as a curative therapy of oral mucosal lesions: a comprehensive review annayat ghuman1, mehar ghuman2, pankaj gupta3 from, 1bds final prof, shaheed kartar singh dental college, 2pg resident pathology, dmc ludhiana, 3private dental practitioner at elite dental clinic, udhampur, india. abstract the term alternative medicine is used to denote a form of medicine that falls outside the mainstream of western medicine. these medicines exist in all cultures to some degrees and terms such as traditional medicines, indigenous medicines, holistic medicines, folk medicines and oriental medicines. when such medicines and related therapies are applied in the dental field, the term holistic dentistry is used. alternative medicines or therapy encompasses a variety of disciplines such as acupuncture, chiropractic treatment, guided imagery, yoga, biofeedback, hypnosis, aromatherapy, herbal remedies, relaxation, massage etc. there is common patient belief that these therapies are safe to use. however, there is lack of sufficient scientifically proven data regarding their safety. the emphasis with most alternative therapies is based upon the natural healing capability of the body. this article emphasis the use of various alternative therapies, their advantages, mechanism of actions, side effects and implications in dentistry. key words: alternative medicine, anti-oxidant, herbal medicines, holistic, oral mucosal lesions. lternative therapy is a generalized term applied to all methods of treating a specific disease or condition. this therapy aims at co-operating with the natural forces and various defensive mechanism of the body against pathogens. world health organization (who) has identified more than 150 systems of alternative medicines. most practiced ones are :ayurveda, homeopathy, naturopathy, unani medicine, magneto therapy, aroma therapy, acupuncture, yoga, biofeedback training, mud therapy, massage therapy etc. complementary and alternative medicine (cam), according to national center for complementary and alternative medicine (nccam), represents a group of diverse medical and health care systems, practices, and products that are not presently considered to be part of the conventional medicine. access this article online received – 25th nov 2023 initial review – 15th dec 2023 accepted – 28th dec 2023 quick response code complementary medicine is the one used together with conventional medicine. example: using aromatherapy to help reduce patient’s discomfort following surgery. alternative medicine is the one used in place of conventional medicine. example: using a special diet to treat cancer instead of undergoing surgery, radiation, or chemotherapy that has been recommended by a conventional doctor. nccam classifies cam therapies into five categories [1,2]. 1. alternative medical systems: these systems are built as a result of various theories and consequently practice and expertise. they are evolved apart from and earlier than the conventional medical approach in treating various diseases. these systems are divided into therapies that fall into western culture (homeopathic and naturopathic medicine) and non-western cultures (traditional chinese medicine and ayurveda) [1,2]. _________________________________________________ correspondence to: pankaj gupta, mds-oral medicine and radiology, shakti nagar, udhampur, j&k, india. email: pankajmds2807@gmail.com, tel.: +91 7006270818 a mailto:pankajmds2807@gmail.com ghuman et al. alternative medicine in dentistry vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 157 2. mind-body interventions: the connection between the mind and the body uses a variety of techniques designed to enhance the mind’s capacity to affect bodily function and symptoms. example: meditation, prayer and therapies that use creative outlets such as art, music, or dance [2]. 3. biologically based therapies: these therapies use natural substances such as herbs and vitamins to cure a disease. example: dietary supplements, herbal products and the use of other natural but as yet scientifically unproven therapies (exampleusing shark cartilage to treat cancer) [2]. 4. manipulative and body-based methods: these methods are based on manipulation and/or movement of one or more parts of the body. example: chiropractic or osteopathic manipulation and massage [1]. 5. energy therapies energy therapies involve the use of energy fields. they are of two types: a) bio-field therapies: the bio-field therapies are intended to affect energy fields that surround and penetrate the human body. however, the existence of such fields has not yet been scientifically proven. some forms of energy therapy manipulate bio-fields by applying pressure and/or manipulating the body by placing the hands in, or through, these fields. examples include qigong, reiki and therapeutic touch [2] b) bio-electromagnetic based therapies: unconventional use of electromagnetic fields such as pulsed fields, magnetic fields, or alternating-current or direct-current fields [2]. the use of alternative medicine aims at prevention of a disease whereas traditional medicine tends to intervene once disease has occurred. alternative medicines when compared with traditional medicine have lesser side effects and are less expensive, since most of them are manufactured using plant extracts. however, there is very little scientifically proven data regarding their safety when compared to traditional medical methods [3]. alternative therapies commonly used in management of oral mucosal lesions. aloe vera the name aloe vera derives from the arabic word “alloeh” meaning “shining bitter substance,” while “vera” in latin means “true.” the botanical name of aloe vera is aloe barbadensis miller. it belongs to liliaceae family. constituents of aloe vera 1. vitamins: it contains antioxidant such as a, c and e. it also contains vitamin b12, folic acid, and choline. 2. enzymes: it contains enzymes such as: alkaline phosphatase, amylase, bradykinase, carboxypeptidase, catalase, cellulase, lipase, and peroxidase. bradykinase helps to reduce excessive inflammation when applied to the skin topically, while others help in the breakdown of sugars and fats. 3. minerals: it contains calcium, chromium, copper, selenium, magnesium, manganese, potassium, sodium and zinc. they are essential for the proper functioning of various enzyme systems in different metabolic pathways. 4. sugars: it contains monosaccharides (glucose and fructose) and polysaccharides (glucomannans, acemannan). 5. anthraquinones: it has12 anthraquinones, which are phenolic compounds traditionally known as laxatives. aloin and emodin act as analgesics, antibacterials and antivirals [3]. 6. fatty acids: it provides 4 plant steroids; cholesterol, campesterol, β-sisosterol and lupeol. all these have antiinflammatory action and lupeolin addition possesses antiseptic and analgesic properties [3] mechanism of action a) healing properties: glucomannan, a mannose-rich polysaccharide, and gibberellin, a growth hormone, interacts with growth factor receptors on the fibroblast, thereby stimulating its activity and proliferation, which in turn significantly increases collagen synthesis [3]. b) effects on skin exposure to uv and gamma radiation: topical application of aloe vera in gel form result in generation of metallothionein (antioxidant) that scavenges hydroxyl radicals and prevents suppression of glutathione peroxidase and superoxide dismutase. this in turn decreases the production and release of il-10 and thus prevents uv-induced suppression of delayed type hypersensitivity (figure 1). c) anti-inflammatory action: aloe vera inhibits the cox pathway and reduces pge2 production from arachidonic acid. d) effects on the immune system: alprogen inhibits calcium influx into mast cells, thereby inhibiting the antigen-antibody mediated release of histamine and leukotriene from mast cells. e) antiviral and antitumor activity: these actions may be due to indirect or direct effects. direct effect is due to anthraquinones. the anthraquinone inactivates various ghuman et al. alternative medicine in dentistry vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 158 enveloped viruses such as herpes simplex, varicella zoster and influenza while indirect effect is due to stimulation of the immune system.3an induction of glutathione s-transferase and an inhibition of the tumorpromoting effects of phorbolmyristic acetate has also been reported which suggest a possible benefit of using aloe gel in cancer chemoprevention [4]. indications in oral mucosal lesions: aloe vera may be indicated in the management of recurrent aphthous stomatitis, lichen planus, radiation mucositis, osmf and leukoplakia. a) oral lichen planus: a randomized controlled trial study was conducted to check the efficacy of aloe vera gel in the treatment of oral lichen planus (olp). the authors concluded that aloe vera gel is statistically more effective than placebo in inducing clinical and symptomatic improvement of olp. therefore, aloe vera gel can be considered as a safe and alternative treatment for patients with olp [5]. b) oral submucous fibrosis: a study was conducted in 2012 to compare the efficacy of antioxidants and aloevera in treatment of osmf, where the subjects under the aloe vera group showed a better treatment response compared to the antioxidants group. it reduced the burning sensation and also improved mouth opening [6]. c) recurrent aphthous stomatitis: in a study the authors conducted a double-blind clinical trial to evaluate the efficacy of topically administered aloevera gel on minor aphthous ulcers. it was concluded that 2% aloevera oral gel was not only effective in decreasing the patient's pain score and wound size, but also decreased the wound healing period [7]. side effects: topical application may cause redness, burning, stinging sensation and rarely generalized dermatitis in sensitive individuals. allergic reactions are mostly due to anthraquinones such as aloin. when administered orally it may cause abdominal cramps, diarrhea, constipation, red urine and hepatitis. figure 1: showing the possible effects of topical application of aloe vera gel on skin. curcumin curcumin (diferuloylmethane) is the chief component of the spice turmeric and is derived from the rhizome of the east indian plant curcuma longa.turmeric contains a class of compounds known as the curcuminoids, comprised of curcumin, demethoxycurcumin and bisdemethoxycurcumin. curcumin has anti-oxidant, antiseptic, analgesic, antimalarial and anti-inflammatory properties. 1. antioxidant activity: curcumin has been shown to inhibit lipid peroxidation using linoleate, a polyunsaturated fatty acid that can be oxidized to form a fatty acid radical. it also scavenges various reactive oxygen species (ros) produced by macrophages (superoxide anions, hydrogen peroxide and nitrite radicals) [8]. it also down regulates the nitric oxide synthase (inos) activity in macrophages, thus reducing the amount of ros generated in response to oxidative stress [9]. 2. anti-inflammatory activity: curcumin supresses the activation of nf-kb, an inducible transcription factor that regulates the expression of a host of genes involved in the inflammation such as cox-2, tnf-a, cyclin –d1, icam-1 [10]. indications a) recurrent aphthous stomatitis: eight studies involving 439 subjects were reviewed. the efficacy of curcumin ghuman et al. alternative medicine in dentistry vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 159 was compared with 1% triamcinolone in four studies, glycerin vehicle in one study, placebo in one study, and honey in one study. overall, these studies reported a good efficacy of curcumin in reducing pain and ulcers size in patients with ras [11]. b) precancerous lesions: curcumin has a crucial role in the management of precancerous conditions like lichen planus, oral submucous fibrosis and leukoplakia. various in vitro and in vivo animal experiments activities of turmeric extract oil have demonstrated that symptoms of pain and burning sensation declined and mouth opening was also partially reversed [11]. dosages: doses of 500-8,000 mg of powdered turmeric per day have been used in human studies. standardized extracts are typically used in lower amounts in the 250-2,000 mg range [12]. adverse effects: may cause gastric irritation, stomach upset, nausea, diarrhea, allergic skin reaction, and anti-thrombosis activity interfering with blood-clot formation in some patients. allicin the word allicin is derived from the latin name of the garlic plant, allium sativum. 1. anti-microbial action: allicin has been reported to inhibit the growth of gram positive, gram-negative, and acid-fast bacterium, including multidrug-resistant bacterium especially the oral bacteria by its chemical reaction on thiol groups of various enzymes in vitro [13]. it also possess some degree of immunomodulatory effect by increasing the activity of natural killer cell. it inhibits tnf-alpha -induced secretion of il-1, il-8, ifn-ɣ in a dose dependent manner. it also suppresses the expression of il-8 and il-1 [14]. 2. indications a) recurrent apthous ulcer: oral adhesive tablets (5 mg allicin): it adhere to the oral mucosa and release allicin slowly at the site of the ulcer in 3 to 4 hours. in this way, a higher drug concentration and a longer-lasting release period could be achieved at the affected site to increase the efficacy [15]. side effects-increases bleedingshould be stopped 2 weeks before surgery. it can irritate gi tract -should be used in caution in patients with digestive problem. it was also found to decreases blood pressure in some patients [16]. green tea green tea is made from un-oxidized leaves and is one of the less processed types of tea and therefore contains one of the most amounts of antioxidants and beneficial polyphenols mainly catechins. constituents: catechins (30 %), caffeine, vitamins (a, b2, e, folic acid, b-carotene), saponins, ɣaminobutyric acid, minerals (potassium, calcium, phosphorus, manganese) and chlorophyll. actions 1. antioxidant: it has antioxidant activity either directly by scavenging of reactive oxygen and nitrogen species or indirectly by inhibition of redox sensitive transcription factors, and induction of antioxidant enzymes [17]. 2. antitumor effect: egcg (epigallocatechingallate) has shown to induce apoptotic cell death and cell cycle arrest in tumor cells [18]. 3. antimicrobial activity: catechins constitute the most important antibacterial agents on methicillin resistant staphylococcus aureus, helicobacter pylori and αhemolytic streptococcus [19]. indications in dentistry a) oral lichen planus: green tea, especially epigallocatechin-3-gallate, possesses anti-inflammatory and chemopreventive properties. it can inhibit antigen presentation, t-cell activation, proliferation and migration, keratinocyte apoptosis, nuclear factor-kappab (nf-кb) activation and mmp-9 activity and can modulate the imbalance between tgf-β and interferon-γ signaling, all of which are involved in the pathogenesis of olp [20]. b) candidiasis: these results indicate that egcg enhances the antifungal effect of amphotericin b or fluconazole against antimycotic-susceptible and resistant c. albicans. combined treatment with catechin allows the use of lower doses of antimycotics and induces multiple antifungal effects [21]. side effects: drinking more than 5 cups a day is unsafe. it can cause side effects because of the caffeine. these side effects can range from mild to serious and include headache, nervousness, sleep problems, vomiting, diarrhea, irritability, irregular heartbeat, tremor, heartburn, dizziness, ringing in ghuman et al. alternative medicine in dentistry vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 160 the ears, convulsions, and confusion [22]. it increases the excretion of calcium in the urine leading to osteoporosis. caffeine should be limited to less than 300 mg per day (2-3 cups) [22]. honey honey, a thick sweet liquid made by bees from the nectar of flowers, is one of the oldest known medicines. properties a) hygroscopic properties: this effect is based on high osmotic properties so it can extract water from bacterial cells and cause them to die. it has been shown that wounds infected with staphylococcus aureus are quickly rendered sterile by honey b) acidic ph: honey is characteristically quite acidic, its ph being between 3.2 and 4.5, which is low enough to be inhibitory to many animal pathogens c) increased lymphocyte and phagocytic activity: honey (at a concentration of 1%) stimulates monocytes in cell culture to release cytokines like tumour necrosis factor (tnf)-alpha, interleukin (il)-1 and il-6, which activate the immune response to infection [23]. uses of honey a) recurrent apthous stomatitis: in a study, honey application can significantly reduce inflammation, accelerate healing process and increase pain free days in those lesions with resolution of the erythema or ulceration without need for steroid therapy. the mean healing period in ras was 4 days which is lower than the previous reported studies.24in the same study, application of honey in the prodromal period, in one case, accelerated healing process with resolution of erythema and tingling sensation and prevented vesicular eruption. when it was applied after eruption of vesicles, in other cases, there was rapid resolution of the vesicles with no crust formation within 8 days [24]. b) denture stomatitis: a study was conducted to evaluate the clinical efficacy of a new brazilian propolis gel in the treatment of denture stomatitis. the results of the study showed that all patients treated with brazilian propolis gel and daktarin had complete clinical remission of palatal edema and erythema [25]. c) anti-tumor effects of honey: a study had reported that sangju honey (simdanjeong, sangju, korea) could suppress cellular migration, metastasis, and cell proliferation in oral squamous cell carcinoma (oscc). the study results further indicated that sangju honey could also induce apoptosis and suppress oscc growth (figure 2) [26]. wang et al. studied the anti-mutagenic effects of different types of honey against a commonly encountered dietary mutagen trp-p-1 and found that all honeys exhibited significant inhibition of mutagenicity against this compound [27]. a study done on tumour development and metastasis in murine tumour models using various honey-bee products showed an important role in controlling tumour growth and metastasis in mammary carcinoma and a methylcholanthrene-induced fibrosarcoma in mouse [28]. figure 2proposed mechanisms underlying inhibition of oral cancer cell growth by sangju honey. spirulina spirulina, or arthrospira platensis, is blue green algae which appear as long, thin, spiral threads. spirulina contains a rich supply of protein (60-70 % dry weight), carbohydrates, iron, vitamins (a, k and b complex), carotenoids (β carotene, zeaxanthin, cryptoxanthin, phycocyanin, lutein, xanthophylls. the nutrients present in ghuman et al. alternative medicine in dentistry vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 161 spirulina boost the immune system and enhance the body's ability to generate new blood cells to prevent disease and cancer [29]. mechanism of action β-carotene has antioxidant and anti-inflammatory activities – it inhibits the production of nitric oxide and pge2 and suppresses the expression of cox-2, tnf-α and il-1. phycocyanin inhibits proinflammatory cytokine formation of tnfα, suppresses cox-2 expression and decreases pge2 production [30]. indications in dentistry a) oral submucous fibrosis: a study was conducted on a group of patients with osmf. the subjects were given spirulina 500 mg along with triamicolone 0.1%. after the end of 3 months the subjects showed significant improvement in burning sensations, mouth opening and painful ulcerations [31]. b) leukoplakia: the evaluated the chemopreventive activity of spirulina fusiformis (sf) (1 g/day for 12 mos) in reversing oral leukoplakia in pan tobacco chewers in kerala, india. complete regression of lesions was observed in 20 of 44 (45%) evaluable subjects supplemented with sf, as opposed to 3 of 43 (7%) in the placebo arm (p < 0.0001) [32]. c) homeopathy: the term homeopathy comes from the greek word homoios, meaning similar, and pathos, meaning suffering or sickness. the basic law of homeopathy is "the law of similars." the law states that disease represents a disturbance in the body’s ability to heal itself. “remedies” are determined by noting the symptoms produced by large doses of a substance in a healthy individual and applying these substances in highly diluted doses to relieve the same symptoms [33]. d) ignatia: it is a homeopathic medicine that is derived from the bean of a small tree that is native to the philippine islands and china. the tree belongs to the loganiaceae family, and has long, twining, smooth branches. ignatia homeopathic 30c in management of oral lichen planus (olp). a single blind randomized control clinical trial was done on 30 consecutive patients with oral lesions consistent clinically and histologically with erosive and/or atrophic olp were recruited. the patients were randomly divided into two groups to receive ignatia or placebo. they were treated for 4 months.mean lesion sizes and mean pain measures differed between control and treatment groups favouringignatia [34]. conclusion the complementory and alternative medicines along with various other extracts from the plants together along with main stream of western medicines can be used as preventive or treatment modalities for oral mucosal and precancerous lesions. the dental health care workers needs to be informed regarding the use of herbal products that may have an impact the delivery of safe and effective dental treatment. in addition, the use of such treatments in dentistry should be based on evidence of effectiveness and safety as demonstrated in randomized clinical trials. references 1. little jw. complementary and alternative medicine: impact on dentistry. oral surg oral med oral pathol oral radiolendod. 2004;98:137-45 2. who, national policy on traditional medicine and regulation of herbal medicines report of a who global 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2002;50(23):6923-6928. 28. orsolic n, terzic s, basic i. honey-bee products in prevention & therapy of murine transplantable tumours. j sci food agric. 2005;85:363-370. 29. mathew b, sankaranarayanan r, nair pp et al. evaluation of chemoprevention of oral cancer with spirulina fusiformis. nutr cancer. 1995;24:197-202. 30. deng r, chow t-j. hypolipidemic, antioxidant and antiinflammatory activities of microalgae spirulina. cardiovascular therapeutics. 2010;28(4):33-45. 31. revant c, ranjit p. clinical improvement in oral submucous fibrosis with spirulina: a newer drug. j oral sign. 2013;5(2):61-64. 32. mathew b, sankaranarayanan r, padmanabhan pet al. evaluation of chemoprevention of oral cancer with spirulina fusiformis. nutr cancer. 1995;24(2):197-202. 33. shinde md, patil sa, mukherjee a, et al. revisiting the therapeutic potential of homeopathic medicine rhustox for herpes simplex virus and inflammatory conditions. j ayurveda integr med. 2023;14(4):100739. 34. mousavi f, sherafati s, mojaver yn. ignatia in the treatment of oral lichen planus. homeopathy. 2009; 98(1):40-4. how to cite this article: annayat ghuman, mehar ghuman, pankaj gupta. application of alternative medicine as a curative therapy of oral mucosal lesions: a comprehensive review. indian j pharm drug studies. 2023; 2(4):156-162. funding: none conflict of interest: none stated shirsath et al. covid-19 survey vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 63 perspective life –style and behavioural changes after covid-19: a perspective nitin rajendra shirsath1, rohit shitole2, vaibhavkumar jagtap1, abhay ramling sagare3, ajaygiri goswami4 from, 1department of pharmaceutics, gangamai college of pharmacy, nagaon, dhule, 2hr patel institute of pharmaceutical education and research, karwand naka, shirpur, 3ies institute of pharmacy, ies university, bhopal, madhya pradesh, 4university institute of chemical technology (uict), kavayitri bahinabai chaudhari north maharashtra university (kbcnmu), jalgaon, maharashtra, india. abstract background and aims: by may 18th 2020, india investigated 1 lakh infected cases from covid-19, and as of 11th july 2020, the cases equaled 8 lakhs. social distancing and lockdown rules were employed in india, which however had an additional impact on the economy, human living, and environment. where a negative impact was observed on the economy and human life, the environment got a positive one. the impact of measures taken to contain covid-19 on lifestyle-related behaviour is undefined in the indian population. methods: the current study was undertaken to assess the impact of covid-19 on lifestyle-related behaviours. an exploratory and anonymous general population-based google survey was conducted among 1000 population. herein, the data for the survey was collected through a well-structured questionnaire and the channel of data collection was google forms. in brief, the study was conducted among the citizens of the dhule district, north maharashtra region, maharashtra, india. results: based on the survey, the participants willingly participated in the study. the majority of participants have seen changes in their life after covid-19. the participants gave priority in their life to both family and health and connections and wealth according to the survey. many participants learn that we need to take mental health seriously to take care of ourselves and devote more time to ourselves. conclusions: there have been too many changes are observed in human life style as a result of covid-19. keywords: covid-19, lifestyle, survey, population, behavioural changes he broad family of viruses known as coronaviruses (covs) is prevalent in many different animal species, including camels, cattle, and bats. coronaviruses are infect and spread among humans only infrequently [1]. by the year 2020, covid-19 has fully grown into a pandemic, endangering both the health of everyone and the global economies [2]. the covid-19 virus, which causes the illness, was initially discovered in december 2019 in wuhan. on march 11th, 2020, the world health organization (who) declared covid-19 to be a pandemic. on february 9th, 2021, there were 105.8 million confirmed cases of covid-19, with more than 2.3 million fatalities [3-4]. due of the unusual covid-19 outbreak, nearly all towns, cities, and villages in the affected countries are under partial or complete lockdown for an extended period of time, ranging from a few weeks to a few months [5]. all local and national governments were instructed to close educational institutions and impose limits on citizens' freedom of movement outside of their homes and non-essential enterprises access this article online received – 14th apr 2024 initial review – 11th may 2024 accepted – 28th may 2024 quick response code in order to avoid communal transmission [6]. there will be no significant religious, social, educational, sports, or political activities. undoubtedly, one of the sectors that has been hit the worst overall is the aviation industry [7]. as a result, covid-19 in india has brought the educational system to a standstill [8]. due to the discrepancy in economic conditions among the students in rural india, web and computer systems were not accessible to them during this lockout, which hindered the whole teaching-learning process and educational system [9]. the covid-19 pandemic has significantly impacted social activities in india. large gatherings, festivals, and cultural celebrations have been scaled down or cancelled [10]. personal interactions have shifted to alternative forms of greeting, and public spaces such as parks and theaters have been closed. online platforms have become popular for virtual socializing, while places of worship have implemented restrictions and virtual services. the pandemic has emphasized the importance of public health measures like wearing masks and maintaining physical distance. additionally, communities have come together to provide support and assistance to those in need [11]. ___________________________________________________ correspondence to: nitin rajendra shirsath, department of pharmaceutics, gangamai college of pharmacy, nagaon, dhule, mh, india. email: nitin.shirsath.5293@gmail.com. t mailto:goswamiakg1@gmail.com shirsath et al. covid-19 survey vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 64 these changes highlight the resilience and adaptability of individuals and the collective responsibility towards public health. staying at home during the lockdown also resulted in bad eating and exercise habits, which in turn led to weight gain, diabetes, and an increased risk of cardiovascular disease. similar to some negative health effects, some positive ones were seen [12]. people can experience significant psychological anguish as a result of covid-19 owing to a number of factors, including isolation, fear, uncertainty, and economic unrest. the covid-19 will make poverty, starvation, and hunger in india an even worse problem. people may become frustrated and self-harm or develop chronic stress, anxiety, or depression as a result of mass unemployment [13]. there have been far too many modifications in human life style as a result of covid-19. in this study, we looked at how people's lifestyles and behaviours have changed in the wake of covid-19. materials and methods study setting: the survey was collected through a wellstructured questionnaire and the channel of data collection was google forms. the survey was accompanied within one month i.e. june 2022. in brief, the study was conducted among the citizens of the dhule district, north maharashtra region, maharashtra, india. study population: 1000 participants (both male and female) from dhule district, north maharashtra region, maharashtra, india, were selected for the study. research were disqualified if they were case reports or series, or if they had no connection to the topic at hand. each author took part in choosing which papers fit into the current review's eligibility criteria. people who claimed to have previously completed the online questionnaire were not included in order to prevent duplicate responses. study tools and technique: the study employed a predesigned, pre-tested, and structured questionnaire. an online google form survey link was used to collect the study subjects' data. the google form link was sent just to the primary contacts that lived in the dhule district of north maharashtra area, maharashtra, india, using various social media platforms like whatsapp, facebook, and gmail. a representative from dhule district in the north maharashtra region was invited to complete the questionnaire till 1000 replies (the limit set) were obtained. those who were contacted but declined to take part were regarded as non-respondents. there were 1000 persons in the final sample. methodology the purpose of the study and the nature of the information which had to be furnished by the study subjects were explained to them. this pre-designed, pretested and structured questionnaire included topics which were related to behavioural, educations, their impacts after covid-19 were studied. the survey questions were as follows (a) will you see any changes in yourself after covid19, (b) what type of changes will you see in yourself, (c) what will you prioritize in your life after covid-19, (d) after covid-19 impact of interrupted education was?, (e) what do you think about students’ future, due to covid-19, and (f) what do you learn from the covid19 pandemic? finally, the data were statistically analysed using descriptive statistics whereas the responses to multiple-choice questions were represented using frequency and percentage for every response received. results effect of covid-19 thoughts on covid-19's effects vary from person to person, but similar motifs do show up. it is acknowledged that the pandemic has had a terrible effect on public health, caused disruptions in daily life, and exposed disparities. people re-evaluate their priorities while reflecting on their resilience and adaptability, placing an emphasis on the value of their health, relationships, and work-life balance. there is a desire for positive change, which includes enhanced healthcare systems, addressing disparities, and redoubling efforts to promote sustainability and societal well-being [14]. overall, covid-19 has sparked serious thought and a demand for a future that is more resilient and just. based on the data gathered, a total of 1000 people chose to voluntarily engage in the study by providing their thoughts on the survey questions. therefore, the first question in our survey was, "have you seen any changes in yourself since covid-19?" from total 1000 peoples in dhule district, (figure 1) shows that 87% of participants have seen changes in their life after covid-19 and only 13% of participants did not see any changes in their life after covid-19. there is no doubt that the covid-19 epidemic has altered society on a global scale in all facets of daily life. as a result of widespread school closures and mandated social isolation measures, one significant change involved the remote delivery of education at all levels [15-17]. figure 1: effect of covid-19 post covid-19 changes the globe has experienced substantial changes in many facets of life since covid-19. the use of remote work and online learning is one of the significant topics. virtual platforms have been adopted by many businesses and educational institutions, shirsath et al. covid-19 survey vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 65 enabling staff members and students to work and study from home. the conventional work-life balance has been reinterpreted as a result of this change, and it may change how people work in the future [18]. the greater emphasis on good cleanliness and health habits is another significant development. hand washing, wearing a mask, and keeping a physical distance from others have developed into routines, raising public awareness of health issues and halting the spread of infections other than covid-19. significant changes have also been made to travel and tourism. the tourism sector has changed as a result of international travel restrictions, quarantine procedures, and health protocols [19]. nowadays, people put safety first and go for off-the-beatenpath locations that are less crowded. in addition, the epidemic has increased attention on mental health and wellbeing. many people now understand how crucial it is to take care of oneself, control their stress, and seek expert assistance when necessary. this mentality change could have a long-term effect on how society approaches mental health concerns. overall, with modifications to work dynamics, health practises, travel habits, and mental health priorities, the post-covid-19 era has brought forth a new normal. beyond the initial phase of recuperation, it is unclear how these changes will develop and influence the next years [20-24]. so, we added a new question that what kind of changes will you notice in yourself? as given in (figure 2) the participants saw that about 31 % behavioural changes were observed after covid-19. physical modifications brought about by covid-19 include the placement of safety barriers in public areas, the implementation of temperature checks at entrances, and the accessibility of hand sanitising facilities. these modifications are intended to increase security and lower the possibility of virus transmission in public areas. according to the survey, 30% population were observed the physical changes in his/her lifestyle. people have experienced a variety of emotional changes as a result of covid-19. there have been widespread increases in anxiety, fear, and worry for one's own and one's loved ones' health. figure 2. life style related changes after covid-19 the social exclusion and isolation tactics have made people feel more isolated, depressed, and frustrated [25]. sadness has also been brought on by the epidemic since so many people have lost friends or family members. the overall effect on mental health has been enormous, emphasising the value of resources and support for mental health in these trying times [26]. as per the survey, about 24% peoples were observed the emotional changes in his/her life followed by 15% population seen other changes in his/her life. other changes brought on by covid-19 include a move to remote work and virtual meetings, a reliance on online shopping and delivery services, alterations in travel habits with less tourism, an increase in telemedicine and virtual healthcare consultations, and a focus on personal and public health hygiene [27]. prioritize in your life after covid-19 prioritising particular facets of life is crucial for general wellbeing after covid-19. prioritising physical fitness, emotional wellbeing, and forming healthy habits should all be part of a person's overall health plan. giving value to family and relationships means valuing time spent together and fostering bonds. it is important to prioritise work-life balance so that you have time for your interests and self-care. social and community involvement should be appreciated since it promotes the general welfare and helps local projects. in order to promote resilience and adaptability, it is also important to prioritise personal development and ongoing learning. in order to maintain our planet for future generations, environmental consciousness should be embraced and sustainable decisions should be made [28-29]. lockdowns in response to the covid-19 outbreak have been blamed for an increase in domestic and intimate relationship violence in numerous nations. aggression at home has increased as a result of greater financial insecurity, stress, and uncertainty, and abusers are now able to exert significant control over their victims' everyday lives. a group of people who live as a single household as members of the same family and interact with one another in their individual social roles, typically that of spouses, parents, children, and siblings. everyone's family is a very significant part of their existence [30-31]. prior to covid-19, the people valued material possessions as essential components of existence [32]. after covid-19, when we asked the next question in survey form, "what would you prioritise in your life?" with respect to covid19, our survey reflects that roughly 45% of participants considered the both family and health as on top priority. about 22% of the population gave priority to health. and 24 % and 9 % of population gave priority to family and relations as well as money respectively (figure 3). figure 3: most important things in life shirsath et al. covid-19 survey vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 66 covid-19 impact on education in addition to having an effect on children, instructors, and families, closing schools has significant negative societal and economic effects. the effects were more severe for underprivileged children and their families, disrupting schoolwork, compromising nutrition, creating childcare issues, and costing money to families who were unable to work [33]. due to the closing of schools and universities in numerous jurisdictions, more than one billion young people are currently not physically enrolled in school. the regular academic session has been interrupted by the lockdown. the majority of children in primary and secondary schools are deprived of intellectual connections with their teachers, which has a negative impact on these kids the most [34]. despite the efforts made by teachers, school administrators, local and national governments to deal with the unprecedented circumstances e-learning, the disruption in education and learning could have medium and long-term effects on the quality of education [35]. education in india has been significantly impacted by the switch from face-to-face to online instruction in the majority of educational institutions. while there have been chances for continuing education during the epidemic, there have also been difficulties with online learning. particularly in rural and economically underdeveloped areas, access to technology and internet connectivity continues to be significant barriers. additionally, a student's interest and comprehension may suffer from a lack of face-to-face interaction and practical learning opportunities. to ensure equal access to high-quality education, efforts are being made to close the digital divide and upgrade online infrastructure. however, this transition has brought to light the need for comprehensive strategies to address the particular difficulties that students and teachers face in the online learning environment [36]. higher education had to serve very different purposes in independent india than it did during the colonial era. building a self-sufficient, growth-oriented economy, a united country, and a progressive, equitable society was clearly necessary. the key tool for modernising tradition was rightfully thought to be higher education. educational planners understood the connection between education and development and the production of a literate and skilled labour force [37-38]. the school system was primarily affected by covid-19. according to the majority of participants (figure 4), when asked whether the pandemic has caused an eruption in the educational pattern, because of some online lectures 25% of them believe that it has had no impact on education system. however, 38% of participants believe that it has an extremely affect education system. while 21% of the study's participants think it had minor impact on education. as per the survey, 27 % of people answered that there are major effect of covid-19 on education system. figure 4: covid-19 impact on education learn from covid-19 pandemic a common perspective on mental health is that it is a behavioural process that enables individuals to balance their needs with those of their surroundings or with other needs. a divergence from the norm that is either undesirable or problematic is indicated by mental health [39]. in few week after covid-19 was declared, there were 1.4 million more unemployed in american country, and 33% of them said that covid-19 had caused them or a member of their family to lose their job, receive a wage cut, or both [40]. the national unemployment rate rose to 14.7% by midapril 2020, the highest level since the great depression [41]. recessions and other times of widespread work insecurity (i.e., permanent job loss or loss of job features) raise the probability of negative mental health consequences [42]. job uncertainty is a stressful situation that causes anxiety and unfavourable emotions [43]. when we ask another question of about ‘what do you learn from covid-19 pandemic?’ as given in (figure 5) the majority of participants i.e. 41% replied that we need a permanent/ secured job. 35 % peoples were replies as we need to focus on our physical as well as mental health. because of emergency health issues, 15% populations answered that good financial condition is very important. in emergency health situations like covid-19, peoples face too much problems like physical health, mental health, job and some other issues. at that time physical facilities are fully waste, peoples are focused on to live only. therefore, only 9% peoples were replied that physical resources are important. future prospects on post covid-19 life style and behavioural changes there are several opportunities for lifestyle and behavioural modifications after covid-19. the epidemic has increased people's awareness of health and hygiene, and this trend is likely to last. people may develop better habits including routinely washing their hands, keeping their surroundings tidy, and placing a high priority on personal hygiene [44]. due to the advantages of flexibility and reduced commuting for both individuals and businesses, the trend towards remote employment and online learning may continue. the work-life balance could change as a result, placing more value on remote and hybrid work arrangements. the significance of social bonds and community shirsath et al. covid-19 survey vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 67 has also been emphasised by the pandemic. spending quality time with family and friends, fostering relationships, and taking part in community activities could become more important to people. local and small companies might experience a comeback as people place a higher priority on helping their communities [45]. positive changes may also occur in terms of mental health assistance and awareness. the pandemic has highlighted the significance of mental health, and there may now be a greater acceptance and willingness to prioritise mental health, seek counselling, and engage in self-care. the pandemic has also increased environmental awareness. more sustainable behaviours could be adopted by people and groups, like cutting back on travel, adopting online meetings, and making ecofriendly decisions. the preservation and resilience of the environment may be receiving more attention. it is crucial to remember that the future is unpredictable and that the pandemic's long-term effects on way of life and behaviour are still developing. continuous monitoring, adaptability, and reactivity to new trends and difficulties will be necessary for adaptation to these changes [46]. the covid-19 pandemic has provided individuals, communities, and governments with a wealth of new information. it has emphasised the value of prepared, wellcoordinated responses to public health crises [47]. the pandemic has made it clearer than ever before how important it is to invest in research and development and have reliable healthcare systems that are accessible to everyone. in order to effectively address global health problems, it has also emphasised the need of international cooperation and information exchange [48]. the epidemic has also taught us the importance of adaptability, inventiveness, and resilience in surviving unpredictable and difficult times. it serves as a reminder to put people's health first, improve the public health system, and be more ready for crisis situations in the future [49]. figure 5: learn from covid-19 pandemic discusions due of the unusual covid-19 outbreak, nearly all towns, cities, and villages in the affected countries are under partial or complete lockdown for an extended period of time, ranging from a few weeks to a few months. all local and national governments were instructed to close educational institutions and impose limits on citizens' freedom of movement outside of their homes and non-essential enterprises in order to avoid communal transmission. staying at home during the lockdown also resulted in bad eating and exercise habits, which in turn led to weight gain, diabetes, and an increased risk of cardiovascular disease. similar to some negative health effects, some positive ones were seen. people can experience significant psychological anguish as a result of covid-19 owing to a number of factors, including isolation, fear, uncertainty, and economic unrest. without a question, the covid-19 pandemic has changed society globally in all aspects of day-to-day living. one notable shift that resulted from the widespread closure of schools and the imposition of social isolation measures was the remote delivery of education at all levels. these days, people prioritize their safety and choose lesstraveled, off-the-beaten-path areas. furthermore, the epidemic has drawn more focus to mental health and wellness. nowadays, a lot of people realize how important it is to look after oneself, manage stress, and seek professional help when needed. this shift in perspective may have a lasting impact on how society views mental health issues. the pandemic has brought attention to the importance of mental health, and people may now be more willing and accepting to prioritize mental health, seek counseling, and take care of themselves. environmental awareness has also grown as a result of the pandemic. individuals and organizations should embrace more environmentally friendly practices such as reducing travel, switching to online meetings, and making more eco-friendly choices. conclusion since the emergence of the covid-19 pandemic in march 2020, it has caused significant loss in human lives and also affected their livelihoods. several efforts have been made to combat the epidemic in this area. moreover, many efforts have been made worldwide to develop sustainable solutions to overcome the epidemic. in this present study, we assessed participants' general attitudes after the post-covid period. from the results, it was observed that most of the participants show positive affirmations towards behavioural changes. many participants 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towards covid-19. inter j envir res pub health. 2021; 18(17):9047-53. 49. liu j, shahab y, hoque h. government response measures and public trust during the covid‐19 pandemic: evidence from around the world. british j manage. 2022; 33(2):571-602. how to cite this article: shirsath nr, shitole r, jagtap v, sagare ar, goswami a. life –style and behavioural changes after covid-19: a perspective. indian j pharm drug studies. 2024; 3(2):63-69. funding: none; conflicts of interest: none stated mhamane s & adat p in vitro anti-inflammatory activity of leaves of cicer arietinum vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 179 original article in-vitro anti-inflammatory activity of leaves of cicer arietinum supriya mhamane1, priyanka adat2 from, 1sahyadri college of pharmacy, methwade, sangola, maharashtra, 2assistant professor, pharmaceutical chemistry, sahyadri college of pharmacy, methwade, sangola, maharashtra, india. abstract cicer arietinum also known as bengal gram. this plant belongs to the family fabaceae. it is cultivated in india in madhya pradesh, uttar pradesh, rajasthan, and maharashtra. this study shows that cicer arietinum leaves anti-inflammatory activity. it is used to decrease inflammation. this work estimates the anti-inflammatory activity of leaves of cicer arietinum by using a uv-visible spectrophotometer at 660nm. the maximum peak of inhibition of protein denaturation by using ethanolic and hydro-alcoholic extract is observed at 500µg/ml. by comparing both the extract ethanolic extract shows maximum percentage inhibition. keywords: cicer arietinum, diclofenac sodium, protein denaturation, inflammation, bovine serum albumin. atural products are compared to pure chemicals, they are less toxic. therapeutic activity is seen in plant or plant extract, but may not be in isolated pure components. one component may decrease the toxicity and increases the usefulness of another. the herbs have a wide range of applications in the medicinal field. from ancient times herbs are used as medicines. herbs are used in the treatment of diseases. ros like superoxide (o2 ●-) and hydroxyl (oh●), hydroperoxyl (ooh●), peroxy (roo●) and alkoxy (ro●) radicals, and non-free radicals, e.g., hydrogen peroxide (h2o2) and hypochlorous acid (hocl), which are constantly produced in the human body during cell metabolism. others are reactive nitrogen species (rns) consisting of nitric oxide (no●), peroxynitrite (onoo●), and nitrogen dioxide (no2). signal transduction, gene expression, and activation of receptors can be regulated by free radicals [1]. excessive free radicals if not eliminated immediately then they can also be toxic to living cells which can lead to oxidative damage to functional macromolecules like dna, proteins, and lipids. as a result, it is critical to look for endogenous antioxidant substances that act as direct scavengers of free radicals or as metal ion chelating agents access this article online received – 21st july 2023 initial review – 26th july 2023 accepted – 1st august 2023 quick response code that catalyze the generation of radical species, which could delay the progression of many chronic diseases or reduce chronic inflammation. antioxidant plant components can decrease the formation of free radicals and also decreases diseases by oxidative stress [2]. the phenolics and flavonoids of plant extract involve in the antioxidant activities of plants and act as antiinflammatory agents. the formation of proinflammatory molecules like tnf-α and nitric oxide (no) can regulate inflammation. free radicals react with inflammatory molecules leading to cell death and tissue damage [3]. chickpea (cicer arietinum l.) is high in protein (17-23%), as well as carbs (70%), lipids (4-10%), vitamins (b group), and minerals such as potassium, phosphorus, magnesium, and calcium. chickpea proteins have excellent bioavailability, and peptides with hypolipidemic, antioxidant, and antibacterial action, among others, have been found due to their amino acid composition after hydrolysis. however, no anti-inflammatory bioactive peptides extracted from enzymatically degraded chickpea proteins have been reported [3]. materials and methods extraction method soxhlet apparatus is used for the extraction of 50gm of _______________________________________ correspondence to: supriya mhamane, korwali, tal. mohol, dist. solapur, maharashtra, india. email: supriyamhamane99@gmail.com tel.: +91 8080244609 n mailto:supriyamhamane99@gmail.com mhamane s & adat p in vitro anti-inflammatory activity of leaves of cicer arietinum vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 180 powdered drug and 200 ml of the solvent system. wet the powder by using a solvent system and take three cycles of solvent on heating mantle at 550c and continuous water supply to condenser for cooling of solvent. due to this it again comes in powdered drug and again goes in the round bottom flask and continues the cycle up to solvent becomes colorless [4]. in-vitro anti-inflammatory activity inflammation: the body’s response (defense) to injury or infection is called inflammation. it shows redness, pain, swelling, and heat-like symptoms [5]. treatment of inflammation 1. acute inflammation: rest, ice, and good wound care. 2. chronic inflammation: vitamin supplements (vitamins a, c, and d), zinc [5]. bovine serum albumin is used to search for antiinflammatory drugs at an early stage without the use of animals. it combines with water, salt, fatty acid, vitamins, and hormones and transfers it to tissues and cells. chickpea is the preferred protein source during inflammation [6]. inhibition of protein denaturation chemicals used for the assay: potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, hcl, bovine serum albumin (bsa), diclofenac sodium, methanol, ethanol, 70% ethanol [1]. reagent preparation 1) phosphate buffer for 200ml buffer, weigh 0.136gm potassium dihydrogen phosphate, 0.176gm disodium hydrogen phosphate, and 0.7gm sodium chloride. mix all the chemicals in a 250ml beaker and add 100ml distilled water. maintain the ph at 6.3 with the help of hcl. make up the volume up to 200ml [1,9]. 2) bovine serum albumin (bsa) weigh 5gm bsa and add in a 100 ml buffer [1]. standard preparation: diclofenac sodium is used as a standard. for the preparation of the stock solution, dissolve 100mg diclofenac sodium in 100ml methanol to produce 1000µg/ml. from this stock solution, 3 different concentrations of 100, 200, and 500µg/ml were prepared by pipetting 1, 2, and 5ml of stock solution and diluting it up to 10ml [1]. test solution preparation: weigh 100mg semisolid drug extract and dissolve it in 100ml solvents (ethanol and 70% hydroethanol) to produce 1000µg/ml. from this stock solution, 3 different concentrations of 100, 200, and 500µg/ml were prepared by pipetting 1, 2, and 5ml of stock solution and diluting it up to 10ml [1,7]. procedure: in different 7 test tubes, add 0.9ml bsa. in the first 3 test tubes add 0.1ml standard i.e. diclofenac sodium of different concentrations (100µg/ml, 200µg/ml, and 500µg/ml), in the next 3 add 0.1ml test solution of cicer arietinum leaves extract of different concentration (100µg/ml, 200µg/ml and 500µg/ml), in remaining 1 test tube add 0.1ml distilled water (figure 1). then the test tubes were incubated at room temperature for 5 minutes and then add 2.5ml phosphate buffer in each test tube. measure the absorbance at 660nm by using a uv visible spectrophotometer [1,9]. figure 1 dilution for anti-inflammatory activity calculation: percentage inhibition was calculated by using the following formula: % inhibition = × 10 results and discussion: inflammation is a physiological response to bacterial infection or damaged vascularized living tissue. although this tissue's response is normally to protect itself, when it becomes uncontrollable, recurring, or chronic, it can be linked to disorders such as asthma, obesity, and rheumatoid arthritis, among others. the most common treatments for inflammation are steroidal and non-steroidal medicines; however, their usage is limited due to their numerous adverse effects; thus, interest is growing in the development of alternative therapies derived from natural sources that do not pose health hazards [10]. cicer arietinum plant shows antimhamane s & adat p in vitro anti-inflammatory activity of leaves of cicer arietinum vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 181 inflammatory activity due to the presence of alkaloids, glycosides, steroids, carbohydrates, amino acids, and inorganic elements. due to injury to the body, the body’s defense system gets activated and releases some mediators which can lead to an increase in blood flow which causes an increase in the size of the blood vessel and then swelling of that body part i.e. inflammation. to overcome the inflammation, antiinflammatory agents are used. inhibition of protein denaturation activity is first performed to study the antiinflammatory activity of the leaves of cicer arietinum. the determination of inhibition of protein denaturation activity by ethanolic and hydro-alcoholic extract was shown by this study. this study gives the percentage inhibition. the ethanolic extract shows high percentage inhibition at 500µg/ml i.e. 66.67%. the percentage inhibition increases as concentration increases, which is shown in the table and graph. the hydro-alcoholic extract shows high percentage inhibition at 500µg/ml i.e. 36.11%. the percentage inhibition increases as the concentration increases, which is shown in the table and graph. these values are compared with the standard (table 1, figure 2) [8]. table 1 in-vitro anti-inflammatory activity of cicer arietinum conc. (µg/ml) percentage inhibition (diclofenac sodium) (%) percentage inhibition (ethanolic) (%) percentage inhibition (hydroalc.) (%) 100 38.89 25 22.22 200 63.89 41.67 30.56 500 77.78 66.67 36.11 figure 2 in-vitro anti-inflammatory activity of cicer arietinum for ethanolic and hydro-alcoholic extract. conclusion based on this study, it is concluded that the ethanolic and hydro-alcoholic extract of leaves of cicer arietinum shows anti-inflammatory activity by inhibition of protein denaturation activity further studies were suggested to isolate the active principles responsible for the activity. the ethanolic extract shows a high percentage of inhibition as compared with the hydro-alcoholic extract. ethanolic extract at 500µg/ml shows high percentage inhibition i.e. 66.67%. the percentage inhibition increases as the concentration increases from 100µg/ml to 500µg/ml and it is compared with the standard. references 1. pradnya shinde et al. ‘in vitro pharmacological investigation of fruit of acacia nilotica’, indian journal of pharmacy and drug studies. 2022;(3):111-115. 2. jukanti ak, gaur pm, gowda cl, et al. nutritional quality and health benefits of chickpea (cicer arietinum l.): a review. br j nutr. 2012;108 suppl 1:s11-s26. doi:10.1017/s0007114512000797 mhamane s & adat p in vitro anti-inflammatory activity of leaves of cicer arietinum vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 182 3. faridy jm, stephanie cm, gabriela mo, et al. biological activities of chickpea in human health (cicer arietinum l.). a review. plant foods hum nutr. 2020;75(2):142-153. doi:10.1007/s11130-020-00814-2 4. karnail singh, kavita gahlot. ‘pharmacognostic evaluation of stem and leaf of cicer arietinum linn’, journal of pharmacognosy and phytochemistry 2018; 7(6): 165-174. 5. inflammation. (n.d.). cleveland clinic. retrieved july 7, 2023, from https://my.clevelandclinic.org/health/symptoms/21660inflammation 6. the best foods for an anti-inflammatory diet. (n.d.). jeffersonhealth.org. retrieved july 7, 2023, from https://www.jeffersonhealth.org/your-health/living-well/thebest-foods-for-an-anti-inflammatory-diet 7. juarez-chairez mf et al. in vitro anti-inflammatory and antioxidant activity of chickpea (cicer arietinum l.) proteins hydrolysate fractions, biotecnia, 2022;24(2):59-68. 8. sharmila dharmadeva et al. in vitro anti-inflammatory activity of ficus racemosa l. bark using albumin denaturation method, ayu. 2018;39(4):239-242. 9. monk jm, wu w, mcgillis lh, et al. chickpea supplementation prior to colitis onset reduces inflammation in dextran sodium sulfate-treated c57bl/6 male mice. appl physiol nutr metab. 2018;43(9):893-901. 10. hassan-ahmed l. e, dahham s. s, fadul s. m., et al. 2016. evaluation of in vitro and in vivo anti-inflammatory effects of (-)-pseudosemiglabrin, a major phytoconstituent isolated from tephrosia apollinea (delile) dc. journal of ethnopharmacology.193:312-320. how to cite this article: supriya mhamane, priyanka adat. in-vitro anti-inflammatory activity of leaves of cicer arietinum. indian j pharm drug studies. 2023; 2(4):179-182. funding: none conflict of interest: none stated https://my.clevelandclinic.org/health/symptoms/21660-inflammation https://my.clevelandclinic.org/health/symptoms/21660-inflammation https://www.jeffersonhealth.org/your-health/living-well/the-best-foods-for-an-anti-inflammatory-diet https://www.jeffersonhealth.org/your-health/living-well/the-best-foods-for-an-anti-inflammatory-diet bhat r & shanbhag p marburg virus: outbreak updates and research review vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 127 review article marburg virus disease: latest outbreak and current research review ramdas bhat1, preeti shanbhag2 from, 1assistant professor, 2pg scholar, department of pharmacology, srinivas college of pharmacy, valachil, mangalore, karnataka, india. abstract the marburg virus disease (mvd) is a severe and deadly viral hemorrhagic fever caused by the marburg virus. recent outbreaks have raised global concerns due to the severity of the disease, high mortality rate, and limited treatment options. this review provides an overview of the latest outbreak and current research on mvd, including efforts to understand the virus's epidemiology, transmission, pathogenesis, and diagnosis, as well as the development of antiviral therapies and vaccines. advances in diagnostic techniques and experimental antiviral drugs show promising results. however, controlling outbreaks remains challenging due to various factors such as limited access to affected areas, inadequate healthcare infrastructure, and community resistance to interventions. therefore, a multidisciplinary approach involving public health measures, clinical care, and research efforts is crucial in mitigating the impact of mvd outbreaks and improving patient outcomes. further research and collaborative efforts are needed to develop effective strategies for preventing and managing mvd and protecting public health. key words: marburg virus disease (mvd), viral hemorrhagic fever, outbreaks, antiviral therapies, vaccines. arburg virus disease (mvd) is a severe and often fatal viral illness caused by the marburg virus (marv), a member of the filovirus family [1]. in august 1967, an epidemic in frankfurt am main and marburg a der lahn, west germany (germany), led to the discovery of mvd for the first time. since then, several outbreaks of mvd have been reported in africa, with the most recent outbreak occurring in 2022. mvd shares similarities with ebola virus disease (evd) in terms of its clinical presentation, transmission, and high mortality rate [2]. the marburg virus is an enveloped, negative-sense rna virus that has a close resemblance to the ebola virus [1]. it is transmitted to humans from infected animals, particularly fruit bats, through contact with bodily fluids or contaminated surfaces. once the virus enters the human body, it can cause severe systemic disease with a wide range of clinical manifestations [3]. mvd is characterized by a sudden onset of fever, headache, myalgia, and malaise, which may progress to more severe symptoms such as hemorrhagic manifestations, including petechiae, access this article online received – 22nd june 2023 initial review – 27th june 2023 accepted – 12th july 2023 quick response code ecchymoses, and mucosal bleeding. gastrointestinal symptoms such as nausea, vomiting, and diarrhea are also common. severe cases of mvd can lead to multiorgan failure, shock, and death, with mortality rates ranging from 24-88% depending on the outbreak and population affected [4,5]. diagnosing mvd can be challenging, as the early symptoms are nonspecific and can resemble other febrile illnesses. laboratory testing, including pcr-based methods, antigen detection, and serological assays, are used for confirmation [6]. however, access to these diagnostic tools may be limited in resource-limited settings where mvd outbreaks often occur. there is no specific antiviral treatment for mvd, and supportive care remains the cornerstone of management. this includes strict infection control measures, fluid and electrolyte management, and organ support as needed. experimental treatments, including antiviral drugs and immunotherapies, have been used in some cases, but their efficacy and safety are still being evaluated through ongoing research efforts [7]. _______________________________________________ correspondence to: ramdas bhat, associate professor, department of pharmacology, karavali college of pharmacy, vamanjoor (post), mangalore, karnataka, india575028, email: ramdas21@gmail.com, tel.: +91 7795772463 m mailto:ramdas21@gmail.com bhat r & shanbhag p marburg virus: outbreak updates and research review vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 128 since the identification of marv, significant research efforts have been directed toward understanding the virus and developing strategies for its prevention, diagnosis, and treatment. advances in our understanding of marv epidemiology, viral pathogenesis, and host immune response have shed light on the complex interplay between the virus and the host, and have informed public health measures to control outbreaks [8]. in recent years, the emergence of new outbreaks of mvd, including the most recent one in 2022, has renewed the focus on this deadly disease. ongoing research efforts are aimed at improving diagnostics, understanding the factors driving the emergence and spread of mvd, developing effective treatments, and advancing vaccine development [9]. in this review, we will provide an overview of the latest outbreak of mvd, and summarize the current understanding of the virus, including its epidemiology, clinical features, diagnostics, treatment, and ongoing research efforts. we will review the existing literature and highlight key findings, discuss challenges and future directions in mvd research, and emphasize the need for continued efforts in understanding and controlling this menacing virus. epidemiology of marburg virus disease the epidemiology of marburg virus disease (mvd) involves studying its distribution, patterns, and determinants within populations. mvd is primarily endemic to africa, with sporadic outbreaks occurring in countries like uganda, angola, kenya, and the democratic republic of congo. fruit bats are believed to be the natural reservoir of the virus, transmitting it to humans through direct contact or exposure to infected animals' bodily fluids or tissues. understanding mvd epidemiology is challenging due to sporadic outbreaks linked to activities such as cave exploration, mining, or handling infected animals. international collaboration, led by organizations like the who, plays a crucial role in responding to mvd outbreaks. ongoing research aims to improve our understanding of the virus's transmission dynamics, and host reservoirs, and develop effective prevention and control strategies [2,10,11]. figure 1: marburg virus outbreaks in the past [10]. pathogenesis marburg virus disease (mvd) is a severe and often fatal illness caused by the marburg virus. the pathophysiology involves the virus entering the body and infecting immune cells, leading to their destruction and facilitating viral spread. the virus causes dysfunction of endothelial cells, increasing vascular permeability and causing leakage of fluid and clotting factors. this results in disseminated intravascular coagulation (dic), abnormal blood clotting, bhat r & shanbhag p marburg virus: outbreak updates and research review vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 129 and bleeding. multiple organ systems, including the liver, spleen, lymph nodes, and adrenal glands, are affected, leading to organ failure. immune dysregulation and a cytokine storm contribute to systemic symptoms. the precise mechanisms are still being studied, but these processes collectively contribute to the severe illness and organ damage seen in mvd [12]. figure 2: pathogenesis of marburg virus disease [13] clinical features of marburg virus disease (mvd) marburg virus disease (mvd) is a severe and often fatal viral hemorrhagic fever, shares similarities with ebola virus disease. the incubation period ranges from 2 to 21 days, with an average of 5-10 days, during which individuals may remain asymptomatic. the disease typically begins with a prodromal phase characterized by sudden onset fever, chills, headache, muscle aches, fatigue, and gastrointestinal symptoms. as mvd progresses, hemorrhagic manifestations occur, including bleeding from gums, nose, gastrointestinal tract, and injection sites, along with petechiae and ecchymosis. multi-organ dysfunction follows, leading to liver damage, kidney failure, and dic, which causes simultaneous clotting and bleeding. severe cases involve central nervous system involvement with confusion, irritability, aggression, delirium, seizures, coma, and neurological deficits. the disease progression can lead to shock, with low blood pressure, rapid heart rate, and poor organ perfusion. despite supportive care, mvd has a high mortality rate ranging from 24% to 88% depending on the outbreak and healthcare resources available [1]. prognosis of marburg virus disease (mvd) the prognosis of marburg virus disease (mvd) remains poor as there is currently no approved treatment available, and only supportive care can be provided. optimal management requires specialized biocontainment units to ensure the safety of healthcare personnel. strict adherence to proper personal protective equipment (ppe), hand hygiene practices, and safe handling of needles and sharps is essential to minimize the risk of occupational exposure. controlling future outbreaks is crucial in preventing primary infections and secondary transmission. the variability in disease severity observed in different outbreaks is believed to be influenced by factors such as the availability of medical care, infectious dose, route of infection, the virulence of the virus strain, and overall population health [1,5]. treatment & management of marburg virus disease the marburg virus has no currently approved therapies. during outbreaks, supportive treatment is the primary strategy, and it's essential to take infection control precautions to stop the virus from spreading. many pharmacological substances are being researched as potential treatments. an evaluation of the safety and pharmacokinetics of the antiviral nucleoside analog bhat r & shanbhag p marburg virus: outbreak updates and research review vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 130 galidesivir (bcx4430), which has shown promise in animal models, was completed. interferon-beta and other antivirals such as favipiravir and remdesivir have also been researched, but neither has shown any appreciable survival advantages [14]. antibody therapy has been researched in animal models and is slated for human use. the monoclonal antibody mr 191-n has demonstrated effectiveness in preventing marburg virus infection in rhesus macaques. due to the similarities between the marburg virus sickness and the ebola virus disease, antibodies have been utilized successfully in the treatment of both diseases [15]. additionally, work is being done to create a vaccine that is effective against filoviruses like the marburg virus. clinical trials are looking into several vaccine modalities, including inactivated viruses, replication-incompetent vaccines, viral vectors, dna-based vaccines, and viruslike particles. after the 2013 ebola virus pandemic, the need for effective immunization grew, but it will take time and more research to assess the value and efficacy of these vaccine candidates [16-18]. future directions future directions for marburg virus disease (mvd) involve key areas of focus, including the development of specific antiviral treatments and vaccines, enhanced surveillance and early detection systems, and investment in public health infrastructure. continued research is needed to develop targeted therapies and safe, effective vaccines against mvd. improving surveillance capabilities and diagnostic methods will enable early detection and timely response to mvd outbreaks. this includes strengthening laboratory capacity for prompt identification of cases and implementing robust surveillance networks to monitor disease trends [19]. in addition, research efforts should prioritize the development of specific antiviral treatments that target the marburg virus. identifying novel drug targets and conducting clinical trials to assess their safety and efficacy are crucial steps in improving treatment options for mvd. this involves collaboration between scientists, pharmaceutical companies, and regulatory agencies to accelerate the development and approval of potential therapies [19,20]. furthermore, the development of safe and effective vaccines is paramount in preventing and controlling mvd. ongoing research should explore different vaccine platforms, such as viral vectors and protein subunits, to elicit robust immune responses and provide long-term protection against the marburg virus. conducting rigorous preclinical and clinical studies, and ensuring regulatory compliance, are essential for advancing vaccine candidates toward licensure and deployment [20,21]. enhanced surveillance and early detection systems are critical in managing mvd outbreaks. strengthening surveillance capabilities involves establishing efficient reporting mechanisms, improving laboratory diagnostics, and enhancing data sharing between countries and international organizations. early detection enables timely implementation of public health measures, such as case isolation, contact tracing, and infection control protocols, to prevent further transmission and contain outbreaks [20,22]. investment in public health infrastructure is vital for effective prevention and management of mvd. this includes establishing specialized treatment centers equipped with isolation units and trained healthcare professionals experienced in handling viral hemorrhagic fevers. adequate resources, including personal protective equipment, laboratory facilities, and medical supplies, must be available to support the response to mvd outbreaks [23]. furthermore, the development of safe and effective vaccines is paramount in preventing and controlling mvd. ongoing research should explore different vaccine platforms, such as viral vectors and protein subunits, to elicit robust immune responses and provide long-term protection against the marburg virus. conducting rigorous preclinical and clinical studies, and ensuring regulatory compliance, are essential for advancing vaccine candidates toward licensure and deployment [18]. thus, the future directions for mvd encompass the development of specific antiviral treatments and vaccines, enhanced surveillance and early detection systems, and investment in public health infrastructure. continued research, collaboration, and investment in these areas are essential for improving the prevention, diagnosis, and management of mvd, ultimately reducing its impact on global public health. conclusion mvd is a highly virulent viral hemorrhagic fever caused by the marburg virus. its diagnosis, treatment, and prevention pose significant challenges. current management primarily involves supportive care, while research focuses on developing specific antiviral treatments and vaccines. early detection, rapid response, and improved healthcare infrastructure are crucial to curtail outbreaks. international collaboration, surveillance strengthening, and public health investment are vital for addressing mvd globally. despite the grim prognosis, bhat r & shanbhag p marburg virus: outbreak updates and research review vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 131 ongoing research, collaboration, and public health efforts offer hope for improving outcomes and minimizing the impact of mvd. advancing knowledge, developing treatments, and implementing possible prevention strategies are very important to lower the threat of mvd and to safeguard the affected individuals/group. references 1. kortepeter mg, dierberg k, shenoy es, et al. marburg virus disease: a summary for clinicians. inter. j of infectious dis. 2020;99:233–42. doi: 10.1016/j.ijid.2020.07.042 2. ristanović es, kokoškov ns, crozier i, et al. a forgotten episode of marburg virus disease: belgrade, yugoslavia, 1967. microbiology and molecular biology reviews. 2020;84(2). doi: 10.1128/mmbr.00095-19 3. amman br, schuh aj, albariño cg, et al. marburg virus persistence on fruit as a plausible route of bat to primate filovirus transmission. viruses. 2021;13(12):2394. doi:10.3390/v13122394 4. brauburger k, hume aj, mühlberger e, et al. forty-five years of marburg virus research. viruses. 2012;4(10):1878– 927. doi :10.3390/v4101878 5. kassa st. 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http://dx.doi.org/10.1136/bmjgh-2022-010984 bhui u et al. anti-tubercular drug-induced vitamin b6 deficiency vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 124 case report unraveling the enigma of anti-tubercular drug-induced vitamin b6 deficiency and its role in provoking convulsive seizures a revelatory case report utpal bhui1, subhajit sarkar1, joy das1, indra kumar ghoshal1, abhik saha2, bimlesh kumar3 from, 1pg student, school of pharmaceutical sciences, lovely professional university, phagwara, punjab, 2pharmacovigilance associate, r. g. kar medical college, kolkata, west bengal, 3professor, department of pharmacology, school of pharmaceutical sciences, lovely professional university, phagwara, punjab, india. abstract the 65-year-old female patient with active tuberculosis who was hospitalized to the primary hospital and was on category 1 antitubercular drugs (cat 1 atd) is the subject of this case report. the patient had complications, a reduced appetite, had stopped eating, was nauseous, and had vomited. eight months prior, the patient has prescribed the first-line anti-tubercular medications rifampin, rifabutin, ethambutol, and isoniazid. three months prior, the patient stopped taking the medicine. now, as a result of an unfavorable drug reaction to anti-tubercular medications, the patient had acquired acute hepatitis and convulsive seizuresdue to vitamin b6 deficiency. key words: cat 1 atd, hepatotoxicity, convulsion, tuberculosis, vitamin deficiency, drug-drug interaction, adverse drug reaction. ycobacterium tuberculosis, which induces tuberculosis (tb), is a potentially serious infectious bacterial disease that primarily affects the lungs. this illness spreads when a person inhales sputum splashes that enter through the mouth and nose, upper respiratory tract, and bronchi before reaching the lungs' alveoli and potentially attacking internal organs. the bacteria that cause tuberculosis can infect a lot of people without any symptoms. the most typical signs of this are fever, weight loss, night sweats, and coughing that occasionally has a bloody tint [1].as a result, even if effective chemotherapy is available, tb remains a serious health concern in the majority of countries. this is owing to low patient adherence, primary multidrug resistance, and interruptions partly brought on by adverse drug reactions (adrs). as a result, more resistant strains evolve, necessitating second-line therapy with more expensive medications and significant adrs. access this article online received – 20th may 2023 initial review – 29th may 2023 accepted – 1st june 2023 quick response code adrs significantly increase patient morbidity and mortality, which drives up healthcare costs and is a major issue for the common people, the pharmaceutical industry, regulatory agencies, and the medical community [2].depending on the severity of the condition, taking medicine for tb might last between 6 and 9 months. the united states food and drug administration (usfda) has currently approved 10 medications for the treatment of tb. isoniazid (inh), rifampin (rif), ethambutol (emb), and rifabutin are recognized medications (rb) [3]. who estimates that tb affects one-third of the population and dies one in every four adult males. primary anti-tb medications have the potential to be hepatotoxic. anti-tb medications inh, rif, and rb produce hepatotoxicity like 2 transaminases and severe hepatitis failure, which are mentioned in the first line. depending on the hepatotoxicity diagnosis criteria, the anti-tb medication can cause hepatotoxicity in 3 to 30% of cases [4]. seizures at standard doses are incredibly rare to be documented. this case study describes a convulsive seizure that happened after receiving a short-term therapeutic dose of inh. _______________________________________ correspondence to: utpal bhui, jhanjhra colony, b type 30/178, paschim bardhaman, west bengal713385 email: bhuiutpal875@gmail.com , tel: +91 7001719578 m mailto:bhuiutpal875@gmail.com bhui u et al. anti-tubercular drug-induced vitamin b6 deficiency vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 125 case report despite having a known case of pulmonary tuberculosis and being on cat 1 atd, a 65-year-old female patient recently presented with the cause of appetite, decreased food intake, nausea/vomiting, and extreme fatigability. she is a known case of tb from eight months ago in the patient's previous medical history. an old high resolution ct scan thorax showed irregular cavities in the right upper and lower lobes, centrilobular nodules in both lungs collapsing with bronchiectasis in the right middle lobe, and a tiny (r) para tracheal pulmonary koch's infection found on the chest. the ecg report overloaded the left atrium excessively, and an ultrasound scan reveals a bladder outflow obstruction. table 1 laboratory investigation on admission, the patient was started on antibiotics, hepatoprotective medications, laxatives, and a single antitubercular drug were used as part of this therapeutic regimen to treat the main symptoms of appetite loss, nausea, and exhaustion. decreasing the levels of sgot/ast, sgpt/alt, direct and indirect bilirubin, and bilirubin. antibiotics, anti-tubercular agents, and hepatoprotective agents for the treatment of severe tuberculosis in the patient and reducing the bilirubin range but the patient developed a disturbance of consciousness and tonic-clonic convulsions (figure 1). on examination, her blood pressure was 180/92 mmhg, her temperature was 36.3ºc, her heart rate was 107 beats/min, her respiratory rate was 26 breaths/min, and her oxygen saturation was 97% on room air. the findings of the systemic examination was normal. no abnormal findings were detected on head ct scans. the user laboratory finding for causes of convulsion was absent, except for the important finding of the patient's serum, vitamin b6 level was low (table 1). this leads us to diagnose the patient with convulsive seizure due to vitamin b6 deficiency is associated with isoniazid which is involved in the cat 1 atd. the justification for therapystops cat 1 atd, causing hepatotoxicity and convulsive seizure to the patient. during follow-up, no medication interactions are discovered after rational drug administration. figure 1: clinical course of the patient. convulsive seizure occurred eleven days after the administration of antitubercular drugs. seizure refractory to standard anticonvulsant therapy were controlled with the administration of pyridoxal phosphate hydrate (vitamin b-6). emb: ethambutol, rif: rifampin, inh: isoniazid, pht: phenytoin. discussion in india, 8-36% of cases of drug-induced hepatotoxicity occur. asian nations have a greater rate of drug-induced hepatotoxicity (dih), which may be attributed to racial vulnerability, the peculiarities of drug metabolism, and the existence of numerous known risk factors such hiv infections, nutritional deficiencies, and alcoholism [5]. the causes of hepatotoxicity in patients range between 1 and 10% across different nations, depending on elements like social and economic situations as well as geographic location. india (8–10%) has the highest rate of druginduced hepatotoxicity, which may be related to genetics, chronic viral hepatitis, malnutrition, and drunkenness [6]. in the intrinsic phase of treatment, which is determined by the etiology of the second-line injectable agent, the who has recommended at least five medications [7]. it is classified as a conditional recommendation with extremely low assurance in the estimate of the effect that name of the test day 2 day 5 day 10 serum bilirubin (0.2-1 mg/dl) 4.6 7.4 2.7 direct bilirubin (0-0.2 mg/dl) 3.5 1.9 1.4 indirect bilirubin (0.3-1.0 mg/dl) 1.9 3.0 1.4 sr. total protein (6-8 g/dl) 5.6 7.2 7.9 albumin (3.4-5.4 g/dl) 2.9 3.0 3.9 globulin (2-3.5 g/dl) 3.0 4.0 2.4 s.g.o.t (4-17 iu/l) 671.8 165 19 s.g.p.t (3-5 iu/l) 132.9 50 18 alkaline phosphatase (44-147 iu/l) 136.4 104 90 bhui u et al. anti-tubercular drug-induced vitamin b6 deficiency vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 126 the recent who moves to prescribe at least 4 effective medications at the beginning of treatment is effective. note that the 2019 who guidelines and our guideline committee both promote the use of newer, more effective oral medicines with better repurposing and downplay the use of injectable treatments [8]. the new who recommendation for the previous approach is multidrug-resistant tb (mdr-tb) and are rifampin resistance tb (rr-tb) in several regards; levofloxacin or moxifloxacin may be used. for fluoroquinolone, levofloxacin is advised because to its safety profile and lower risk of medication interactions [9]. isoniazid can cause various adverse events. central nervous system (cns) effects, such as headache seizure, dysphoria, and irritability, have been reported. cns effects occur due to vitamin b-6 deficiency; in the patient's case, a deficiency of vitamin b-6 of regarded to be a cause of patient convulsions and seizures because the serum vitamin b-6 level was administration was low [10]. conclusion the case is unique because the patient developed hepatotoxicity and convulsion seizures due to an adverse drug reaction of cat 1 atd. the supplementation of vitamin b-6 is recommended in patients with other conditions indicative of subclinical vitamin b-6 deficiency to prevent the central nervous system effect. the patient was diagnosed with pulmonary koch’s infection, where standard treatments are provided to a patient to reduce the level of alt/ast. clinical advancements and positive patient outcomes are future goals that we can accomplish. references 1. tuberculosis symptoms, and causes [internet]. mayo clinic publications; 2021 [cited 22 march 2023]. available from: https://www.mayoclinic.org/diseasesconditions/tuberculosis/s ymptoms-causes/syc-20351250. 2. tesfahuneygn g, medhin g, legesse m. adherence to antituberculosis treatment and treatment outcomes among tuberculosis patients in alamata district, northeast ethiopia. bmc res notes. 2015;8(1):503. doi: 10.1186/ s13104-0151452-x, pmid 26420164. 3. center for global health stories of hope sorted by date [internet]; 2021[cited 16 may 2023]. available from: https://www.cdc.gov/globalhealth/ stories/date-list.html. 4. khan ms, sunny b, krishnan rm. case report on antitubercular drug induced hepatoxicity/modified att. indian journal of pharmacy practice. 2021;14(4). 5. tost jr, vidal r, caylà j, et al. study group for severe hepatotoxicity due to anti-tuberculosis drugs and the study group for severe hepatotoxicity. severe hepatotoxicity due to anti-tuberculosis drugs in spain. int j tuberc lung dis. 2005;9(5):534-40. pmid 15875925. 6. nahid p, mase sr, migliori gb, et al. treatment of drugresistant tuberculosis. an official ats/cdc/ers/idsa clinical practice guideline. am jrespircrit care med. 2019; 200(10):e93-e142. 7. mase sr, chorba t. treatment of drug-resistant tuberculosis. clinics in chest medicine. 2019 dec 1;40(4):775-95. 8. temmerman w, dhondt a, vandewoude k. acute isoniazid intoxication: seizures, acidosis and coma. acta clin belg 54: 211-216, 1999. 9. minns ab, ghafouri n, clark rf. isoniazid-induced status epilepticus in a pediatric patient after inadequate pyridoxine therapy. pediatremerg care. 2010 may;26(5):380-1. 10. gokhale ya, vaidya ms, mehta ad, rathod nn. isoniazid toxicity presenting as status epilepticus and severe metabolic acidosis. j assoc physicians india. 2009 jan;57:70-1. how to cite this article: utpal bhui, subhajit sarkar, joy das, indra kumar ghoshal, abhik saha, bimlesh kumar. unraveling the enigma of anti-tubercular drug-induced vitamin b6 deficiency and its role in provoking convulsive seizures a revelatory case report. indian j pharm drug studies. 2023; 2(3) 124126. funding: none conflict of interest: none stated https://www.mayoclinic.org/diseasesconditions/tuberculosis/symptoms-causes/syc-20351250 https://www.mayoclinic.org/diseasesconditions/tuberculosis/symptoms-causes/syc-20351250 mhamane & adat an investigational study towards use of leaves of cicer arietinum as an anti-oxidant vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 35 original article in-vitro anti-oxidant activity of leaves of cicer arietinum supriya mhamane1, priyanka adat2 from, 1sahyadri college of pharmacy, methwade, sangola, maharashtra, 2assistant professor, pharmaceutical chemistry, sahyadri college of pharmacy, methwade, sangola, maharashtra, india. abstract background: cicer arietinum also known as chickpea. chickpea plant belongs to family fabaceae. chickpeas are an essential food plant in india, africa and central and south america. this study concludes the cicer arietinum leave’s anti-oxidant activity. it shows anti-oxidant activity and it is used to reduce oxidative stress. materials and methods: this work conceived the anti-oxidant activity of leave’s of cicer arietinum via dpph scavenging activity and measuring the absorbance at 517nm. result: the maximum peak of free radical scavenging activity by using ethanolic and hydro-alcoholic extract is observed at 400µg/ml. by comparing both the extract hydro-alcoholic extract shows maximum percentage inhibition. conclusion: thus, the high percentage inhibition of hydroalcoholic extract which is 51.31% can be more effective as an antioxidant than the ethanolic extract. keywords: cicer arietinum, dpph, ascorbic acid, free radical scavenging activity, oxidative stress. ll over the world, traditional medicine has a long history of serving people. as ancient as human civilization, there is use of natural products with therapeutic properties. main sources of drugs for a long time are mineral, plant and animal products. in almost all ancient civilizations, there is an evidence of herbs being used in the treatment of diseases and for revitalizing body systems. free radicals included in ros (reactive oxygen species) can cause liver cirrhosis, atherosclerosis, cancer, diabetes. to protect body from damage due to reactive oxygen species, antioxidants are used. in the diabetic state, oxidative stress is increased. oxygen free radical activity can lead to peroxidation of lipids, which in turn activates glycation of protein, inactivation of enzymes and alterations in the structure and function of collagen, basement and other membranes and play a role in the long team of diabetes [1,7]. materials and methods extraction method: in soxhlet apparatus, add 50gm of powdered drug and pour the solvent system. pour sovent upto which three cycles were complete. then start the heating mentle at 550c and start water supply to condenser. the solvent pass from powder and along with constituents present in powder it settles in round bottom flask. keep this process upto the solvent comes colourless [2,8]. access this article online received – 24th jan 2024 initial review – 02nd feb 2024 accepted – 08th feb 2024 quick response code in-vitro anti-oxidant activity oxidative stress: the imbalance between formation and accumulation of reactive oxygen species (ros) in the body cells and tissues is called as oxidative stress [3]. oxidative stress can lead to: cancer, cardiovascular disease, neurologic disease, respiratory disease, rheumatoid arthritis, kidney disease, delayed sexual maturation, etc. [3,5]. antioxidants: exogenous antioxidants: vitamin e, flavonoid. prooxidant agents: ascorbic acid, polyphenols, radiation [4]. dpph free radical scavenging activity: principle: dpph i.e. 2,2-diphenyl-1-picrylhydrazyl is a deep violet colored stable free radical. it has unpaired electron. antioxidant donate electron to dpph, so non radical of pale yellow color form from free radical of deep violet color. due to this, absorbance get decreased which is observed by uv spectrophotometer at 517 nm wavelength [1,6]. chemicals used in the assay: dpph (2,2-diphenyl-1picrylhydrazyl), methanol, ascorbic acid, ethanol. procedure: methanolic solution of dpph was used to test free radical scavenging action of cicer arietinum leaves __________________________________________________ correspondence to: supriya mhamane, korwali, tal. mohol, dist. solapur, maharashtra, india. email: supriyamhamane99@gmail.com. tel.: +91 8080244609. a mailto:supriyamhamane99@gmail.com mhamane & adat an investigational study towards use of leaves of cicer arietinum as an anti-oxidant vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 36 extract. in this assay as a free radical, dpph is used. antioxidants reduce dpph which is observed at 517 nm wavelength. in this assay, as a standard antioxidant, ascorbic acid is used. in this assay, reaction mixture is used, which contain 0.1 ml leaves extract of 6 different concentrations in 6 different test tubes and add 3.9 ml methanolic dpph solution in each test tube as shown in (figure 1). as a standard, 0.1 ml ascorbic acid was used and as a control 0.1 ml methanol was used instead of extract. at a room temperature for 30 minutes it is incubated. observe the absorbance at 517 nm wavelength [1,6]. figure 1: dilution of anti-oxidant activity calculation: percentage inhibition were calculated by using following formula: results in-vitro anti-oxidant activity of standard (ascorbic acid), having percentage inhibition was found to be 96.83%. table: in-vitro antioxidant activity of cicer arietinum conc. (µg/ml) percentage inhibition (ethanolic) (%) percentage inhibition (hydro-alc.) (%) 25 5.26 5.17 50 7.09 6.5 100 7.27 8.01 150 7.61 10.57 200 9.62 12.99 400 22.92 51.31 discussion cicer arietinum plant includes plant constituents like alkaloids, glycosides, steroids, carbohydrates, amino acids, inorganic elements. these constituents are responsible for showing anti-oxidant activity. in this study, the antioxidant activity was determined using dpph free radical scavenging activity in which the purple color of dpph gets reduced to yellow color due to transfer of electrons. due to this, decrease in value of absorbance depicts that constituents of leaves of cicer arietinum are able to reduce free radicals. due to free radicals of oxygen, the number of reactive oxygen species (ros) increases, so that the oxidative stress increases this can lead to various diseases. to overcome it, the antioxidants are used. among two samples ethanolic and hydro-alcoholic extract, the highest percentage inhibition was observed in hydro-alcoholic extract at 400µg/ml which was 51.31%. the percentage inhibition increases as concentration increases, which is as shown in table and graph. ethanolic extract shows high percentage inhibition at 400µg/ml i.e. 22.92%. thus it could be concluded that hydro-alcoholic extract shows good antioxidant property. conclusion the conclusion of this study shows that, the ethanolic and hydro-alcoholic extract of leaves of cicer arietinum shows anti-oxidant activity by dpph free radical scavenging activity. the hydro-alcoholic extract shows high percentage inhibition as compared with ethanolic extract. hydroalcoholic extract at 400µg/ml shows high percentage inhibition i.e. 51.31%. it was observed as the concentration of extract increases from 25µg/ml to 400µg/ml the percentage inhibition also increases. from given table, it can be concluded that hydro-alcoholic extract has the highest antioxidant activity or good scavenging activity. references 1. pradnya shinde et al, ‘in vitro pharmacological investigation of fruit of acacia nilotica’, indian journal of pharmacy and drug studies, 2022; 3(3): 111-115. 2. ali esmail al-snafi., the medical importance of cicer arietinum -a review. j pharmacy. 2016; 6(3):29-40. 3. karnail singh, kavita gahlot. pharmacognostical and pharmacological importance of cicer arietinum linn -a review. res j pharm technology. 2018; 11(10): 4755-4763. 4. karnail singh, kavita gahlot. pharmacognostic evaluation of stem and leaf of cicer arietinum linn. j pharmaco phytochemi. 2018; 7(6): 165-174. 5. gabriele pizzino, natasha irrera, mariapaola cucinotta, et al. oxidative stress: harms and benefits for human health. oxid med cell longev. 2017; 8416763. doi: 10.1155/2017/8416763. 6. vadnere gp, patil av, wagh ss, et al. in vitro free radical scavenging and antioxidant activity of cicer arietinum l. (fabaceae). inter j pharmtech res. 2012; 4(1):343-350. 7. k jukanti, pm gaur, cll gowda et al. nutritional quality and health benefits of chickpea (cicer arietinum l.): a review. cambridge university press: 23 august 2012. 8. khandelwal kr, practical pharmacognosy. nirali prakashan. 2007; 9-161. how to cite this article: supriya mhamane, priyanka adat. in-vitro anti-oxidant activity of leaves of cicer arietinum. indian j pharm drug studies. 2024; 3(1):35-36. funding: none; conflicts of interest: none stated https://www.cambridge.org/core/search?filters%5bauthorterms%5d=a.%20k.%20jukanti&eventcode=se-au https://www.cambridge.org/core/search?filters%5bauthorterms%5d=p.%20m.%20gaur&eventcode=se-au https://www.cambridge.org/core/search?filters%5bauthorterms%5d=c.%20l.%20l.%20gowda&eventcode=se-au mandumpala amitriptyline induced high blood pressure vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 87 case report a case report on tricyclic antidepressant induced increased blood pressure janice jacson mandumpala from, pharm d intern, department of pharmacy practice, nirmala college of pharmacy, muvattupuzha, ernakulam, kerala, india. correspondence to: janice jacson mandumpala, pharm d intern, department of pharmacy practice, nirmala college of pharmacy, muvattupuzha, kerala, india. email: janice.jacson@gmail.com abstract this is a case of antidepressant-masked high blood pressure. administration of tricyclic antidepressants that cause raised blood pressure becomes particularly important in resistant hypertension cases. in this case, we report a case of raised blood pressure despite multiple antihypertensives. the patient also presented with electrolyte imbalances also raising the suspicion of drug side effects due to unnecessary drugs administered. therefore, arriving at a clinical decision with minimal side effects and focused treatments requires identifying every detail of the patient, including their medical and medication histories. key words: amitriptyline, resistant hypertension, anti-hypertensive, electrolyte imbalance esistant hypertension is defined as ‘an above goal blood pressure despite adherence to at least three antihypertensives one of which should be a diuretic’. although dietary sodium restriction is an important aspect in the control of blood pressure. in the given case, a geriatric patient has presented with hyponatremia and an above goal blood pressure level [1]. while addressing hypertensive episodes among individuals on anti-depressants, it seems important to understand the rise in blood pressure due to antidepressant treatment. antidepressants can cause variations in blood pressure through the adrenergic, cholinergic, serotonergic, histaminergic, and dopaminergic systems. selective serotonin reuptake inhibitors have a mild effect on blood pressure and are considered relatively safe among the geriatric population. other antidepressants, such as venlafaxine, bupropion, nefazodone, trazodone, and tricyclic antidepressants, are associated with a rise in blood pressure. tricyclic antidepressants affect blood pressure levels by various mechanisms [2]. amitriptyline is a tricyclic antidepressant with multiple side effects and is more prominent within the geriatric population. the common side effects of amitriptyline are weight gain, generalised weakness, hypotension, sleepiness, alopecia, blurred vision, dry mouth, vomiting, constipation, diarrhoea, urinary retention, and dyscrasias. hypertension is a rare side effect of amitriptyline use, but it can occur. in the given case, the patient is already diagnosed with resistant hypertension, and introducing a molecule such as amitriptyline with the potential to cause blood pressure elevations makes the clinical situation even more challenging. it is proposed that amitriptyline works by blocking norepinephrine reuptake or increasing vascular reactivity [3]. here we describe an elderly woman with resistant hypertension who received amitriptyline for depression and then developed high blood pressure. all procedures performed in the case report involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. informed consent was obtained from the individual included in this case report. the participant has also consented to the submission of a case report to this journal. clinical case a 68 year old female was admitted at 8 pm to the general medicine department of a primary care hospital. the patient complained of abdominal bloating for the past few days. the past medical history of the patient included hypertension (since 10 years), asthma (since 30 years) and depression. the patient's previous medication history included metoprolol succinate prolonged release tablet 50 mg (1-0-1), telmisartan 40 mg (1-01), torsemide 5 mg (1-0-0), moxonidine 0.3 mg (1-0-1), cilnidipine 10 mg (0-0-1), amitriptyline 10 mg (0-0-1) and inhaler formoterol fumarate 6 mcg and budesonide 200 mcg (2 puffs). the patient also underwent a hysterectomy and hemorrhoidectomy ten years ago. the patient was also previously admitted with hyponatremia and hypokalemia following usage of tablet chlorthalidone 6.25 mg (1-0-1), which was subsequently discontinued and replaced with tablet torsemide 5 mg (1-0-0). after examination of the lab reports on day 1 it was found that the sodium and potassium levels were 128 meq/l and 3.3 meq/l, respectively. the erythrocyte sedimentation rate (esr) was elevated with a value of 38 mm/hr. likewise, the haemoglobin (hb) level was 10.4 g/dl. urine albumin was nil, sugar was nil, pus cells were 1-2; and rbcs in urine were 2-4. on admission, the patient had a blood pressure (bp) of 170/80 mm hg. the bp monitoring chart has been tabulated in table 1 below. r mailto:janice.jacson@gmail.com mandumpala amitriptyline induced high blood pressure vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 88 table 1 – bp monitoring chart of the patient during the hospital stay and other lab investigations bp monitoring chart day 1 day 2 day 3 8pm 170/80* 6am 10am 1pm 6pm 9pm 120/70 130/80 120/70 130/70 160/90 6am 130/70 lab investigations parameters day 1 day 3 hb (g%) 10.4 esr (mm/hr) 38 neutrophils (%) 52% lymphocytes (%) 43% eosinophils (%) 5% sodium (meq/l) 128 135 potassium (meq/l) 3.3 4.4 *all the bp measurements are in mm/hg on admission, the patient was started on tablet amlodipine 5mg to quickly control the elevated bp level. the following day, the patient was instructed to take tablet tolvaptan 30 mg once daily (1-0-0) for sodium correction, syrup potassium chloride 2 tablespoons thrice daily (1-1-1) for potassium correction, and all previous medications (metoprolol succinate prolonged release tablet 50mg (1-0-1), tablet telmisartan 40 mg (1-0-1), tablet moxonidine 0.3mg (1-0-1), tablet cilnidipine 10mg (00-1),the bp measurements were regularly monitored, and another routine lab investigation was conducted to examine the electrolyte level. the values have been indicated in table 1. the patient was diagnosed with resistant hypertension with hyponatremia and hypokalemia. a suspicion of acute gastritis was also raised, for which the patient was advised to take a syrup containing a combination of magaldrate (400 mg/5 ml) and simethicone (20 mg/5 ml) thrice daily (1-1-1). complaints of bloating resolved post-consumption of the syrup. upon clinical improvement, the patient was discharged with the following list of medications: – tablet tolvaptan (30 mg 1-0-0 * 4 days), tablet pantoprazole (40 mg 1-0-0*10 days), tablet amitriptyline (10 mg 0-0-1*15 days), tablet metoprolol succinate (50 mg 1-0-1 *continue), tablet telmisartan (40 mg 10-1 *continue), tablet moxonidine (0.3 mg 1-0-1 *continue), and tablet cilnidipine (10 mg 0-0-1*continue). on follow up after 15 days, the patient had an in-office bp of 160/90 mm hg. following further investigation, it was determined that tablet amitriptyline 10 mg (0-1) could have resulted in increased blood pressure levels. amitriptyline is a tricyclic antidepressant and has the potential to cause elevations in bp. during follow-up, tablet amitriptyline 10 mg 0-0-1 was discontinued and tablet etizolam 0.25 mg 0-0-1 was added to overcome generalised anxiety and depressive symptoms. discussion this study highlights the effect of antidepressants in the context of resistant hypertension. often, resistant hypertension is labelled based on the number of antihypertensives prescribed and the corresponding uncontrolled blood pressure levels. upon further examination, such as a postmenopausal woman, suggesting the involvement of sex hormones there's a chance of getting higher bp (2), other factors that can lead to an increased level of blood pressure should be investigated. otherwise, this could result in unnecessary treatment, charges, electrolyte imbalances, and other adverse drug reactions. in a study conducted by breeden et al., the author has highlighted the role of antidepressants in elevating the chances of incident hypertension. tricyclic antidepressants, in particular, have been linked to an increase in blood pressure. it is also argued that this class of antidepressants also causes orthostatic hypotension [4]. therefore, large scale studies are necessary to validate the findings obtained from an individual patient. this case also highlights the need to promptly capture the instances of electrolyte imbalance among hypertensive patients. therefore, if there is no need for anti-hypertensive such as this case where possibly amitriptyline could have caused the raised blood pressure, the patient is exposed to a drug with no indication. tricyclic antidepressants are not only used for depression but also for anxiety, chronic pain, insomnia, and migraine. there are multiple sections of the population that are exposed to the threat of tricyclic antidepressants. a thorough understanding of the risks and benefits of medication prescribed for each patient can help improve the clinical decision making process. although a nonjudgmental approach was used during the medication reconciliation process, it was difficult to determine whether the patient was truly adhering to the antihypertensive medications prescribed. a formal assessment of medication adherence to the existing antihypertensives was not done; therefore, a conclusive diagnosis of resistant hypertension may be doubtful. increasing age is also a factor that can make an individual resistant to treatment; thus, it cannot be ruled out in this case. conclusion mandumpala amitriptyline induced high blood pressure vol 2 | issue 2 | apr – jun 2023 indian j pharm drug studies | 89 this case highlights a common clinical situation where multiple causes of a complaint can remain masked or unnoticed. in this case, a geriatric patient arrives with elevated blood pressure despite prompt treatment, but the condition remains unresolved. amitriptyline-induced hypertension could be one explanation. such scenarios must be highlighted in practice for better patient outcomes. references 1. carey rm, calhoun da, bakris gl, et al. resistant hypertension: detection, evaluation, and management a scientific statement from the american heart association. hypertension. 2018; 72(5): 5390. 2. calvi a, fischetti i, verzicco i, et al. antidepressant drugs effects on blood pressure. fcm. 2021;8:704281.. 3. hmoud m, al-husayni f, alzahrani a, et al. hypertension secondary to amitriptyline use as prophylactic for migraine in a 26-year-old man. cureus. 2021; 13(1):e12848. 4. breeden m, brieler j, salas j, et al. antidepressants and incident hypertension in primary care patients. jabfm. 2018;31(1):22-28. how to cite this article: mandumpala j j. a case report on tricyclic antidepressant induced increased blood pressure. indian j pharm drug studies. 2023: 2(2) 87-89. funding: none conflict of interest: none stated bhirich et al. plants and essential oils against bedbugs vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 1 review article a review of plants and essential oils effective as natural remedies against bedbugs nihal bhirich1, ghita salime meknassi1, mohamed yafout2, hicham elhorr2, amal ait haj said2, brahim mojemmi1 from, 1laboratory of analytical chemistry, faculty of medicine and pharmacy, mohammed v university, rabat, 2laboratory of drug sciences, biomedical research, and biotechnology, faculty of medicine and pharmacy, hassan ii university of casablanca, morocco. abstract called cimex lectularius and cimex hemipterus, the bedbug has made its return to daily life in many developed countries since the 1990s, although it had practically disappeared since the 1950s. the resurgence of bedbug infestations can be explained partly by the evolution of the lifestyle and by the mode of consumption favoring second-hand purchasing. one of the main factors behind their resurgence is the excessive use of synthetic insecticides with similar modes of action, which has led to the development of insecticide resistance. the application of synthetic insecticides indoors or in the environment is a public health concern due to the toxic effects that can result from prolonged exposure to these chemicals. it is for this reason that there is an increased demand from the public for the use of effective “green” products for urban pest control. natural insecticides, including eos are considered safe due to their low toxicity to humans and animals. thanks to a search in ethnobotanical studies, we identified a list of 10 plants used in the fight against bedbugs: lavandula latifolia spica, corymbia citriodora, syzygium aromaticum (l), cymbopogon winterianus jowitt, thymus vulgaris, chrysanthemum cinerariaefolium, phaseolus vulgaris, oreganum vulgare l., tagetes paluta l. and cinnamomum camphora. the study of the literature that we subsequently carried out showed that these plants/essential oils/components act according to 4 mechanisms, namely: topical, neurological, mechanical or fumigant effects. we therefore conclude that the use of preparations of natural origin has the advantage of avoiding the toxic and polluting harm of substances of chemical and synthetic origin and that it is desirable to continue research and investigation of the composition details of its plants and essential oils. keywords: cimex lectularius, cimex hemipterus, bed bugs, plants, essential oils, green product, natural insecticides. he bed bug has made a comeback in daily life in many developed countries since the 1990s, although it had practically disappeared since the 1950s. the increase in bedbug infestations is partly explained by changes in lifestyle and consumption patterns favoring second-hand purchases [1]. one of the main factors behind their resurgence is the excessive use of synthetic insecticides with similar modes of action, which has led to the development of insecticide resistance [2-5]. called cimex lectularius l. and cimex hemipterus, the bedbug belongs to the cimicidae family, exclusively hematophagous throughout its life, it has always cohabited with humans. it is an insect that has been known for thousands of years, specimens found in egyptian tombs bear witness to this. without functional wings, brown to reddish in color. it can measure 5 to 8 mm as an adult, its body is flattened dorso-ventrally, with a rounded shape [6,7]. access this article online received – 20th dec 2023 initial review – 10th feb 2024 accepted – 12th feb 2024 quick response code after a blood meal, fertilized female bed bugs lay, throughout their adult life, around five eggs per day in a place protected from light. at room temperature, the eggs hatch into first instar larvae in approximately 7 to 15 days. insects pass through four larval stages and one nymphal stage, each requiring a blood meal before they can progress to the next stage, the fifth instar molting into an adult. the lifespan of bed bugs is approximately 12 months in total. they are nocturnal, photophobic and gather during the day in aggregates located near the resting sites of human beings, sheltered from light [1]. the bedbug must feed every three to fifteen days on average; this duration varies depending on the digestion time, egg laying and temperature and humidity conditions and the availability of the host. certain populations of bed bugs have been shown to be highly resistant to various chemicals including neonicotinoids and to possess reduced sensitivity to pyrrole compounds [4,5]. __________________________________________________ correspondence to: mohamed yafout, pharm d, 19, rue tarik ibnou ziad b.p. 9154, casablanca, maroc, laboratory of drug sciences, biomedical research, and biotechnology, faculty of medicine and pharmacy, hassan ii university of casablanca, morocco. email: yafoutm@yahoo.fr t mailto:yafoutm@yahoo.fr bhirich et al. plants and essential oils against bedbugs vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 2 an amino acid substitution mutation, f348y (or f331y in standard numbering), occurring at an acyl binding site of the ache paralog gene ( p-ace ), was identified among achei resistant colonies of common bedbugs [8]. the difficulty of eliminating resistant bed bug populations requires pest management that uses both chemical and non-chemical or alternative treatments [9-11]. substances of natural origin such as plant essential oils, which are secondary metabolites derived from the outer glandular cells of the leaves and stems of aromatic plants [12], are one of the treatment alternatives used to combat bedbugs. and many other urban and agricultural insect pests [13–20]. review of plants and essential oils (eo) used against bedbugs the application of synthetic insecticides indoors or in the environment is a public health concern due to the toxic effects that may result from prolonged exposure to these chemicals [21–23]. it is for this reason that there is an increased demand from the public for the use of effective “green” products for urban pest control. natural insecticides including eos are considered safe due to their low toxicity to humans and animals [12-13]. theses eos is marketed under a lot of brand names as: sereni-d® (bouclier® punaise de lit), ecowidow® (bed bug repellent®), anbe® (bed bug spray®), ready steady defend® (bed bug killer® spray) and other. in this context, eos of plant origin in particular have emerged as an interesting option in the fight against harmful insects [24-25]. to identify plants and eos used to treat bedbugs, we searched scientific article or book databases as well as google scholar for publications containing ethnobotanical data on plants and eos. used to treat these parasites. this research allowed us to establish a list of the ten most cited plants or eos and their constituents (table 1). table 1: list of identified plants and essential oils used against bedbugs: plant / source of he botanical family english common name active molecule lavandula latifolia spica lamiaceae aspic lavender linalool corymbia citriodora myrtaceae lemon eucalyptus eucalyptol geraniol syzygium aromaticum (l) myrtaceae clove eugenol cymbopogon winterianus jowitt poaceae java lemongrass citronellol thymus vulgaris lamiaceae thyme thymol chrysanthemum cinerariaefolium asteraceae pyrethrum flowers pyrethrin phaseolus vulgaris fabaceae common bean oreganum vulgare l. lamiaceae wild oregano carvacrol tagetes paluta l. asteraceae french marigold terthyenyl cinnamonum camphora lauraceae camphor tree camphor, monoterpene lavandula latifolia spica: spike lavender is a shrub with a woody, square stem from the lamiaceae family that can reach 80cm high. its stems branch into forks and its narrow, linear leaves can reach 10cm. its spike-shaped inflorescences have small purple flowers with a shape characteristic of lamiaceae [26]. the extraction of lavender essential oil is done by steam distillation, the resulting oil is very light yellow, almost colorless [27], its composition is not constant, this variation is probably due to various conditions including environment, genotype, geographic origin, drying location, temperature and extraction method. eo is rich in linalool (25-50%), 1,8cineole (20-35%) and camphor (8-20%) [28,29]. lavender owes its repellent activity and mortality rate to its predominant component linalool which provides neuro-excitatory effects on the nervous system of bedbugs [30]. corymbia citriodora: also called eucalyptus citriodora, is a species of the genus corymbia. it is a large tree 50 meters high whose foliage gives off a lemony smell. its bark is fibrous, gray or gray brown coming off in long ribbons. the small branches are green in color. the leaves are green, concolorous, narrow, lanceolate, pointed and measure 7 to 15 centimeters long and 0.7 to 1.5 centimeters wide. they give off a peppermint smell when kneaded. the eo is extracted from the leaves and branches, which has medicinal properties thanks to the known active ingredients: aldehydes (citronellal), eucalyptol (3%), alcohols: citronellol, geraniol [31]. eucalyptus essential oil is composed of an average of 70% citronellol, effective against insects and against bedbugs [32]. syzygium aromaticum (l): the clove or clove tree is a large tree with a light gray trunk 12 to 15 meters high which can reach up to 20 meters high. it has an erect and pyramidal port. its foliage is aromatic, leathery, evergreen, dark green and glazed with a lighter underside. its leaves are opposite, entire, elliptical. it is a tree native to the moluccas islands in the indonesian archipelago. the nails are actually flower buds. green in color then red once ripe, the nails take on their pretty brown color during the drying phase, which takes place in the open air for a month [33]. the biochemical composition is likely to change depending on the production conditions and the quality of the oil. however, we can rely on this composition to evaluate the quality of an oil: main chemical compound: phenols 70 to 85% (eugenol) [34]. clove essential oil appears to exhibit insecticidal activity against bedbugs. however, its application requires direct spraying on the insect, which can prove difficult in practice [16]. cymbopogon winterianus jowitt: java lemongrass is a genus of herbaceous plants of asian species, many crops of which have been introduced throughout the world. java bhirich et al. plants and essential oils against bedbugs vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 3 lemongrass is robust, aromatic, and persistent. a tuft of erect leaves arises from a short rhizome and can reach a height of 2 meters. it is widely cultivated in java and south-east asia. the fresh aerial parts, from which java lemongrass essential oil is extracted, are rich in terpene aldehydes (35 to 65%): citronellal alcohols (30 to 40%): geraniol, citronellol and esters (10%): citronellal acetate [35]. a recent study demonstrated the toxic effects of lemongrass against various insects including bedbugs. the authors reported that some compounds contained in this plant have strong activity as membrane channel activating cations. they observed that this effect is similar to the mode of action of pyrethrin [36]. thymus vulgaris: native to the mediterranean basin, thymus vulgaris l. is an aromatic shrub with branched stems, which can reach 40 cm in height. it has small leaves curved on the edges, dark green in color, and which are covered with hairs and glands (called trichomes). the trichomes contain the essential oil mainly composed of monoterpenes. the color of its small flowers varies from white to purple to pink. thymus vulgaris is characterized by floral and therefore chemical polymorphism as well [37]. the essential oil of t. vulgaris has a high content of oxygenated monoterpenes (56.53%) and low contents of monoterpene hydrocarbons (28.69%), sesquiterpene hydrocarbons (5.04%) and oxygenated sesquiterpenes (1.84%) [4]. the predominant compound among the components of essential oils is thymol (51.34%) [38]. maximum mortality and repellency of up to 100% have been reported for thymus vulgaris in previous studies [30]. chrysanthenum cinerariafolium: perennial flower native to mountainous regions of europe, with the appearance of a large daisy. the term "pyrethrum" is the powder produced from the flowers harvested and air-dried to avoid fermentation and loss of pyrethrin. the dried flowers are crushed and extracted with hexane or other suitable solvent, the plant undissolved and the material is filtered and the solvent is flashed to leave a crude oleoresin which generally contains about 30% pyrethrins: pyrethrins i and ii, cinerins i and ii, jasmoline i and ii [39]. pyrethrins act by contact or ingestion on the nervous system of bedbugs. it is the most economically important natural insecticide, comprising a group of six closely related monoterpene esters. industrial production is based on the extraction of chrysanthemum flower heads cinerariaefolium (pyrethrum) [40]. these alkaloids are toxic to the central nervous system of insects, but also to that of cold-blooded animals. their toxicity for humans is much lower. these biodegradable natural products are quickly inactivated by light, their effectiveness is therefore quite limited and also cause resistance [41]. pyrethrum is still found today in organic insecticides in the form of dry powder or combined with other components such as lavender essential oil [41]. phaseolus vulgaris: the bean is an herbaceous, annual plant, which can take several types of growth depending on the variety. there are two large groups, climbing beans (called pole beans), with a twining habit. the bean has a nondominant main root which is very quickly supplemented by lateral roots. the roots can reach a depth of one meter if the soil is suitable [42]. in the past, common bean leaves were used by dropping them in infested areas to capture bed bugs after using them the leaves were destroyed or burned. this capturing power was analyzed, by microscopy, it revealed the presence of tiny sharp needles on the surface capable of piercing the integument of the legs of bedbugs, and these are the trichomes). the imprisonment and death of bedbugs is therefore purely physical [43]. origanum vulgaris: a perennial herbaceous plant that measures 30 to 80 cm high, with fragrant foliage and flowers when crushed. it is thus recognizable by its odor and its phenolic, spicy and hot flavor [44]. it grows from sea level to 4000 m altitude, mainly on limestone substrates and flowers from may to october. the aerial parts die during the bad season, and the plant can therefore start again from renewal buds [45]. it is a plant that is often slightly reddish-purple and is covered with hairs. oregano is a plant with erect stems, generally hairy, sometimes glabrous. they have leaves with entire or toothed edges. oregano essential oil contains carvacrol (61.9%), which is the majority component followed by pcymene (25.2%) and gterpinene (2.24%) [46]. this 39% oil gives 99% bed bug repellency in the petri dish test, it has been used against 4th and 5th instar nymphs and bed bug adults using a rabbit and human host [47]. tagetes patula l: marigold is an annual or perennial plant. its stems are erect, glabrous, or hispid, with opposite branches erect in the upper part. leaves all stem opposite, external flowers feminine or absent with short yellow, white or orange ligule, tubular internal flowers. in the essential oil of t. patula, 21 compounds were identified, and αterthienyl (43.1%), pentatriacontane (23.9%) and 2-ethyl-l-dodecanol (7.9%) %) are the main constituents [48]. the phytochemical constitution of the essential oil attests to a rich composition of monoterpenes, sesquiterpenes, and secondary metabolites closely linked to the insecticidal potential [49]. most reports indicate that these compounds exhibit inhibitory effects, growth retardation, maturation damage, reduction of reproductive capacity, appetite suppression or direct toxicity [49]. thus, we find that its essential oil is reported in studies giving maximum mortality and repellency of up to 100% [30]. cinnamonum camphora: also called camphor, it is a tree that can reach 15 to 40 meters high, although it rarely exceeds 20 meters in europe. its longevity is of the order of a thousand years [50]. the trunk is branched at the base, and has rough, chapped bark. the fragrant camphor tree (cinnamomum camphora) and its products, such as camphor essential oil, have been coveted since ancient times. having a rich history of traditional use, it was particularly used as a fumigant [51]. bhirich et al. plants and essential oils against bedbugs vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 4 camphor essential oil is extracted from the bark of the camphor tree. the distillation of this bark gives an essence. this is sublimated a certain number of times, to obtain a very aromatic white crystallized product: crystallized camphor. the latter must be mixed with other vegetable oils to be able to use it. the benefits of camphor essential oil are mainly explained by its high content of camphor, monoterpenes, and terpene oxides [52]. indeed, camphor essential oil owes its action to monoterpenes which give it toxic effects (fumigants and contact toxicity), repellent, anti-feedant, ovicidal and sterilizing effects against bedbugs [53]. figure 1: chemical structures of certain molecules discussion the analysis of the data that we collected concerning the plants and eos most cited for their effectiveness studied against bedbugs allowed us to classify their effects into four main mechanisms of action summarized in (table 2). we subsequently discuss each of these mechanisms. topical toxicity: according to the studies that we have cited for oregano and thyme, it clearly appears that their common mechanism of action is exerted by topical activity due to the most predominant components, namely carvacrol and thymol, which have two properties major [13.54]: saturated compounds (carbon-carbon single bonds outside the benzene cycle) presence of functional hydroxyl groups, on the benzene ring these structural properties allowed thymol and carvacrol, after undergoing slight detoxification, to quickly penetrate through the cuticle and interact effectively with their target sites [13,54,55]. lipophilicity of essential oil compounds is another important property that plays a role in penetration through the insect cuticle [50]. therefore, the plant essential oils of thyme (thymus vulgaris l.) and oregano (origanum vulgare l.), which contain large amounts of thymol and carvacrol, respectively, can be included in the formulation of natural insecticide products. eucalyptol and eugenol also in one study showed topical toxicity towards bedbugs, but with resistance for adult males [56]. physical toxicity: artichoke leaves have tiny hairs that attach to the exoskeleton of bed bugs. a bean leaf barrier therefore traps bedbugs, which eventually die. researchers are trying to reproduce by synthesis this type of trap naturally present in bean leaves but have not succeeded in reproducing its effectiveness [43]. if researchers could synthesize a comparable leaf, it could be an effective alternative biological tool to use in combination with other techniques. neurological toxicity: we can differentiate between two types: 1. a neuro-inhibition is exerted by the essential oils of plants (oregano, thyme, clove) due to the action at the level of possible target sites for the components: thymol, eugenol and carvacrol, respectively on gamma -amino butyric acid (gaba), octopamine / tyramine and nicotinic acetylcholine (nach) receptors [57-58]. 2. neuroexcitation has been reported in studies for eos from lavender, pyrethrum flower and camphor plants containing linalool, pyrethrum derivatives and camphor respectively [18]. linalool produced neuroexcitatory effects on the nervous system of bedbugs. studies report that linalool acts as a reversible competitive inhibitor of the acetylcholinesterase enzyme [59]. synthetic pyrethroid derivatives modify the gating characteristics of potential-sensitive sodium channels which result in a delay in their closure and thus cause a bhirich et al. plants and essential oils against bedbugs vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 5 neuroexcitatory effect on the insect nervous system [60]. the same effect was noticed in lemongrass [36]. camphor has been shown to inhibit catecholamine secretion by blocking acetylcholinesterase nachr in bovine adrenal chromaffin cells [61]. finally, molecular modeling studies suggest that the main compounds in the essential oils of tagetes patula l. such as terthyenyl competitively inhibit acetylcholinesterase of c. lectularius [19]. toxicity by fumigation: a study on the constituents of essential oils of plants: thyme, oregano, lavender, camphor shows that thymol was the most powerful, followed by carvacrol, linalool and camphor [17]. indeed, thymol was more potent as a fumigant than any other essential oil constituent tested in this study. therefore, thymol or thymolcontaining essential oils have the potential to be used as fumigants to control bedbugs in the field. the behavior of adult females, which lay eggs in isolated places, justifies the adoption of a more practical approach such as fumigating or fogging bedbug-infested objects by enclosing them in plastic bags with paper or a cloth impregnated with essential oils containing thymol [17,18]. camphor has great potential for development as an alternative green commercial insect repellent to replace harmful synthetic agents currently in use [62]. monoterpenes exert toxic (fumigant and contact toxicity), repellent, antifeedant, ovicidal and sterilizing effects against bedbugs [53]. a synergistic interaction between these monoterpenoids has been demonstrated by previous studies [63], which may provide information on the oily constituents that can be formulated together in botanical insecticide products, such as increased cuticular penetration and also great alteration of the nerve triggering activity [64]. table 2: summary of mechanisms of action of plants or he used to eliminate bedbugs. mechanism of action of bedbug toxicity plant or he active molecule references topical oreganum vulgare l. thymus vulgaris corymbia citriodora carvacrol thymol eucalyptol, geraniol 47,13,54,55 13,30,54,55 31 mechanics (physics) phaseolus vulgaris 43 neurological neuro-inhibitory oreganum vulgare l. thymus vulgaris syzygium aromaticum (l) carvacrol thymol eugenol 57.58 30,57,58 16,57,58 neuro-excitatory lavandula latifolia spica chrysanthenum cinerariafolium cinnamomum camphora tagetes patula l. cymbopogon winterianus jowitt linalool pyrethrin camphor alphaterthyenyl citronellol 18,30,59 40,18,60 18,53,61 30,49,19 31.36 fumigant thymus vulgaris oreganum vulgare l. lavandula latifolia spica cinnamomum camphora cymbopogon winterianus jowitt thymol carvacrol linalool camphor, monoterpene citronellol 18,17,30 17 17.30 17,51,53,62 31.17 conclusion this present review summarizes the current knowledge available on the toxic potential: neurological, physical, fumigant and topical of the plants and eos most studied for their effects against bed bugs. the studies indexed on the scopus database, and which we have included in this review provide relevant scientific data which make it possible to elucidate the precise mechanism of action of the action observed against the bed bugs of these plants or he. the major interest of our development is therefore to guide the research and development of effective insecticides of natural origin against these harmful insects. the use of preparations of natural 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drug studies. 2024; 3(3):1-7. funding: none; conflicts of interest: none stated https://hal.science/tel-01894678 dave role of pharmacists in opioid addiction management vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 57 review article the role of pharmacists in opioid addiction management pallav dave from, regulatory compliance analyst, 604 la fontenay ct, louisville, ky 40223, usa abstract opioid addiction claims thousands of lives every year in the united states. the prevalence of misuse, abuse, and overdose continues to rise despite the measures and initiatives taken by the federal government and other government agencies to manage the problem. these efforts have led to a re-evaluation of the role different healthcare providers can play to ensure the problem is adequately addressed. being one of the most accessible healthcare providers to patients under opioid therapy, pharmacists are better positioned to deal with opioid addiction. they dispense millions of opioids every year making them better positioned to screen for patients who are likely to develop an addiction problem and refer them to treatment resources before the abuse escalates to opioid use disorder (oud). the aim of this review is to explore the role of pharmacists in opioid addiction management including prevention, screening, education, naloxone prescribing, counselling, and referral to treatment resources. the review also provides pertinent information regarding the current state of the opioid crisis in the us, including recent data on prevalence and opioid-related deaths. exploring the role of pharmacists is instrumental in coming up with effective measures that can help in opioid addiction management. keywords: opioids, opioid addiction management, pharmacists, opioid misuse, opioid abuse, opioid use disorder pioids are crucial in the management of pain. various studies have established that prescription opioids are instrumental in managing pain, particularly in the short term [1,2]. however, despite their significance in the management of pain, prolonged use without medical supervision can increase the risk of dependence and lead to addiction. the recent 2022 data by national survey on drug use and health is an indication of the opioid problem in the us with 8.9 million people who are 12 years and older having misused opioids while another 6.1 million being diagnosed with oud in the last year [3]. managing opioid addiction is paramount considering the number of deaths attributed to opioid addiction. in the past two decades alone, opioid overdose deaths in the us have gone up significantly from around 21,089 in 2010 to approximately 70,000 in 2020 [4,5]. in 2021, deaths linked to synthetic opioids other than methadone were approximately 70,601.4 on average, 1 death in every 22 in the us in 2021 was attributed to opioid toxicity [6] of these deaths, the most affected are younger adults aged 25 to 34 years [7]. the early loss of life means years of loss of productivity. abuse and misuse of opioids also increase health risks and access this article online received – 03rd jul 2023 initial review – 30th jan 2024 accepted – 08th mar 2024 quick response code complications which has a major toll on public health. in 2017 alone, the opioid epidemic cost the us $1.02 trillion, with approximately $35 billion being on healthcare and treatment [8]. considering the magnitude of opioid misuse, abuse, and oud as exemplified by available population data, there is a need for measures to increase awareness of the risks associated with opioids and put proper addiction management strategies in place. an interdisciplinary approach to addiction management including involving different professionals in prescribing and managing opioid addictions can enhance clinical outcomes [9]. pharmacists can be instrumental in addiction management because of their role in opioid dispensing and the training they have in medication safety and management [10]. this review explores the role of pharmacists in opioid addiction management including how they can help in harm reduction, prescription of naloxone for overdose treatment, and referral to addiction management resources. prevalence of opioid misuse, abuse, and deaths in the us recent data from the substance abuse and mental health service administration (samhsa), national institute on drug abuse, and centers for disease control and prevention (cdc) show that opioid dependence and deaths are increasing in the us despite the reduction in the number of opioid prescribing [3,4,11]. the last two decades have recorded __________________________________________________ correspondence to: pallav dave, regulatory compliance analyst, 604 la fontenay ct, louisville, ky, usa 40223. email: pallavdav@gmail.com o mailto:pallavdav@gmail.com dave role of pharmacists in opioid addiction management vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 58 significant numbers of individuals affected by the opioid epidemic with the most being young adults [7]. according to the cdc, the number of opioids prescribed to patients increased significantly from the 1990s which corresponds to the rise in the number of overdoses and deaths [12]. approximately, 280,000 people died from prescription-related opioids between 1999 and 2021 [12]. in 1999, only 3,442 deaths were reported compared to 16, 416 deaths reported in 2021 which is an indication of the escalation in the number of deaths attributed to opioids [4]. deaths related to other opioids other than prescription opioids also increased significantly during this period. the cdc reported approximately 645,000 deaths between 1999 to 2021 while another study reported 422 605 deaths between 2011 and 2021 [12,6]. in addition to deaths attributed to opioids, the number of people who misused or abused opioids also increased significantly in the last two decades. in 2022, 8.9 million people who were 12 years and older misused opioids [3]. among these, 1.1 million were aged 18 to 25 years while 7.4 million were adults aged 26 years and older [3]. another 6.1 million people had oud with adults aged 26 years and older accounting for 5.4 million while those aged 18 to 25 accounting for 424,000 [3]. the high number of people misusing, abusing, and dying from opioid-related deaths is attributed to several factors key among them being highvolume prescribing and ease of accessibility of the drugs. the number of opioid prescribing has gone up in the last two decades with 2012 recording a prescription rate of 81.3 per 100 persons [13]. between 2013 to 2016, the us accounted for the largest per-capita consumption of opioids in the world [14,15]. although opioids play a significant role in managing pain, a large number of patients prescribed opioids end up abusing or misusing them. a study by vowles et al [16] established that 21% to 29% of patients who were prescribed opioids ended up misusing them and another 8% to 12% developed oud. despite the decline in the rates of prescribing in recent years, the number of people dying from opioid-related deaths continues to rise showcasing a discrepancy between safe prescribing and opioid assess. the discrepancy could also mean that the number of people abusing other forms of opioids including heroin and synthetic opioids has increased. considering the high rate of misuse and abuse associated with opioids, there is a need to identify individuals at risk of opioid addiction to ensure healthcare professionals are knowledgeable and can be able to intervene before the abuse turns to oud. risk factors for opioid misuse and abuse identifying people who are at risk of opioid misuse or abuse is vital in fighting opioid addiction. identifying these risks makes it possible to assess them and put measures in place to manage them effectively. various factors increase the risk of opioid misuse and abuse including individual factors, demographic factors, pain-related factors, sociodemographic factors, psychosocial factors, drug-related factors, and factors related to alcohol and substance use disorders [17]. although independently these factors are not likely to increase the risk of misuse, a combination of two or three increases the risk significantly. demographically, young, white, and non-urban american males are more likely to have a prescription opioid misuse and abuse problem compared to other population groups [17,18]. however, this could be changing. the recent 2022 national survey showed that rates of misuse were higher among multiracial groups at 4.5%, followed by blacks at 4.1%, and hispanics at 3.4% [3]. whites had a misuse percentage of 3.0% while asians were at 1.5% [3]. one of the factors why prescription opioid abuse is more associated with white men is because the rate of prescription for opioids in emergency departments and primary care practices is much higher [19]. emotional issues and affective distress were also identified as factors that increased the risk of misuse and abuse with women who had these problems being more likely to misuse or abuse opioids [17]. socioeconomic factors also play a crucial role in determining if an individual is likely to have an opioid abuse problem. individuals who do not have health insurance, are unemployed, have work-related accidents or disability, and those who do not graduate high school have a higher risk of abuse [20,21]. one of the most consistent factors associated with the opioid abuse problem is a history of substance use disorders (sud). various studies have found that individuals who have a history of sud are at a higher risk of opioid abuse [22,23]. coincidentally, individuals who experience chronic pain have higher rates of suds [17]. according to a national epidemiologic survey, these rates are higher than those observed in the general population [24]. considering the rates of suds are higher in individuals struggling with chronic pain, it leaves a dilemma on how patients who struggle with pain can be managed effectively using opioids without the risk of addiction. other risk factors that were associated with opioid misuse and abuse were mental health diagnoses including psychotic and anxiety disorders [22,24], pain severity [17], genetic factors [17], and geographical factors. with regard to geographical factors, a high number of opioid prescriptions were mostly concentrated in counties in the western and southern states [25]. besides, hospitalizations related to opioid overdoses between 2000 to 2012 were also concentrated in the south region [18]. controlling for risk factors associated with misuse and abuse is vital in opioid addiction management. for pharmacists, identifying these risk factors is vital in ensuring they are knowledgeable and can be able to intervene to manage opioid addictions. dave role of pharmacists in opioid addiction management vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 59 role of pharmacists in addressing the opioids epidemic addressing the opioid epidemic requires the involvement of different healthcare providers who are in contact with patients being prescribed opioids. in the us, involving community pharmacists in dealing with opioid addiction is instrumental considering they dispense approximately 131 million prescription opioids every year [26]. besides, pharmacists are knowledgeable about the mechanism of action of medications and are in a better place to identify patients who are at risk of addiction. the cdc clinical practice guideline for prescribing opioids for pain recognizes the crucial role of pharmacists in opioid prescription, especially their role in opioid dispensing and naloxone prescribing [27]. various studies highlight the importance of pharmacists in ensuring opioid safety including prevention, surveillance, and treatment of oud [28-30]. with the help of prescription drug monitoring programs (pdmps), pharmacists are better positioned to control prescriptions and identify patients who have a higher risk of opioid addiction [31]. the pdmp enables pharmacists to screen for opioid misuse and discuss opioid-related treatment with patients [32]. in addition to pdmp, pharmacists have access to naloxone which is instrumental in reversing opioid overdose. a study done in canada established that pharmacists play a crucial role in the distribution of naloxone as part of the public health initiative in dealing with the opioid crisis [33]. community pharmacies were accessible to patients and being a key point of naloxone distribution they were instrumental in preventing overdoses. other than emergency treatment, pharmacists can play a crucial role in prevention, which is recognized as instrumental in preventing the likelihood of developing abuse or oud. role of pharmacists in addiction management pharmacists play a crucial role in opioid prescription which makes them better positioned to prevent misuse and abuse. prevention is recognized as instrumental in dealing with substance use disorder and addiction management and can be used as an early intervention measure to deal with opioid addiction [31]. prevention is recognized as instrumental in delaying both the onset and progression of substance use disorders which makes it instrumental in addiction management. successful prevention of opioid addiction and abuse can involve a number of measures including addressing risk factors, screening, education, and discouraging use among other measures. using models such as the screening, brief intervention and referral to treatment (sbirt) model as a guide to prevention can enable pharmacists to achieve optimal outcomes [31]. the model provides a comprehensive approach that allows healthcare providers to carry out screening, prevention, and timely referral to patients who have sud [34]. the model is vital in the management of people with sud including identifying those at risk of developing the problem. by using the sbirt model, pharmacists can be able to screen for people who have high oud risk, initiate discussions about the problem, and provide interventions if necessary. screening is recognized as an instrumental measure in preventing opioid use disorder, especially in clinical and community settings [28]. using screening as an intervention measure for opioid addiction management has a lot of potential with some surveys showing that community pharmacists have a positive attitude towards screening and are interested in using screening to help patients that have oud [35,36]. the sbirt model also recommends brief intervention before referring patients for treatment. the main aim of the brief intervention is to explore an individual’s motivation to change by increasing their insight and awareness about risks associated with substance abuse [31]. pharmacists can play this role effectively considering they are knowledgeable about the risks associated with opioid abuse. they are also better positioned to give advice to patients on the negative effects of prolonged opioid use. pharmacists are also better positioned to know patients who need a referral to treatment resources for oud even though they cannot do the referral themselves [30]. they are more knowledgeable about outpatient treatment centers and can provide this information when necessary. the sbirt model has been proven instrumental in screening, initiation of treatment, and referral of patients that have a substance use disorder but its efficacy is limited because results of clinical trials are mixed [37,38]. despite these limitations, it has various areas of strength that can prove instrumental in addressing opioid use disorder. for instance, it is easy to use and adaptable meaning it can be applied in different healthcare settings [31]. providing training to pharmacists on its proper use can ensure positive outcomes in opioid addiction prevention. education is an additional measure that is reported in the literature as being vital in addiction management. various studies have found a positive correlation between educationrelated activities and positive outcomes when it comes to the prevention of opioid misuse and abuse [10,39,40]. pharmacists are better positioned to provide education to patients because of the important role they play in opioid dispensing. different studies have noted improvements in patient outcomes following education-based interventions [30,39]. in addition to providing education to patients, pharmacists can also be a valuable resource for training and educating healthcare providers who are involved in opioid addiction management [10]. considering their knowledge in pharmacology, they are better placed to provide training and continuing education to other healthcare providers involved in opioid addiction management such as counsellors. their role in providing training is documented in research, particularly dave role of pharmacists in opioid addiction management vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 60 on the proper use of naloxone for patients to law enforcers and other community members [41,42]. pharmacists also play a crucial role in addiction management through safe dispensing. using pdmps is recognised as one measure that can ensure safe opioid dispensing to prevent misuse or abuse. pdmp is a risk mitigation practice that is recognized as instrumental in the effective monitoring of opioid use by healthcare providers [43]. pdmps ensure safe prescribing because they track how controlled substances are prescribed [10]. cdc recognises pdmps as instrumental in ensuring safe prescribing because they provide healthcare providers with timely information about patients’ prescriptions and behaviours that can contribute to misuse or abuse [27]. having access to timely information and patients’ history of prescription opioids can enable pharmacists to have targeted responses to prevent misuse or abuse. considering that more than 50 states have operational pdmps, then it is a critical avenue for timely opioid addiction management. besides, it is recommended by the cdc clinical practice guideline as a measure that can help to determine whether patients are receiving opioid doses that put them at higher risk of overdose. however, when using risk mitigation practices such as pdmp it is important to acknowledge the challenges that pharmacists may face especially when dealing with customers in a customer-service setting and put measures in place to minimize them to be able to achieve optimal outcomes. an additional role that pharmacists can play in addiction management is providing medication-assisted treatment (mat). mat entails the use of the food and drug administration (fda) approved prescription medications to treat opioid addiction [10]. examples of such medications include methadone, naltrexone, and buprenorphine [44]. the use of mat in treating opioid addiction is proven in the literature. different studies have established that the use of these medications is effective in reducing and treating opioid overdoses [44,45]. mat helps to reduce illegal opioid use by reducing the craving for opioids and providing relief for individuals experiencing withdrawal syndrome. when used in treating opioid addiction, the medication has been shown to reduce the number of fatal overdoses [10]. because of their role in opioid dispensing, pharmacists can provide mat and support in substance abuse treatment centers. pharmacists can also deal with opioid addiction through the distribution of naloxone and opioid rescue kits. naloxone plays an instrumental role in opioid overdose reversal and is recognised as an effective measure that can help in addressing the opioid epidemic [31]. the role of pharmacists in naloxone distribution is recognised in research. community pharmacies are recognised as key distribution points for naloxone because of their accessibility to patients [33]. in addition to being key distribution points for naloxone, pharmacists are recognised as instrumental in providing education on their proper use to patients and support persons including law enforcement and members of the community [10,31,41]. through effective naloxone distribution and dispensing, pharmacists can be able to deal with addiction management by preventing overdoses. other additional measures that can help pharmacists deal with opioid addiction include providing medication therapy management (mtm), counselling, and referral to addiction treatment resources. the main aim of mtm is to optimize patient outcomes especially when it comes to effective medication use [46]. this is done by identifying and addressing problems related to the prescribed drug problems such as misuse or mismanagement. pharmacists are in a unique position to do mtm for opioids because of their role in dispensing. by using tools such as pdmp, pharmacists can know patients who are at risk of opioid misuse and abuse and intervene by either recommending medication review or suggesting measures that can help to address the misuse before it escalates. providing counseling is also a measure that can help with opioid addiction management. counseling is mainly done in addiction treatment and rehabilitation centers but pharmacists are in a unique position to provide counselling because of their unique role that allows them to be in contact with patients [47,48,49]. providing counselling on the risks of overdose, for example, can ensure that patients are more knowledgeable on why overdosing is risky. pharmacists can also address the safe disposal of opioids and safe storage [50]. pharmacists are also uniquely positioned to provide referrals to addiction treatment resources. because they often interact with patients, they are in a unique position to identify patients who may have undiagnosed oud and provide referrals to addiction treatment resources [28]. pharmacists are also in a better position to identify resources that are available to patients within the community making them vital in opioid addiction management [50]. chisholm-burns et al. [10] also recognise that pharmacists could be instrumental in referring patients to mat programs and making sure they adhere to treatment. the following review contributes significantly to the existing research by identifying the role of pharmacists as instrumental in dealing with opioid addiction. it provides different interventions that pharmacists can engage in to prevent opioid misuse, abuse, and oud. identifying different ways in which pharmacists can help in dealing with opioid addiction management, cements their role as crucial in dealing with the opioid epidemic being experienced in the us. though the role of pharmacists is recognised as instrumental, several barriers exist that may hinder the effective implementation of the interventions discussed above. for instance, pharmacists may not be better positioned to provide screening and counseling because of the limited duration of interaction with the patients. dave role of pharmacists in opioid addiction management vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 61 pharmacists may also not be better positioned to provide medication-assisted treatment (mat) because their role is limited to dispensing. however, considering the enormous potential that pharmacists possess in opioid addiction management, creating an enabling environment that can increase their readiness to provide the above-discussed interventions is paramount in the opioid epidemic. providing education, training, and putting guidelines in place, for example, are some of the measures that can ensure pharmacists' readiness to reduce opioid misuse and abuse. although this paper clearly outlines the role that pharmacists can play in opioid addiction management, it has its limitations. the scope of this paper mainly focuses on the role of pharmacists in the us which makes it challenging to generalize results to other regions and countries affected by the opioid epidemic. the paper is also limited by the fact that rigorous data extraction is not followed. the paper only includes articles that the author deems applicable to inform this research. conclusion the us is still grappling with the opioid epidemic necessitating measures to manage addiction and prevent overdose deaths. pharmacists are recognised as instrumental in opioid addiction management because of the crucial role they play in opioid dispensing and their direct involvement with patients. this is particularly the case for community pharmacists who are in a position to identify signs of misuse and provide interventions before patients reach the critical point of addiction. pharmacists can provide various interventions including prevention, screening, education, overdose prevention through naloxone prescribing, counseling, and referral to treatment resources. using models such as sbirt can help pharmacists screen for patients who are at risk of addiction, provide brief interventions, and refer them to proper treatment resources. using tools such as pdmp can ensure pharmacists have access to data on patients and are able to identify those who are at an increased risk of opioid misuse and abuse. overall, implementing these measures can play a crucial role in helping to reverse the worrying statistics about opioid prevalence and morbidity. abbreviations: oud: opioid use disorder, samhsa: substance abuse and mental health service administration, cdc: centers for disease control and prevention, pdmp (s): prescription drug monitoring programs, mat: medication-assisted treatment, fda: food and drug administration, sbirt: screening, brief intervention and referral to treatment, sud: substance use disorder, mtm: medication therapy management. references 1. busse jw, wang l, kamaleldin m, et al. opioids for chronic noncancer pain: a systematic review and meta-analysis. jama. 2018; 320(23):2448-2460. 2. trescot am, datta s, lee m, et al. opioid pharmacology. pain physician. 2008; 11(2s): s133. 3. substance abuse, mental health services administration. key substance use and mental health indicators in the united states: results from the 2022 national survey on drug use and health, nsduh series h-58, hhs publication no. pep23-07-01-006. rockville, md: center for behavioral health statistics and quality, substance abuse and mental health services administration; 2023. 4. national institute of drug abuse. drug overdose death rates. 2022. available from: https://nida.nih.gov/research-topics/trendsstatistics/overdose-deathrates#:~:text=opioid%2dinvolved%20overdose%20deaths%20r ose 5. spencer mr, miniño am, warner m. drug overdose deaths in the united states, 2001-2021. nchs data brief, no 457. hyattsville, md: national center for health statistics. 2022. doi: doi.org/10.15620/cdc:122556 6. gomes t, ledlie s, tadrous m, et al. trends in opioid toxicityrelated deaths in the us before and after the start of the covid19 pandemic, 2011-2021. jama. 2023; 6(7): e2322303. 7. gomes t, tadrous m, mamdani mm, et al. the burden of 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https://www.incb.org/documents/narcotic-drugs/technical-publications/2016/narcotic_drugs_publication_2016.pdf https://www.incb.org/documents/narcotic-drugs/technical-publications/2017/narcotic_drugs_technical_%20publication_2017.pdf https://www.incb.org/documents/narcotic-drugs/technical-publications/2017/narcotic_drugs_technical_%20publication_2017.pdf https://www.incb.org/documents/narcotic-drugs/technical-publications/2017/narcotic_drugs_technical_%20publication_2017.pdf dave role of pharmacists in opioid addiction management vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 62 18. unick gj, ciccarone d. us regional and demographic differences in prescription opioid and heroin-related overdose hospitalizations. inter j drug policy. 2017; 46:112-9. 19. pletcher mj, kertesz sg, kohn ma, et al. trends in opioid prescribing by race/ethnicity for patients seeking care in us emergency departments. jama. 2008; 299(1):70-8. 20. han b, compton wm, jones cm, et al. nonmedical prescription opioid use and use disorders among adults aged 18 through 64 years in the united states. jama. 2015; 314(14):1468-78. 21. altekruse sf, cosgrove cm, altekruse wc, et al. socioeconomic risk factors for fatal opioid overdoses in the united states: findings from the mortality disparities in american communities study (mdac). plos one. 2020; 15(1):e0227966. 22. klimas j, gorfinkel l, fairbairn n, et al. strategies to identify patient risks of prescription opioid addiction when initiating opioids for pain: a systematic review. jama. 2019; 2(5):e193365. 23. turk dc, swanson ks, gatchel rj. predicting opioid misuse by chronic pain patients: a systematic review and literature synthesis. the clini j pain. 2008; 24(6):497-508. 24. grant bf, stinson fs, dawson da, et al. prevalence and cooccurrence of substance use disorders and independentmood and anxiety disorders: results from the national epidemiologic survey on alcohol and relatedconditions. archives of general psychiatry. 2004; 61(8):807-16. 25. centers for disease control and prevention. opioid prescribing. where you live matters. 2017. available from: https://www.cdc.gov/vitalsigns/pdf/2017-07-vitalsigns.pdf 26. centers for disease control and prevention. united states dispensing rate maps. 2023. available from: https://www.cdc.gov/drugoverdose/rxrate-maps/index.html 27. dowell d, haegerich tm, chou r. cdc guideline for prescribing opioids for chronic pain-united states. jama. 2016; 315(15):1624-1645. 28. bach p, hartung d. leveraging the role of community pharmacists in the prevention, surveillance, and treatment of opioid use disorders. addiction science & clinical practice. 2019; 14(1):30. 29. hartung dm, johnston ka, hallvik s, et al. prescription opioid dispensing patterns prior to heroin overdose in a state medicaid program: a case-control study. j gen int med. 2020; 35:3188-96. 30. compton wm, jones cm, stein jb, at al. promising roles for pharmacists in addressing the us opioid crisis. research in social and administrative pharmacy. 2019; 15(8):910-6. 31. iwuchukwu of, kim d. prevention better than cure: expanding on pharmacists’ role in prescription opioid overdose and addiction management. american j pharmaco pharmace sci. 2023; 2. 32. hartung dm, hall j, haverly sn, et al. pharmacists’ role in opioid safety: a focus group investigation. pain medicine. 2018; 19(9):1799-806. 33. so r, al hamarneh y, barnes m, et al. the status of naloxone in community pharmacies across canada. canadian pharmacists journal/revue des pharmaciens du canada. 2020; 153(6):352-6. 34. babor tf, mcree bg, kassebaum pa, et al. screening, brief intervention, and referral to treatment (sbirt): toward a public health approach to the management of substance abuse. alcohol/drug screening and brief intervention. 2023; 4:7-30. 35. cochran g, field c, lawson k, et al. pharmacists' knowledge, attitudes and beliefs regarding screening and brief intervention for prescription opioid abuse: a survey of utah and texas pharmacists. j pharmace health servi res. 2013; 4(2):71-9. 36. cochran g, rubinstein j, bacci jl, et al. screening community pharmacy patients for risk of prescription opioid misuse. j addiction med. 2015; 9(5):411. 37. d’onofrio g, chawarski mc, o’connor pg, et al. emergency department-initiated buprenorphine for opioid dependence with continuation in primary care: outcomes during and after intervention. j gen intern med. 2017; 32:660-6. 38. hargraves d, white c, frederick r, et al. implementing sbirt (screening, brief intervention and referral to treatment) in primary care: lessons learned from a multi-practice evaluation portfolio. public health reviews. 2017; 38(1):1-1. 39. gondora n, sanyal c, carter c, et al. the role of pharmacists in opioid stewardship: protocol. res in social and administra pharm. 2021; 17(5):993-6. 40. bratberg jp, smothers zp, collins k, et al. pharmacists and the opioid crisis: a narrative review of pharmacists' practice roles. j american college of clini pharm. 2020; 3(2):478-84. 41. akers jl, hansen rn, oftebro rd. implementing take-home naloxone in an urban community pharmacy. j american pharmac associa. 2017; 57(2): s161-7. 42. duvivier h, gustafson s, greutman m, et al. indian health service pharmacists engaged in opioid safety initiatives and expanding access to naloxone. j american pharmac associa. 2017; 57(2): s135-40. 43. chaudhary s, compton p. use of risk mitigation practices by family nurse practitioners prescribing opioids for the management of chronic nonmalignant pain. substance abuse. 2017; 38(1):95-104. 44. volkow nd, jones eb, einstein eb, et al. prevention and treatment of opioid misuse and addiction: a review. jama psychiatry. 2019; 76(2):208-16. 45. connery hs. medication-assisted treatment of opioid use disorder: review of the evidence and future directions. harvard review psychiatry. 2015; 23(2):63-75. 46. viswanathan m, kahwati lc, golin ce, et al. medication therapy management interventions in outpatient settings: a systematic review and meta-analysis. jama intern med. 2015; 175(1):76-87. 47. cobaugh dj, gainor c, gaston cl, et al. the opioid abuse and misuse epidemic: implications for pharmacists in hospitals and health systems. american j health-system pharm. 2014; 71(18):1539-54. 48. cochran g, gordon aj, field c et al. developing a framework of care for opioid medication misuse in community pharmacy. res social adm pharm. 2016; 12:293-301. 49. tewell r, edgerton l, kyle e. establishment of a pharmacist-led service for patients at high risk for opioid overdose. am j healthsyst pharm. 2018; 75:376-83. 50. thakur t, frey m, chewning b. pharmacist services in the opioid crisis: current practices and scope in the united states. pharmacy. 2019; 7(2):60. how to this article: pallav dave. the role of pharmacists in opioid addiction management. indian j pharm drug studies. 2024; 3(2):57-62. funding: none; conflicts of interest: none stated https://www.cdc.gov/vitalsigns/pdf/2017-07-vitalsigns.pdf https://www.cdc.gov/drugoverdose/rxrate-maps/index.html saralaya an overview on the synthesis of rasagiline and its related scaffolds vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 46 review article a comprehensive review of disclosures in academic journals regarding the synthesis of rasagiline and its closely related compounds along with major biological activity advancements sanjay sukumar saralaya from, assistant professor, department of chemistry, sri dharmasthala manjunatheshwara institute of technology (sdm it), [affiliated to visvesvaraya technological university (vtu), belagavi], ujire, belthangady taluk, dakshina kannada, karnataka, india. abstract this review article was primarily focused to unwind the prior art disclosures about the synthetic strategies adopted for the preparation of rasagiline and its closely related scaffolds. additionally, some of the major biological activity advancements of rasagiline and its derivatives were also given the priority. it was observed that, propargyl part/ indane part/ or both of rasagiline was extended through numerous synthetic pathways to achieve enhanced biological activity. additionally, numerous resolution routes were also executed to isolate the intended product with high chiral purity. hence, this review contribution can be a good platform for the worldwide researchers to design new routes to synthesize rasagiline and its structurally close resemblance derivatives. key words: rasagiline, indane moiety, propargyl moiety, mesylation, resolution, synthesis, isolation. an overview of the drug information, medical use and (maob) inhibition. rasagiline r is a propargylamine based. rasagiline r is a propargylamine based drug prescribed for the treatment of idiopathic parkinson's disease [1, 2]. it has been marketed around the globe with the trade names as azipron®, azilect® tablets, having the active pharmaceutical ingredient rasagiline (as mesylate) along with other associated excipients [3]. it is a renowned irreversible inhibitor of monoamine oxidase-b (mao-b) [4-6]. rasagiline mesylate rm has the iupac nomenclature as 1h-inden-1amine,2,3-dihydro-n-2-propynyl-(1r)-methanesulfonate with an empirical of (c12h13n)ch4so3 and molecular weight (mw: 267.34 g/mol). it is a white to off-white powder, freely soluble in water or ethyl alcohol and sparingly soluble in isopropyl alcohol [7, 8]. the compound agn-1135 (n-2-propynyll-indanamine-hydrochloride) was reported to be an irreversible mao-b inhibitor with selectivity in the rat in-vivo [9, 10]. systematic exfoliation of past disclosures numerous synthetic routes were disclosed in various patent publications to isolate r and its associated compounds. access this article online received – 29th mar 2024 initial review – 04th apr 2024 accepted – 07th may 2024 quick response code a recent review article was published with details of all those patent disclosures towards the synthetic aspects of r/rm/rh and its closely related compounds (salts/impurities/forms) [11]. this work was selective towards the prior art disclosures in academic journals about the synthesis and biological activity studies towards r and its closely related compounds. youdim mbh and coworkers had disclosed a commercial synthetic route to isolate r and its mono-fluorinated derivatives along with an extensive biological application studies as patent applications in united states patent and trade mark office [12, 13]. lawson wb & rao gjs, had reported the resolution and configurations studies of 1-aminoindan or 2,3-dihydro-1h-inden-1-amine 1 using l-malic acid or 2-hydroxybutanedioic acid 2 in absolute ethanol to obtain its optical isomers [14] or the same can be achieved by the formation of diastereomeric salts [15]. nh ch h nh ch h s o o oh ch3 r rm nh ch h rh hcl figure 1: rasagilne r-form and its popular salts. __________________________________________________ correspondence to: sanjay ss, assistant professor, department of chemistry, sdm it, opposite to siddhavana gurukula, dharmasthala main road, ujire, belthangady taluk, dakshina kannada, karnataka, india. email: sanjayss@sdmit.in mailto:sanjayss@sdmit.in saralaya an overview on the synthesis of rasagiline and its related scaffolds vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 47 nh2 1 oh oo oh oh hh h 2 figure 2: key starting material 1 and the resolving agent 2 maruyama w & coworkers had reported the antiapoptotic function of r and (1s)-n-(prop-2-yn-1-yl)-2,3dihydro-1h-inden-1-amine 3. this initiative had established the role of these compounds to suppress the activation of caspases and dna fragmentation [16, 17]. nh ch 3 figure 3: structure of rasagiline s-form youdim mbh & coworkers had demonstrated the role of r as a selective and potent inhibitor of mitochondrial mao-b. additionally the work reports the metabolite/s of r (metabolite is 1) and n-methyl-n-(1-phenylpropan-2-yl)prop2-yn-1-amine; selegiline 4 (metabolites are 1-phenylpropan-2amine 5 and n-methyl-1-phenylpropan-2-amine 6 [18]. n ch3 ch3 ch nh2 ch3 4 5 nh ch3 ch3 6 + metabolites r metabolite 1 figure 4: structure of rasagiline & selegiline metabolites youdim mbh & coworkers had demonstrated the molecular basis of neuroprotective activities of r and three other related compounds {((3r)-3-(prop-2-yn-1-ylamino)-2,3dihydro-1h-inden-5-yl ethyl (methyl) carbamate 7, (3s)-3(prop-2-yn-1-ylamino)-2,3-dihydro-1h-inden-5-yl ethyl (methyl) carbamate 8 and 3-[(1r)-1-(dimethylamino) ethyl]-2, 3-dihydro-1h-inden-5-yl ethyl (methyl) carbamate 9; rivastigmine} [19]. akao y & coworkers had illustrated the antiapoptotic potential of r [20]. nh ch on o ch3 ch3 7 nh ch on o ch3 ch3 8 on o ch3 ch3 ch3 n ch3 ch3 9 figure 5: rasagiline related compounds as per [18] maruyama w & coworkers had illustrated the antiapoptotic capabilities of four aliphatic compounds like {(2r)n-methyl-n-(prop-2-yn-1-yl)heptan-2-amine 10, (2s)-nmethyl-n-(prop-2-yn-1-yl)heptan-2-amine 11, (2r)-n-(prop2-yn-1-yl)heptan-2-amine 12 and 3-[(2r)-heptan-2ylamino]propanoic acid 13} [21]. this work was influenced by the initiatives from yu ph & coworkers [22] and berry md & boulton aa [23]. ch3 n ch3 ch3 ch ch3 n ch3 ch3 ch ch3 n h ch3 ch ch3 n h ch3 o oh 10 11 12 13 figure 6: aliphatic amines as per [21] sterling j & coworkers had reported the synthesis, characterization and biological activity studies (against alzheimer’s disease) of carbamate derivatives of npropargylaminoindans and n-propargylphenethylamines. the work had established the fact that, n-methylation of propargylamine enhanced the mao-a & b inhibitory activities and decreased the ache inhibitory activity [24]. maruyama w & coworkers had illustrated the anti-apoptotic action determination of r and its derivatives like 7, 8, 9, (3r)3-(prop-2-yn-1-ylamino)-2,3-dihydro-1h-inden-5-ol 14 and 2,3-dihydro-1h-inden-5-yl ethyl(methyl)carbamate 15. the experimental outcomes suggested that, anti-apoptoticneuroprotective activity was found to reside in the propargylamine extension and certainly not in the carbamate moiety. in the context, both 7 & 8 were found to be as effective as r to protect dopaminergic sh-sy5y cells against apoptosis induced by the peroxynitrite donor. the donated compounds used for the experimentation were prepared by teva pharmaceuticals (netanya, israel) [25]. nh ch oh 14 on o ch3 ch3 15 figure 7: rasagiline related compounds as per [25] yogev-falach m and coworkers had demonstrated the use of 7 & 8 for the treatment of alzheimer's disease [26]. guillon j & coworkers had demonstrated the multi-step synthesis, configurational studies and the preliminary reports for mao-b inhibition by some novel n-propargyl-3-pyrrol-1ylindanamine derivatives [27]. binda c & coworkers had reported the complexation, binding mode studies and highresolution crystal structure details of four compounds such as saralaya an overview on the synthesis of rasagiline and its related scaffolds vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 48 r, 3, 13 and (1r)-n-methyl-n-(prop-2-yn-1-yl)-2,3-dihydro1h-inden-1-amine 16 with mao-b inhibition studies [28]. n ch ch3 16 figure 8: rasagiline related compound as per [28]. hubálek f & coworkers had illustrated the spectroscopic and the kinetics of inhibitory activity of five compounds such as r, 3, 11, 13 & 16. the study outcome suggests that, maob and mao-a are more selective towards r than s-enatiomer 3 by 2500-fold and 17-fold, respectively [29]. waibel s & coworkers had reported the clinical combination of r and riluzole to enhance neuroprotective treatment strategies of amyotrophic lateral sclerosis (als) [30]. bar-am o & coworkers had illustrated the in-vivo regulation of protein kinase-c by r and its derivatives 7 & 8 [31]. parkinson study group had established the importance of r based on clinical trials to treat parkinson’s disease [32, 33]. rascol o & coworkers had reported the clinical study details of r in comparison with the catecol-o-methytransferase inhibitor, entacapone, for the treatment of parkinson’s disease [34]. youdim mbh & coworkers had compiled a comprehensive review article on the pharmacological activity of r and the fundamental molecular mechanism behind its neuroprotective and neurorescue activities [35]. o o n ch3 ch3 n ch3 ch o o n ch3 ch3 nh ch nh2 nh ch3 nh ch3 on o ch3 ch3 n ch3 ch 17 18 1-r isomer 1-s isomer 19 20 21 nh2 nh ch oh 22 figure 9: rasagiline related compounds as per [36]. binda c & coworkers had illustrated the binding of r and its related compounds to human maos. the work had established the crystallographic analysis of complexes and the kinetics of inhibition process. the compounds used for the studies are r, 1-r isomer, 1-s isomer, (1r)-1-[methyl(prop-2yn-1-yl)amino]-2,3-dihydro-1h-inden-4-yl ethyl(methyl)carbamate 17, (1r)-1-(prop-2-yn-1-ylamino)2,3-dihydro-1h-inden-4-yl ethyl(methyl)carbamate 18, (1r)n-methyl-2,3-dihydro-1h-inden-1-amine 19, (1s)-n-methyl2,3-dihydro-1h-inden-1-amine 20, (3r)-3-[methyl(prop-2-yn1-yl)amino]-2,3-dihydro-1h-inden-5-yl ethyl(methyl)carbamate 21 and (1r)-1-(prop-2-yn-1-ylamino)2,3-dihydro-1h-inden-4-ol 22. all the compounds used for the tests were received from teva pharmaceuticals [36]. youdim mbh & coworkers had reported a collective review contribution on the therapeutic potential of mao inhibitors, which includes many molecules along with r and its related compounds [37]. oldfield v & coworkers had prepared a review article covering all the disclosures of r towards its effectiveness to treat parkinson’s disease [38]. gallagher da & schrag a had compiled a review work covering the newer pharmacological treatments on the quality of patients with parkinson’s disease. in that coverage, r had improved hr-qol as monotherapy in early parkinson's disease (based on one study). meanwhile, it was not completely in more advanced state of the disease (based on one study) [39]. to improve the yield and purity of rm, tatendra rk & coworkers had reported a new process to manufacture rm using (1s)-2,3-dihydro-1h-inden-1-ol 23. the condensation of 23 with 4-methylbenzenesulfonyl chloride 24 was achieved in the presence of triethylbenzylammonium chloride (tebac), sodium hydroxide solution and toluene to isolate (1r)-2,3-dihydro1h-inden-1-yl 4-methylbenzenesulfonate 25. it was treated with prop-2-yn-1-amine hydrochloride 26 (to be made free base by the addition of ammonia solution) under the catalytic impact of di-potassium hydrogen phosphate (kh2po4) and tebac in toluene to get the residue (crude r). to the residue, added isopropyl alcohol and treated with methanesulfonic acid (ch3so3h) to isolate rm. this process was efficient to manufacture enatiomerically pure rm in reasonably high yield [40]. oh + s ocl o ch3 toluene, h 2o o s o o ch3 ch nh2 .hcl kh2po4, tebac, toluene ipa, ch3so3h, h2o nh ch .ch3so3hrm 23 24 25 26 92.0% 79.0% tebac, naoh scheme 1: route of synthesis to isolate rm from 23 as per [40]. numerous synthetic routes were reported in prior arts for the isolation of r and its salts but the recovery and saralaya an overview on the synthesis of rasagiline and its related scaffolds vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 49 reconversion of unwanted enatiomer 3 was not attempted. in this context, kapubalu sk & coworkers had reported an efficient one-pot racemization of 3 with high conversion rate and purity. the reported process involves the addition of potassium hydroxide and n,n-dimethylsulfoxide (dmso) to the residue comprising the unwanted 3 and heated to 800c. it was maintained at the same temperature under agitation for 2 h and then cooled, quenched to water and extracted with dichloromethane (dcm). upon solvent distillation, light brown colored oily mass of r was isolated. this work had adopted the process optimization strategy to finalize the above mentioned base, solvent and temperature for the efficient racemization [41]. this sort of racemization was attempted for the first time since the process to isolate the racemic mixture of r (free base or as hydrochloride salt) and other related compounds were reported by gittos mw & coworkers [42, 43]. nh ch 3 koh, dmso, h 2o, dcm nh ch r 85.0% scheme 2: disclosed resolution pathway of 3 to form r as per [41]. alonso n & coworkers had demonstrated the synthesis, characterization and the interesting neuroprotective effects of some novel carbamate derivatives of r. as per the disclosure, (1s,3r)-3-(prop-2-yn-1-ylamino)-2,3-dihydro-1h-inden-1-ol 27 or (1r,3r)-3-(prop-2-yn-1-ylamino)-2,3-dihydro-1hinden-1-ol 32 or (1s,3r)-3-[di(prop-2-yn-1-yl)amino]-2,3dihydro-1h-inden-1-ol 35 or (1r,3r)-3-[di(prop-2-yn-1yl)amino]-2,3-dihydro-1h-inden-1-ol 38 in acetonitrile was treated with dimethylcarbamic chloride 28 or diethylcarbamic chloride 29 in the presence of sodium hydride (nah) to isolate eight carbamate derivatives of r. the novel compounds isolated are (1r,3s)-3-(prop-2-yn-1-ylamino)-2,3-dihydro-1hinden-1-yl dimethylcarbamate 30, (1r,3s)-3-(prop-2-yn-1ylamino)-2,3-dihydro-1h-inden-1-yl diethylcarbamate 31, (1s,3s)-3-(prop-2-yn-1-ylamino)-2,3-dihydro-1h-inden-1-yl dimethylcarbamate 33, (1s,3s)-3-(prop-2-yn-1-ylamino)-2,3dihydro-1h-inden-1-yl diethylcarbamate 34, (1s,3r)-3[di(prop-2-yn-1-yl)amino]-2,3-dihydro-1h-inden-1-yl dimethylcarbamate 36, (1s,3s)-3-(hepta-1,6-diyn-4-yl)-2,3dihydro-1h-inden-1-yl diethylcarbamate 37, (1r,3r)-3[di(prop-2-yn-1-yl)amino]-2,3-dihydro-1h-inden-1-yl dimethylcarbamate 39 and (1r,3r)-3-[di(prop-2-yn-1yl)amino]-2,3-dihydro-1h-inden-1-yl diethylcarbamate 40. these compounds were tested for the high-throughput screening of multitarget drugs in chemical neurosciences. three compounds (34, 36 & 39) had exhibited reasonably high neuroprotective effects [44]. this work was inspired from gonzález-díaz h & coworkers, since they disclosed the synthesis, characterization and assay of mao-b inhibitors. they synthesized 3-hydroxy, acetate or benzoate derivatives mono/di-propargyls starting from benzaldehyde [45]. the same team (luan f & coworkers) had extended their work towards the study of 1,3-derivatives of r which are potentially useful in neurodegenerative diseases. the derivatives were synthesized from benzaldehyde through the multistep pathway, they are 27, 32, 35, 38, (1s,3r)-3-[di(prop-2-yn-1yl)amino]-2,3-dihydro-1h-inden-1-yl acetate 41, (1r,3r)-3[di(prop-2-yn-1-yl)amino]-2,3-dihydro-1h-inden-1-yl acetate 42, (1s,3r)-3-[di(prop-2-yn-1-yl)amino]-2,3-dihydro-1hinden-1-yl benzoate 43 and (1r,3r)-3-[di(prop-2-yn-1yl)amino]-2,3-dihydro-1h-inden-1-yl benzoate 44 [46]. nh ch oh no cl ch3 ch3 nah, acn no cl ch3 ch3 nh ch o o n ch3 ch3 nh ch o o n ch3 ch3 nh ch oh nah, acn nh ch o o n ch3 ch3 nh ch o o n ch3 ch3 27 28 29 30 31 32 28 29 33 34 scheme 3a: synthesis of rasagilne related compounds as per [44-46]. rodríguez-borges j & coworkers had reported the synthesis and characterization of novel propargylated 1-pyrindane derivatives. the isolated compounds having close structural resemblance to r are 6,7-dihydro-5h-cyclopenta[b]pyridine 1oxide 45, 6,7-dihydro-5h-cyclopenta[b]pyridin-7-yl acetate 46, 6,7-dihydro-5h-cyclopenta[b]pyridin-7-ol 47, 7-(prop-2yn-1-yloxy)-6,7-dihydro-5h-cyclopenta[b]pyridine 48, 5,6dihydro-7h-cyclopenta[b]pyridin-7-one 49, n-(prop-2-yn-1yl)-6,7-dihydro-5h-cyclopenta[b]pyridin-7-amine 50 and n,ndi(prop-2-yn-1-yl)-6,7-dihydro-5h-cyclopenta[b]pyridin-7amine 51 [47]. saralaya an overview on the synthesis of rasagiline and its related scaffolds vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 50 n ch oh ch nah, acn n ch o o n ch3 ch3 ch ch o o n ch3 ch3 ch n ch oh ch nah, acn n ch o o n ch3 ch3 ch nch o o n ch3 ch3 ch 35 36 37 38 39 40 28 29 28 29 scheme 3b: synthesis of rasagiline related compounds as per [44-46]. nh ch oh nh ch oh n ch oh ch n ch oh ch n ch oac ch n ch oac ch n ch obz ch n ch obz ch 27 32 35 38 41 42 43 44 figure 10: rasagiline related compounds as per [44-46]. n + o n o o ch3 n oh n o ch n o n nh ch n n ch ch 45 46 47 48 49 50 51 figure 11: rasagiline related compounds as disclosed by [47]. ma g & coworkers had illustrated the novel seven-step synthetic route to prepare rm via kinetic resolution (kr) or dynamic kinetic resolution (dkr), catalyzed by candida antarctica lipase b (calb) and palladium-nanocatalyst. 3phenylpropanoic acid 52 was treated with poly phosphoric acid (ppa) to isolate 2,3-dihydro-1h-inden-1-one 53. it was treated with hydroxylamine hydrochloride (nh2oh. hcl) in alkaline medium to get (1e,z)-n-hydroxy-2,3-dihydro-1hinden-1-imine 54. it was reduced under the impact of pd/c to get 1. it was condensed with isopropyl 2-methoxyacetate and racemized under special conditions to isolate n-[(1r)-2,3dihydro-1h-inden-1-yl]-2-methoxyacetamide 55 and then 1-r isomer. it was condensed with propargyl bromide in the presence of potassium carbonate (k2co3) and acetonitrile to form r. it was treated with methanesulfonic acid in isopropyl alcohol to get rm (overall yield: 25.0%) with an excellent enantioselectivity [48]. the reported process was much superior to the previous disclosures. the racemization of 1 is much superior to the disclosures by colyer jt & coworkers. as per the disclosure, numerous n-tert-butanesulfinyl imines were reduced in the presence of sodium borohydride (nabh4) in tetrahydrofuran and water to obtain the corresponding secondary sulfinamides in high yield and diastereoselectivity. by the use of l-selectride instead of nabh4 the stereoselectivity was effectively reversed to provide the opposite diastereomer product in high yield and selectivity. the condensation of 53 with 2-methylpropane-2-sulfinamide 56 was achieved in the presence of titanium(iv)ethoxide {ti(oet)4} to isolate n-[(1e,z)-2,3-dihydro-1h-inden-1ylidene]-2-methylpropane-2-sulfinamide 57. it was reduced by nabh4 to isolate n-[(1r)-2,3-dihydro-1h-inden-1-yl]-2methylpropane-2-sulfinamide 58. the use of l-selectride for the reduction of 57 had resulted in the formation of n-[(1s)2,3-dihydro-1h-inden-1-yl]-2-methylpropane-2-sulfinamide 59. both 58 and 59 distinctly can be brought under the influence of hydrochloric acid to isolate respective 1-r isomer and 1-s isomer [49]. numerous synthetic pathways were disclosed to prepare rm through the formation of 1-r isomer or r. the adopted methodologies includes many patents, a few sectorial pathways are; the resolution driven chiral acids [41, 50], hydrosilylation by the use of chiral rhodium and ruthenium catalysts [51-53], asymmetric synthesis using chiral moieties [49, 54-57], biocatalyst based deracemization with cyclohexylamine oxidase (chao) [58], kinetic or dynamic kinetic enzymatic resolution [48, 59-62] and configuration inversion [40]. to overcome the past chemistry and process related issues, fonseca t de s & coworkers had reported a lipase driven chemoenzymatic route to prepare rm. the reduction of 53 under the impact of nabh4 in methanol gave (r,s) 2,3-dihydro-1h-inden-1-ol 60. it was acetylated and hydrolyzed in the presence of lipase in hexane to obtain 23. further reaction of 23 gave (1r)-1-azido-2,3-dihydro-1hindene 62, it was then converted to 1-r isomer. addition of saralaya an overview on the synthesis of rasagiline and its related scaffolds vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 51 propargyl chloride to 1-r isoler in the presence of k2co3 and acetonitrile gave r. it was treated with methanesulfonic acid in isopropyl alcohol resulted in formation of rm. the disclosed biocatalytic process was very effective to impart rconfiguration by lipase-mediated kinetic resolution [63]. this work was more inspired by the initiatives earlier by fernández r & coworkers [64] and lin fl & coworkers [65]. a review article by carvalho acl de m & coworkers had covered all the disclosures from 2007-2015 regarding the lipase mediated synthesis of drug molecules [66]. oho ppa 98.2% o nh2oh. hcl naoh, etoh, h 2o 89.9% n oh nh2 pd/c, h2, etoh 93.5% calb, pd, toluene acyl moiety 80.1% nh o o ch3 naoh triethanolamine 76.2% nh2 propargyl bromide k2co3, acn 59.7% nh ch rm ipa methanesulfonic acid 83.1% 52 53 54 1 551-r isomerr scheme 3a: disclosed route of synthesis to obtain rm from 52 as per [48]. o 53 s nh2 ch3 ch3 ch3 o + 56 ti(oet) 4 n so ch3 ch3 ch3 nabh4 nh so ch3 ch3 ch3 hcl 1-r isomer 57 58 84.0% 57 l-selectride nh so ch3 ch3 ch3 59 90.0% hcl 1-s isomer scheme 3b: disclosed route of synthesis to isolate pure isomeric forms if 1 as per [49]. o oh oac oh n3nh2 + rrm nabh4 meoh 86.0% lipase hexane vinoac dppa, dbu toluene 70.0% pph3, koh thf, h 2o 70.0% propargyl chloride k2co3, acn 79.0% ch3so3h ipa 98.0% 53 60 61 23 621-r isomer scheme 4: disclosed route of synthesis to isolate rm from 53 as per [63]. sousa cad & coworkers had reported the chemical/enzymatic resolution driven synthetic route to prepare enantiomerically pure propargyl ethers {(7r)-7-(prop2-yn-1-yloxy)-6,7-dihydro-5h-cyclopenta[b]pyridine} 63 and {(7s)-7-(prop-2-yn-1-yloxy)-6,7-dihydro-5hcyclopenta[b]pyridine} 64. the importance of chiral center in r for the biological activity had influenced the isolation of these enatiomers in pure form [67]. the same team had earlier disclosed the synthesis of racemic mixture of the present disclosed compounds [47]. n o ch n o ch 63 64 figure 12: rasagiline related compounds as per [67]. rizzo-aguiar f & coworkers had demonstrated the synthesis and characterization of some analogues of r. the isolated compounds had 1-pyrindane moiety at c-7 position to have the respective propargyl 48 and isobutyl 67 ethers, propargyl 50, n-propyl 68, isopropyl 69 and cyclopropyl 70 amines and n,n-dimethyl 65 and diethylcarbamoyl 66 derivatives [68]. n o n o ch3 ch3 n o n o ch3 ch3 n o ch n o ch3 ch3 n nh ch n nh ch3 n nh ch3 ch3 n nh 65 66 48 67 50 68 69 70 figure 13: rasagiline related compounds disclosed as per [70]. aguilar n & coworkers had reported the synthesis of the r,r-tartrate salt of r using simple, abundant and inexpensive chemicals. it was developed with an aim for the practice at routine lab sessions (4 h) of undergraduates. the work involves the condensation of 1 with propargyl benzenesulfonate 71 under alkaline medium in the presence of phase transfer catalyst to obtain the racemic oil of drug. the crude oily mass was dissolved in methanol/isopropyl alcohol and treated with r,r tartaric acid to isolate the solid r-r,r tartarate and the other diasteriomer 3-r,r tartarate will be in the mother liquor [69]. nh2 + .hcl s oo o ch naoh, tbab, h 2o nh ch tartaric acid resolution r-r,r tartarate + 3-r,r tartarate avrg yield: 45.0% racemic oil1 71 ppt scheme 5: simple route of rasagiline synthesis from 1 as per [69]. saralaya an overview on the synthesis of rasagiline and its related scaffolds vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 52 an extended work of the past disclosure [70], sun y & coworkers had disclosed the identification, characterization and synthesis prop-2-yn-1-yl (1r)-2,3-dihydro-1h-inden-1ylcarbamate (impurity-a) and prop-2-yn-1-yl (1r)-2,3dihydro-1h-inden-1-yl(prop-2-yn-1-yl)carbamate (impurityb) of r. it was prepared and isolated by the reaction of 1-r isomer with prop-2-yn-1-yl methanesulfonate 71 [71]. nh2 s o o o ch3 ch + k2co3 co2 r + nh o o ch + n o o ch ch 1-r isomer 71 impurity-a impurity-b scheme 6: disclosed route of synthesis to isolate rasagiline impurities from 1-r isomer as per [71]. brenna d & coworkers had illustrated the flow process to isolate 1-r isomer from 53 via imine formation 72r and its stereo-selective metal free reduction 73r [72]. o 53 n r hsicl 3, lb nh r 1-r isomer 72r 73r scheme 7: preparation of 1-r isomer from 53 as per [72]. matzel p & coworkers had reported the synthesis of r, 3 and other compounds (selegiline and pramipexole) in a single step asymmetric synthesis by reductive amination in the presence of imine reductases (irs). this method is an efficient route for the synthesis of pharmaceutically active scaffolds comprising chiral secondary and tertiary amines [73]. o 53 nh2 ch+ ir-14 58% r o 53 nh2 ch+ ir-sip 81% 3 scheme 8: synthesis of rasagiline & its s-isomer from 53 as per [73]. raju nm & coworkers had reported the synthesis and structural elucidation of some major impurities of r. treatment of 53 with ammonium formate (hco2nh4) and reduction from zinc dust gave 1. the addition of 3bromoprop-1-ene to 1 in the presence of potassium carbonate in acetonitrile had resulted in the formation of n-(prop-2-en-1yl)-2,3-dihydro-1h-inden-1-amine 74 (low yield and purity). in another example, 53 in chloroform was treated with nbromo succinamide in the presence azobisisobutyronitrile to get 3-bromo-2,3-dihydro-1h-inden-1-one 75. it was condensed with prop-2-yn-1-amine hydrobromide in the presence of potassium carbonate in acetonitrile to obtain 3(prop-2-yn-1-ylamino)-2,3-dihydro-1h-inden-1-one 76. in another demonstration, rs was treated with lindlar’s catalyst in the presence of pyridine to isolate 74 (good yield and purity). similarly, the treatment of rs with d-tartaric acid in alcohol medium resulted in the isolation of 3. in line to the context, treatment of rs with concentrated hydrochloric acid gave two products like n-(2-chloroprop-2-en-1-yl)-2,3dihydro-1h-inden-1-amine 77 and n-[(2e,z)-3-chloroprop-2en-1-yl]-2,3-dihydro-1h-inden-1-amine 78 [74]. o 53 o 53 hco 2nh4/zn/ch 3oh nh2 k2co3/ch3cn ch2 br nh ch2 nbs/aibn, chcl 3 o br k2co3/ch3cn ch nh2 . hbr nh ch o 1 74 75 76 scheme 9a: synthesis of rasagiline related compounds as per [74]. nh ch rs lindlar catalyst pyridine/ch 3oh 74 d-tartaric acid ipa/ch3oh 3 lindlar catalyst pyridine/ch 3oh nh ch2 cl + nh cl 77 78 scheme 9b: synthesis of rasagiline related compounds as per [74]. sun h & coworkers had reported a review article about the use of various biocatalysts like reductases, oxidases, hydrolases, lyases, isomerases and transaminases for the preparation of active pharmaceutical ingredients including r [75]. this work includes the details furnished in the previous disclosure regarding the chemo-enzymatic pathway to synthesize r [63]. albarrán-velo j & coworkers had saralaya an overview on the synthesis of rasagiline and its related scaffolds vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 53 contributed a review article regarding the use of bio-catalysts for the stereo-selective asymmetric synthesis of active pharmaceutical ingredients [76]. this work had highlighted some of the past initiatives [66, 67, 73] towards the synthesis of r via bio-catalysm pathway. galvão ws & coworkers had reported the kinetic resolution of secondary alcohols (including 60 to isolate the intermediate for r synthesis) using nano-hydrid bio-catalysts [77]. oh oh + oac fe3o4@aptes-glu-pf vin oac, hexane 60 23 61 scheme 10: resolution pathway to isolate 23 and 61 from 60 as per [77]. xiao x & coworkers had demonstrated the design, synthesis and biological activity estimation of several derivatives of r having various linkers like -och2-, -sch2-, och2ch2-, -och2ch2o-, -och2ch2ch2oetc. this work had established 79 (d14) as the promising derivative with a similar inhibitory activity as r with an improved iso-form selectivity [78]. nh o ch nh o cl 79 figure 14: rasagiline related compound 79 disclosed as per [78]. pérez-venegas m & coworkers had the use of candida antarctica lipase b (calb) in the kinetic resolution of racemic chiral amines. this work had contributed to an efficient and easily scalable process to manufacture r with high chiral purity. this disclosure involves the conversion of 1 to 3 and n-[(1r)-2,3-dihydro-1h-inden-1-yl]acetamide 80 by the use of ethyl acetate and prop-2-yn-1-yl methanesulfonate. aqueous hcl treatment to 80 and then the reaction with prop2-yn-1-yl methanesulfonate gave r [79]. avila-ortiz cg & coworkers had reported a review article regarding the utility of mechanochemistry in enantioselective synthesis. this work had emphasized the role of enzymes for the resolution of amino acids and amines [80], thus covering the past disclosure to prepare r [79]. elshorbagi a-n & coworkers had contributed a comprehensive review article on the management of parkinson’s disease (pd), enriched with drug discovery and pharmacological approaches of numerous pd specific drugs [81]. guieu b & coworkers had demonstrated the synthesis, characterization and biological activity studies of racemic transpropargylamino-donepezil 81 [82]. nh2 candida antarctica lipase b (calb) acoet, dioxane ch o s ch3 o o nh ch3 o + nh ch 1 380 aq. hcl, reflux 1-r isomer r scheme 11: synthesis of r from 1 as per [79]. o ch3 o ch3 o nh ch n 81 figure 15: rasagiline related compound 81 disclosed as per [82]. li j & coworkers had illustrated the synthesis of chiral helic[1]triptycene[3]arenes and their enantio-selective recognition towards chiral aminoindan groups like 1 r-isomer, 1 s-isomer, r and 3 [83]. ramachandran pv & coworkers had reported the synthesis of racemic r from 53 using the specific catalyst system {(ch3o)3b and ammonia-borane} [84]. o + ch nh2 (ch3o)3b, ammonia-borane 92% racemic r 53 scheme 12: synthesis of racemic rasagiline from 53 as per [84]. ying p & coworkers had reported a review article regarding the pathway of liquid-assisted grinding mechanochemistry in the synthesis of active pharmaceutical ingredients. this article had covered the preparation of r and its s-isomer 3 from 1 [85]. zhang k & coworkers had demonstrated the use of acredam through rational design to obtain highly stereo-selective mutants. the best mutant formed could synthesize r from 53 in moderate yield with high enantiomeric purity [86]. dugarte-dugarte aj & coworkers had illustrated the characteristic hydrogen bonding patterns and c-h....π interactions in the structure of rm was saralaya an overview on the synthesis of rasagiline and its related scaffolds vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 54 determined using laboratory and synchrotron x-ray powder diffraction data aided with dft calculations [87]. summary this review contribution was empowered with twelve reaction schemes (scheme 1 to 12) being furnished for a better understanding of the synthetic pathways disclosed by various researchers. similarly, eighty one compounds (figure 1 to 15) were sequentially numbered to provide a systematic flow for the disclosed compounds related to r. in most of the circumstances, academic journal disclosures are given the priority during the literature survey by excluding the contents published in patents. hence, an exclusive patent focused contribution was furnished towards the synthesis of r [11]. this review article was focused mainly on the academic disclosures towards the synthesis of r and its close resemblance scaffolds. conclusion this review article provides a glimpse of disclosed details in prior arts towards the synthesis of rasagiline and some of its close resemblance scaffolds. this work had given the importance to exfoliate the disclosed information from the academic journals and hence patents are intentionally excluded. this work could serve as a vital template for the researchers around the globe to design the synthesis of scaffolds related to rasagiline core moiety. in recent times, special emphasis was given by the researchers to adopt green chemistry pathway for the synthesis of rasagiline with high enantio-selectivity. references 1. sterling j, veinberg a, lerner d, et al. 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using laboratory and synchrotron x-ray powder diffraction data. acta crystallogr b struct sci cryst eng mater. 2023; 79(6):462–72. how to cite this article: saralaya ss, a comprehensive review of disclosures in academic journals regarding the synthesis of rasagiline and its closely related compounds along with major biological activity advancements. indian j pharm drug studies. 2024; 3(2):46-56. funding: none; conflicts ofinterest: none stated https://patents.google.com/patent/%20cn103804200a/en?oq=+cn103804200a https://patents.google.com/patent/%20cn103804200a/en?oq=+cn103804200a http://dx.doi.org/10.7324/%20japs.2020.1010015 http://dx.doi.org/10.7324/%20japs.2020.1010015 http://dx.doi.org/10.1002/adsc.202001245 http://dx.doi.org/10.3389/fbioe.2021.%20798147 shanbhag et al. cancer therapy: an overview of pharmacogenomics strategies vol 2| issue 2 | apr – jun 2023 indian j pharm drug studies | 63 review article the potential of sirna-mediated oncogene silencing in cancer therapy: an overview of pharmacogenomics strategies preeti shanbhag1, ramdas bhat2, a r shabaraya3 from 1pg scholar, 2assistant professor, department of pharmacology, 3principle and head of the department of pharmaceutics, srinivas college of pharmacy, valachil, post farangipete, mangalore, karnataka, india-574143. correspondence to: ramdas bhat, assistant professor, department of pharmacology, srinivas college of pharmacy, valachil, post farangipete, mangalore, karnataka, india. email: ramdas21@gmail.com tel.: +91 7795772463 abstract sirna-mediated silencing of oncogenes holds great promise as a potential cancer therapy. by selectively silencing genes involved in cancer progression, sirna-based therapies have the potential to be highly specific and effective, while minimizing off-target effects and toxicity. however, several challenges need to be addressed before sirna-based therapies can be widely used in clinical settings. one of the major challenges is the development of efficient and safe delivery systems for sirnas. additionally, more clinical trials are needed to evaluate the safety and efficacy of sirna-based therapies for cancer treatment. despite these challenges, ongoing research and development in the field of sirna-based therapies are promising. new delivery systems, personalized medicine approaches, and combination therapies are being explored, which could potentially lead to more effective and personalized cancer treatment. with continued research and development, it is hoped that sirna-based therapies will eventually become cancer treatments, providing patients with a more targeted and effective treatment option. keywords: sirna, oncogenes, cancer therapy, gene silencing, targeted therapy, delivery systems. potent biological mechanism called rna interference (rnai) enables the post-transcriptional selective suppression of gene expression. this mechanism has undergone substantial research and has emerged as a potentially useful therapeutic tool for the management of a number of illnesses, including cancer [1]. small interfering rnas (sirnas) are double-stranded rna molecules that target and degrade particular mrna regions to silence genes. oncogenes, which are genes that promote the growth and proliferation of cancer cells, have been discovered as promising targets for sirna-mediated gene silencing in cancer therapy [2]. an enormous amount of work has gone into creating sirna-based cancer medicines in recent years. the capacity of sirnas to specifically target oncogenes offers a promising strategy for the creation of highly effective cancer treatments with little offtarget side effects. moreover, sirnas have the potential to address the drawbacks of conventional cancer therapies, such as chemotherapy medicines, which frequently exhibit low selectivity and high levels of toxicity [3]. an overview of the present status of sirna-mediated oncogene silencing for cancer therapy will be given in this thorough review. the review will go over the various sirna delivery methods, the difficulties faced by sirna-based therapies, and the development of sirna-based cancer therapy in the clinic [4]. future developments in this area as well as the possibilities of sirna-based cancer therapeutics will be covered. ultimately, the goal of this study is to provide a thorough grasp of the status and potential applications of sirna-mediated oncogene silencing for cancer therapy [5]. fig no.1: delivery systems for sirna drug development in cancer therapy [6] a mailto:ramdas21@gmail.com shanbhag et al. cancer therapy: an overview of pharmacogenomics strategies vol 2| issue 2 | apr – jun 2023 indian j pharm drug studies | 64 different approaches used for sirna delivery for cancer therapy the development of sirna-based cancer therapeutics faces significant challenges in the efficient delivery of sirna to cancer cells. delivering sirnas to target cells can be difficult since they are generally big, negatively charged molecules that are prone to nuclease destruction [5]. biological obstacles like the extracellular matrix, cellular membranes, and endosomal compartments must also be surmounted by the sirna delivery mechanism. to get over these obstacles and make sirna distribution to cancer cells easier, a number of strategies have been devised [7]. lipid-based delivery systems one of the most widely used methods for sirna distribution is lipid-based delivery systems. a hydrophobic core that can enclose sirnas is often surrounded by a lipid bilayer in lipid nanoparticles [8]. these nanoparticles can passively gather in tumor tissues due to the improved permeability and retention impact because their size ranges from 20 to 100 nm (epr). by integrating targeting moieties like antibodies, peptides, or aptamers, lipid-based delivery systems can also be designed to target particular cell types [9]. polymer-based delivery systems are biodegradable polymers used to encase sirnas in polymer-based delivery systems. these systems can be made to have different characteristics, such as size, charge, and hydrophobicity, which can influence how they are taken up by cells and transported within them. by adding targeting moieties, polymer-based systems can also be created to target particular cell types [10]. delivery methods based on inorganic nanoparticles for the transport of sirna, and inorganic nanoparticles like gold, silica, and iron oxide have been researched. to make these particles easier for cancer cells to absorb, they can be functionalized with sirnas and/or target molecules [11]. the special physicochemical characteristics of inorganic nanoparticles can be used to promote cellular absorption, endosomal escape, and gene silencing [12]. viral vectors have been used to distribute sirna because they are particularly effective in delivering genes to cells. however, because of safety issues such as immunogenicity and mutagenicity, their use is restricted. usually, viral vectors are altered to lose their pathogenicity and designed to express sirnas for certain targets [13]. cell-penetrating peptides (cpps) are short peptides that can penetrate cell membranes and facilitate the delivery of sirnas into cells. cpps can be fused to sirnas to enhance their cellular uptake and endosomal escape. however, the delivery efficiency of cpps is often low and is dependent on the type of cpp used [14]. overall, each delivery system has its advantages and limitations, and the choice of delivery system will depend on the specific application and target. to overcome the challenges associated with sirna delivery, researchers are continuing to develop new and innovative delivery strategies for sirna-based cancer therapies. fig no.2: different approaches used for sirna delivery for cancer therapy [15] challenges associated with sirna-based therapies while sirna-based therapies have shown great promise in preclinical studies and have advanced to clinical trials, there are still several challenges that need to be overcome to ensure their clinical success. as was already indicated, one of the main difficulties with sirna-based therapeutics is getting sirnas into the target cells. it is difficult to deliver sirnas because they are big, negatively charged molecules that are vulnerable to nuclease destruction. a delivery mechanism that can shield sirnas from deterioration, promote their cellular uptake, and guarantee their intracellular trafficking is necessary for effective sirna delivery [16]. although sirnas are made to target particular genes, they can potentially have unwanted side effects. when sirnas bind to undesired mrna targets that shanbhag et al. cancer therapy: an overview of pharmacogenomics strategies vol 2| issue 2 | apr – jun 2023 indian j pharm drug studies | 65 have partial sequence complementarity to the sirna, off-target effects may result. unwanted side effects may happen if undesired genes are silenced as a result of this [17]. sirnas have the ability to elicit an immune response, which can result in the release of cytokines and chemokines that can cause cytotoxicity and inflammation. although the immune system might become adapted to the sirna delivery mechanism, this immune response can be particularly problematic for repeated sirna treatment [18]. because of their vulnerability to nuclease-mediated degradation, sirnas have a short half-life in the bloodstream. moreover, sirnas' bioavailability can be decreased by the kidneys' quick clearance of the molecules. to enable the optimal distribution of sirnas to target cells, the pharmacokinetics of these molecules must be adjusted [19]. tumors are diverse, and various tumor cells may exhibit unique patterns of gene expression. because of this, achieving uniform and efficient gene silencing across all tumor cells may be challenging. because of the intricacy of sirna production and delivery systems, sirna-based medicines can be expensive to produce. the cost of production may restrict the accessibility of sirna-based medicines and the scope of their extensive clinical use [20]. overall, while sirna-based therapies have great potential, there are still several challenges that need to be overcome to ensure their clinical success. researchers are continuing to develop and refine sirna delivery systems and optimize the pharmacokinetics of sirnas to address these challenges and advance sirna-based therapies to clinical practice. clinical progress of sirna-based cancer therapeutics sirna-based cancer therapeutics have shown great promise in preclinical studies, but their translation to clinical practice has been challenging. despite these challenges, several sirnabased cancer therapeutics have advanced to clinical trials [21]. several sirna-based therapies are currently being tested in humans for a range of cancer types, including ovarian, pancreatic, and liver cancer [21]. patisiran can be used for the treatment of hereditary transthyretin-mediated amyloidosis (hattr), a rare genetic condition that affects the heart and nervous system, patisiran is a sirna-based therapy. in clinical trials, the drug patisiran—which inhibits the transthyretin gene—promisingly improved patient quality of life and nerve function [22]. a sirna-based therapy called inclisiran is used to treat hypercholesterolemia. pcsk9, a protein that controls blood levels of ldl cholesterol, is the target of inclisiran. clinical trials on inclisiran have yielded positive outcomes, with notable drops in ldl cholesterol levels [23]. a sirna-based therapy called aln-ttrsc02 is being developed to treat attr amyloidosis. in clinical studies, the transthyretin gene-targeting drug aln-ttrsc02 demonstrated encouraging outcomes with notable improvements in patient quality of life and nerve function [24]. qpi-1002 is used to avoid acute kidney injury, qpi-1002 is a sirna-based therapy. in clinical studies, the p53 gene-targeting drug qpi-1002 had encouraging outcomes, significantly lowering the risk of acute renal damage in patients having heart surgery [25]. although sirna-based cancer treatments are currently in the early phases of clinical research, they have the potential to completely change the way cancer is treated. it is hoped that sirna-based cancer treatments would advance and eventually become a standard component of cancer treatment with continued research and clinical studies [5]. sirna-based therapies for cancer treatment. the potential of sirna-based therapies for cancer treatment is significant, and these therapies hold great promise for improving the effectiveness of cancer treatment. targeted gene silencing is a very specific and exact sirna-based therapeutics can be created to target particular genes that contribute to the development of cancer. this method of targeted gene silencing may help to lessen the toxicity and side effects of conventional cancer therapies like chemotherapy [26]. potentially synergistic with other therapies where sirna-based cancer therapies may be used in conjunction with other cancer therapies, such as chemotherapy or immunotherapy, to increase the efficacy of both. for example, sirna-based therapeutics could be used to target genes that give resistance to chemotherapy, thus enhancing the efficacy of the chemotherapy [27]. sirna-based therapies can be designed to target a wide range of genes that play a role in cancer progression, including oncogenes, tumor genes, and genes involved in angiogenesis and metastasis. sirna-based therapies can be customized for individual patients based on their genetic profile , which could potentially lead to more effective and personalized cancer treatment. sirna-based therapies can be customized for individual patients based on their genetic profile, which could potentially lead to more effective and personalized cancer treatment [28]. sirna-based therapies can be customized for individual patients based on their genetic profile, which could potentially lead to more effective and personalized cancer treatment [27]. sirna-based therapies are highly specific, which means that they have the potential to be less toxic than traditional cancer treatments, which can have significant side effects [5]. despite these potential benefits, there are still several challenges associated with sirna-based therapies, as discussed earlier. the delivery of sirnas to target cells is one of the major challenges associated with sirna-based therapies. additionally, the high cost of manufacturing and the potential for off-target effects are also significant challenges that need to be addressed. overall, sirna-based therapies have the potential to significantly improve cancer treatment, but further research and development are needed to optimize their efficacy and safety. with ongoing research and clinical trials, it is hoped that sirna-based therapies will continue to advance and eventually become a routine part of cancer treatment. shanbhag et al. cancer therapy: an overview of pharmacogenomics strategies vol 2| issue 2 | apr – jun 2023 indian j pharm drug studies | 66 fig no.3: targeted gene silencing [29] fig no.4: synergistic chemotherapy towards breast cancer [30] future directions in the field of sirna-mediated silencing of oncogenes for cancer therapy the field of sirna-mediated oncogene silencing for cancer therapy is quickly developing, and it is moving in fascinating directions. combining sirna-based therapies with other cancer treatments, such as chemotherapy or immunotherapy, is one of the most promising areas of research. it might be possible to improve therapeutic outcomes by combining sirna-based medicines with other cancer medications [31]. a key area of research focuses on delivering sirnas to cancer cells in certain tissues. to deliver sirnas to cancer cells in certain organs while limiting toxicity and off-target effects, researchers are creating innovative delivery mechanisms [32]. sirna-based therapies can be customized for individual patients based on their genetic profiles. developing methods to identify patient-specific targets and designing sirnas that specifically target these genes is an area of active research [33]. combating drug resistance is one of the main obstacles in the fight against cancer. to increase the efficiency of chemotherapy and other cancer treatments, researchers are investigating the potential of sirna-based therapeutics to target genes that are involved in drug resistance [34]. the development of innovative delivery technologies that might increase the effectiveness of sirna distribution is a key area of scientific interest. to improve the transport of sirnas to target cells, researchers are experimenting with the use of nanoparticles, liposomes, and other technologies [35]. further clinical trials are required to assess the security and effectiveness of sirna-based treatments for the treatment of cancer. to maximize the clinical translation of sirna-based therapeutics, ongoing research, and development are required [36]. overall, the field of sirna-mediated silencing of oncogenes for cancer therapy is rapidly advancing, and there is great potential for these therapies to significantly improve cancer treatment. with continued research and development, it is hoped that sirna-based therapies eventually become integral to cancer treatment. advantages of sirna-based therapies for cancer treatment very targeted and precise sirna-based therapeutics can be created to target particular genes that contribute to the development of cancer [2]. sirna-based therapies have the potential to be utilized in conjunction with other cancer treatments like chemotherapy or immunotherapy to increase the efficacy of both [37]. sirna-based cancer therapies can be created to target a multitude of genes, including oncogenes, shanbhag et al. cancer therapy: an overview of pharmacogenomics strategies vol 2| issue 2 | apr – jun 2023 indian j pharm drug studies | 67 tumor suppressors, and genes involved in angiogenesis and metastasis, that are implicated in the development of cancer [38]. sirna-based therapies have the potential to advance personalized medicine since they can be tailored for specific patients based on their genetic profiles, which may result in more efficient and specialized cancer treatment [39]. sirnabased therapies are highly specific, which means that they have the potential to be less toxic than traditional cancer treatments, which can have significant side effects [40]. limitations of sirna-based therapies for cancer treatment include sirnas are substantial, negatively charged molecules that are difficult to get through cell membranes. the creation of effective and secure sirna delivery methods is a difficult task [41]. possibility for harmful off-target effects: sirna-mediated gene silencing may have unintended consequences on genes that are not intended targets [42]. sirna-based medicines still have a high production cost, and patients have less access to them [43]. sirnas have a short bloodstream half-life; regular dosing may be required [44]. only a small number of sirnabased cancer medicines have moved to clinical trials, and none have received approval for use in humans despite extensive preclinical research [4]. overall, while sirna-based therapies hold great potential for improving cancer treatment, several challenges still need to be addressed before they become widely used in clinical settings. ongoing research and development are necessary to optimize the efficacy, safety, and delivery of sirna-based therapies. conclusion sirna-mediated cancer therapy has great potential for highly specific and effective treatment. however, efficient and safe delivery systems need to be developed, and more clinical trials are required for the evaluation of safety and efficacy. researchers are working on developing new delivery systems, personalized medicine approaches, and combination therapies to overcome these challenges. continued 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none stated dyade et al. qbd and chemometric method for estimation of metoprolol and cilnidipine online first indian j pharm drug studies | 1 original article qbd approach and chemo metric method development for the estimation of metoprolol succinate and cilnidipine in combined solid dosage form g. k. dyade1, r. b. jadhav2, sanket repal3, shital uparwad3, prashant suryawanshi3, aashish taware3, sonali khapne3, shweta kokare3, anuse dnyaneshwar3 from, 1asso. professor, 2principal, 3m. pharma students, dept of post graduate studies in pharmaceutical quality assurance, svpm’s college of pharmacy, malegaon (bkii)-413115 baramati dist pune, maharashtra, india. abstract background: development of several pharmaceutical processes including analytical methods by applying quality by design assists in ensuring the robustness of the method. usfda and other regulatory agencies have recommended implementation of quality by design (qbd) a systematic process for pharmaceutical development along with its significance. objective: chemo metric assisted uv-vis spectrophotometric analytical method based on qbd was developed for the estimation of metoprolol succinate (met) and cilnidipine (cil) from the combined dosage forms. method: nature of spectra focused applicability of absorbance correction and amplitude modulation methods for estimation of both drugs from the formulations; and 50 % alcohol was being the common solvent. for both this method 221 nm and 242 nm was the wavelength for measurement of absorbance of metoprolol and cilnidipine respectively. effect of input variables on spectrum characteristics were studied for selection of critical parameters and developed method was validated as per ich q 2 r1 regulatory guidelines. result and conclusion: linearity of the drugs was ascertained over the conc range 1-36 μg/ml (microgram/ml) for met and 1-16 μg/ml for cil. the percentage purity of assay in method ii was found 103.773 % for met and 96.825 % for cil; and the accuracy study data of method i were varied 2.04859 for met and 1.26321 for cil. precision study was shown acceptable data as % rsd in method i data varied 0.58894 for met and 1.15116 for cil. the developed method is rigid, robust and efficient for the estimation of met and cil from the composition of dosage form. keywords: metoprolol succinate, cilnidipine, qbd, ich, absorbance correction method, amplitude modulation method ilnidipine (cil) chemically 1, 4-dihydro-2,6dimethyl-4-(3-nitrophenyl)-3,5-pyridine dicarboxylic acid 2-methyloxyethyl (2e)-3-phenyl-2-propenyl ester [1] is a dihydropyridine calcium channel blocker given orally in the management of hypertension [2] reported analytical methods for estimation of cilnidipine were includes alone by uv spectrophotometric method [3], uv spectrometric methods with metoprolol [4, 5] alone uv-hplc method [6] , stability indicating hplc [7] qbd based hplc [8] chromatographic methods [9-12] and have been found in the literature. metoprolol succinate (met) is a beta blocker; used in the treatment of hypertension, angina and to reduce myocardial infarction [2]. chemically it is (rs)-1-(isopropy access this article online received – 20th may 2024 initial review – 13th june 2024 accepted – 15th july 2024 quick response code lamino)-3-[p-(2methoxyethyl) phenoxy]-2-propan-2-ol succinate [1]. literature survey revealed that various analytical methods have been reported for estimation of met such as alone uv spectrophotometric method [13-16], with other drugs uv spectrophotometric method [17-22], bio analytical method [23], met alone by rp-hplc [24], with other drug by rphplc [25-30], stability indicating hplc [31, 32], designed and eco-friendly tlc densitometry [33] and hptlc [34] in combination with other drug. cilnidipine and metoprolol succinate are official in indian pharmacopoeia [35], whereas metoprolol is official in bp [36]. chemical structures of both drugs are shown in (fig 1). quality by design concept is applied for the development of pharmaceutical processes to assure a predefined product quality. __________________________________________________ correspondence to: g. k. dyade, dept of post graduate studies in pharmaceutical quality assurance, svpm’s college of pharmacy, malegaon (bkii)-413115 baramati dist pune, maharashtra, india. email: pharmacyresearchsvpmcop@gmail.com c pharmacyresearchsvpmcop@gmail.com dyade et al. qbd and chemometric method for estimation of metoprolol and cilnidipine online first indian j pharm drug studies | 2 fig 1: chemical structure of drug molecule qbd concepts are mentioned in ich guidelines q 8(r2) (pharmaceutical development), q9 (quality risk management), and q10 (pharmaceutical quality system) [3739] shown in fig 2. ich guidelines q8 (r2) [40] defines qbd as a “a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management”. qbd approach in analytical method summarizes a complete understanding of how the analytical technique attributes and operating conditions affect the analytical performance. factors to study in analytical quality by design (aqbd) approach may include the type of analytical technique chosen, reagents used and instrument parameters. qbd was applied to build rigid robust method through risk assessment at early stage and defining the design space at the later stage. fig 2: analytical qbd approach there are similar advantages of applying qbd principles to analytical methods as to manufacturing processes and product [41]. a qbd approach can be beneficial in the development of suitable, robust, low cost and eco-friendly (eco-friendly solvent, chemicals) method which is applicable at any stage of the lifecycle of the product. also some regulatory guidelines have mentioned flexibility of changing analytical method without revalidation if the aqbd approach has been implemented during analytical method development. the first stage of aqbd approach is to fix an analytical target profile (atp) for the method. atp defines the goal of the analytical method development process and it is the sign of method performance [42, 43]. for analytical method validation ich q2 (r1) has given various method performance characteristics for an analytical method. thus a qbd based uv spectrophotometric was developed, qbd approach was implemented with the study of the effect of method input variables on spectral shape, intensity of absorbance, and absorbance maxima λmax and critical parameters were selected for the proposed method and method was validated as per ich guidelines q2 (r1). materials and methods instrumentation: analysis was performed with a shimadzu double beam uv-visible spectrophotometer 1900i and 1700 (shimadzu, kyoto, japan) with spectral bandwidth of 2 nm and wavelength accuracy of ± 1 nm with 10 mm matched quartz cells was used. electronic balance afcoset balance (the bombay burmah trading corpo ltd) with accuracy ±0.1 mg model no. er 200a was utilised for weighing and for degassing the solution digital ultrasonic cleaner 1.8 ltr (labman scientific instruments chennai) was used. reagents and chemicals: pharmaceutically pure sample of met was procured from macleods pharmaceuticals ltd. mumbai and cil from swapnroop drugs and pharmaceuticals, aurangabad, maharashtra, india procured as a gift samples and the commercial formulation cilacar-m manufactured by j b chemicals and pharmaceuticals containing cilnidipine 10 and metoprolol 50 mg was procured from the local market. aqbd approach application in method development: aqbd approach was applied to study the influence of input variable parameters on spectrophotometric analytical method performance shown in (fig 3). solvent selection: cil is very soluble in n, n-dimethyl acetamide, freely soluble in acetone, soluble in methanol and practically insoluble in water, whereas met is freely soluble in water, ethanol, methanol and ethyl acetate;. although the solubility of the procured drugs were studied in alcohol 90%, 0.1 n hcl and 0.1 n naoh separately; and found that cil is insoluble in both these solvents however met is soluble in 0.1 n naoh. alcohol 90% was selected as common solvent as both drugs have solubility. each solution with known conc of analyte was scanned in uv range of 400 nm to 200 nm. it dyade et al. qbd and chemometric method for estimation of metoprolol and cilnidipine online first indian j pharm drug studies | 3 was found that suitable solvent is alcohol 90% with respect to stable, robust and precise in producing result. fig 3: diagram showing the relationship between input variable parameters and the spectrophotometric method performance characteristics preparation of stock solutions and standard solutions: 10 mg each of drug cil and met were separately and accurately weighed; and transferred into separate 25 ml volumetric flask. dissolved into alcohol 90% solvent and volume was made to 25 ml with solvent. subsequent standard solution of drug met with conc 16μg/ml and cil with conc 8μg/ml were prepared by diluting aliquot of stock solution to 10 ml with 50% alcohol into 10 ml capacity volumetric flask. selection of method, wavelength and conc range: from uv spectra it was found that cil has measurable absorbance at 242 nm (fig 4) and less interference was observed by met; similarly met has maximum absorbance at 221 nm (fig 4) and measurable interference having constant absorptivity by cil was accounted. chemo metric method i.e. absorbance correction method was applied and which was reasonable remedy to overcome interference at each other’s absorbance, and other method was amplitude modulation method. from the nature of spectra to study linearity, working conc range 1 to 16 μg/ml for cil and 1 to 36μg/ml for met was selected. also combined drug solution was prepared simulated to marketed formulation. selected critical parameters based upon above discussion, observations are listed in and by using these; method was validated as per ich guidelines and by analysing marketed preparations. experimental method for estimation: from the overlain spectra absorbance correction method was applicable for estimation of both the analytes from the combined dosage form. method-i: absorbance correction method for estimation of met and cil: met was shown maximum absorbance i.e. λmax at 221 nm where measurable interference with constant absorptivity by cil found and cil has maximum absorbance i.e. λmax at 242 nm where negligible interference observed by met. at 242 nm the λmax of cil, met was shown consistency in the absorptivity; hence two wavelengths 221 and 242 nm were considered as 1 and 2 respectively for the said method to estimate met and cil. the equation a= abc was applied for x (met) and y (cil) determination. conc of working standard solutions of met and cil containing 28 mcg/ml and 8 mcg/ml conc respectively were separately prepared and used for the method. where cx = conc of mps in sample solution a1 and a2 = absorbance of sample solution at 1 and 2 wavelength ay1 and ay2 = absorptivity of cil at 1 and 2 wavelength of standard solution ax1 and ax2 = absorptivity of met at 1 and 2 wavelength of standard solution cy = conc of cil in sample solution as = absorbance of sample solution at 2 wavelength method-ii: amplitude modulation method: this method comprises the conversion of zero order/normal spectra to its first, second or higher derivative spectrum. the amplitude is directly proportional to the conc of solution provided beer’s law is obeyed by spectrum. in derivative method zero crossing wave length for both drugs is found such that at the zero crossing of one drug the other drug should show substantial absorbance. here standard solutions 16 µg/ml each of metoprolol and cilnidipine were prepared in 10 ml volumetric flask and scanned from 400 to 200 nm wavelength range against ethanol 50% as blank. absorption spectra of both drugs were recorded and the found λmax of met and cil were stated in earlier discussed method. from overlain spectra cil has substantial interference due to absorption at wavelength 221 nm and met shows negligible absorption at 242 nm, hence it was decided to apply derivative method. dyade et al. qbd and chemometric method for estimation of metoprolol and cilnidipine online first indian j pharm drug studies | 4 zero order spectra of both drug was modulated and converted to first order derivative spectra. from overlain spectra, it clearly shows that no interference or zero absorbance of met was found at 263 nm hence it was selected λmax of cil and 229.5 nm was zero crossing of cil, so it was selected as λmax of met. both drugs were obeying beer’s law in first order derivative mode at the respective wavelength. fig 4: uv spectra of cilnidipine and metoprolol succinate in ethanol validation of the method: selected critical parameters should meet the performance characteristics of the analytical method so as to attain analytical target profile of the method. an ich guideline q2 r1 was applied to study methods performance with critical parameters in order to implement aqbd approach. the method was validated as per ich guidelines. system suitability: system suitability is studied to demonstrate the suitability of the developed procedure under consideration for the analytical method. six replicates of working standard solutions with conc16 μg/ml and 8 μg/ml each of met and cil respectively were prepared separately and absorbance was recorded, sd and % rsd of the response was calculated. linearity: the linearity of an analytical method is its ability to obtain response i.e. absorbance which is directly proportional to the conc of analyte. series of working standard solutions were prepared in conc. range of 1-36μg/ml for met and 1-16 μg/ml for cil and scanned in 400 to 200 nm range in spectrum mode of the spectrophotometer, absorbance of the standard solutions were recorded at their respective wavelength; i.e. 221 for met and 242 nm for cil in spectrum order. microsoft office excel software tool was used to obtain the standard regression curve and its analysis as slope, intercept, and correlation coefficient. assay of formulation: assay was carried out by proposed methods and assay was validated by statistical parameters. estimation of formulations by absorbance correction and amplitude modulation method: tablet powder equivalent to 12.5 mg met and 2.5 mg cil was weighed and transferred into 25 ml volumetric flask. dissolved into 90% alcohol, mixed well for 10 mins and volume was made to 25 ml with the solvent. solution was filtered through what man filter paper and aliquots of solution were further diluted with the 50% alcohol to obtain tablet sample solution. solution was scanned in the range of 400 to 200 nm to obtain absorbance of tablet solution at 221 nm and 242 nm in spectrum order. also spectrum converted to first order derivative and absorbance was recorded at 229.5 and 263 nm. obtained absorbance were utilised to estimate unknown conc of formulation; and results were statistically validated to obtain % of nominal conc, standard deviation and % of rsd. accuracy and precision: the accuracy of an analytical method expresses the closeness of an agreement between test result and true result. accuracy study was performed by recovery study i.e. standard addition method; diluted standard solutions of met and cil were prepared and standard solutions added in 100 % proportionate to the tablet solution. three replicates at this level was prepared, absorbance measured and % of conc, sd and rsd were calculated. the precision study was carried out by performing assay of tablet six times; also the reproducibility in result was studied by inter day and intraday precision. limit of detection (lod) and limit of quantitation (loq): the lod and loq of met and cil by the proposed method were determined using calibration graph method and calculated as 3.3σ/s and 10 σ/s for lod and loq respectively; σ is the standard deviation of calibration curve and s is the slope of regression line. robustness and ruggedness: it is measure of capacity of analytical procedure to remain unaffected by small but deliberate variations in method parameter. results method development comprises numerous steps, and of which solvent selection, selection of method for measurement are significant one. uses of aqueous solvents, eco-friendly solvents like hydrotropic have got remarkable weightage due to low cost, readily available and environmentally sound. drugs underlying analysis must have appreciable solubility in the selected solvent. chemical structure of the drug and physico-chemical properties available in the literature guides about use of appropriate solvent in the method. from uv spectra two wavelengths were selected as 221nm dyade et al. qbd and chemometric method for estimation of metoprolol and cilnidipine online first indian j pharm drug studies | 5 (λmax of met) and 242 nm (λmax of cil) shown in fig 5 for calculation of both drugs in combined solution shown. fig 5: overlaid spectra of met and cil system suitability: the absorbances of six replicates of standard solutions of tabulated respective conc are reported in table no 1. the sd and % rsd was found for met and cil and meets the system suitability requirements indicates method was suitable for analysis. table no 1: system suitability study of met and cil sr no conc in μg /ml absorbance of met* conc in μg /ml absorbance of cil* 1 16μg/ml 0.5157 8 μg /ml 0.6519 2 sd rsd % 0.004676 0.9060 sd rsd 0.004809 0.73774 *mean of six determinations linearity: the calibration curve of both drugs was found to be linear in the conc range of 1-36 μg/ml for met and 1-16 μg/ml for cil in normal spectrum as shown in fig 6 and in first order derivative mode fig 7. the regression equation of line and its parameters slope, r2 value and intercept are tabulated in table no 2, which proved the linear relationship between conc and obtained response. assay: the assay was carried out by the proposed method. the overlaid spectra obtained in amplitude modulation method are shown in fig 8. the assay of formulation was carried out by proposed method and calculated % of nominal conc and rsd was found within acceptable limits are summarized in table no 3. the results indicated applicability of the method for estimation of formulation. fig 6: normal spectrum kind calibration curve of met and cil fig 7: calibration curve of met and cil in first order derivative accuracy and precision: the results of accuracy are summarised in table no 4, the obtained results were within acceptable limit; and methods accuracy was justified by calculating % drug content. the precision study was carried out by performing assay of solutions; further the reproducibility in result was studied by interday and intraday precision. the values obtained sd and % rsd was shown methods precision and are summarised in table no 4. limit of detection (lod) and limit of quantitation (loq) and robustness and ruggedness: the lod and loq of cil and met were found in acceptable limits by the proposed method. robustness was studied and capacity of analytical procedure to measure analyte was remain unaffected by small but deliberate variations in method dyade et al. qbd and chemometric method for estimation of metoprolol and cilnidipine online first indian j pharm drug studies | 6 parameter. the analytical method was found rugged during development; similarity the result was produced by performing the analysis by different analyst. table no 2: parameters of regression equation obtained in microsoft excel parameters met cil met cil linearity study in normal linearity in derivative detection wavelength 221 242 229.5 263 beer’s law limit (μg/ml) 1–36 μg/ml 1–16 μg/ml 1–36 μg/ml 1–16 μg/ml correlation coefficient (r2) 0.9984 0.9994 0.9991 0.9972 regression equation (y = mx + c) y = 0.0317x + 0.0097 y = 0.0797x + 0.0145 y=0.0006x 0.0001 y=0.0015x0.0003 table no 3: results of assay of formulation by proposed method formula tion dr ug label claim (mg/ta blet; n=6) amou nt found/ mg drug cont ent % std deviat ion % rsd methodi me t 50 47.084 94.16 7 4.1614 6 4.419 2 ci l 10 9.681 96.78 7 1.7606 3 1.809 67 methodii me t 50 51.886 103.7 73 1.8865 1 1.817 91 ci l 10 9.682 96.82 5 2.7493 4 2.839 49 table no 4: results of accuracy and precision sr. no. parame ter level of study dru g na me % mean s.d. % rsd meth od i precisi on intrada y precisi on me t 94.877 % 0.588 94 0.620 74 cil 97.744 % 1.151 16 1.177 12 accura cy 100% me t 96.308 % 2.048 59 2.127 12 cil 94.845 % 1.263 21 1.331 88 meth od ii precisi on intrada y precisi on me t 102.06 6% 1.665 33 1.622 08 cil 99.476 % 2.260 24 2.272 15 accura cy 100% me t 97.646 % 1.176 75 1.205 11 cil 96.667 % 1.159 71 1.194 52 fig 8: overlaid spectra obtained in amplitude modulation method dyade et al. qbd and chemometric method for 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metoprolol succinate and hydrochlorothiazide in tablet dosage form. international journal of pharmaceutical technology letters. 2023;1(1):17-26. 33 patel k, macwan p, prajapati a, et. al. box‒behnken designassisted development of an eco-friendly thin-layer chromatography‒densitometry method for the quantification of amlodipine besylate, metoprolol succinate, and telmisartan in synthetic mixture using agree tool. jpc–journal of planar chromatography–modern tlc. 2023;36: 265-277. 34 patil v p. simulteneous hptlc analysis of hydrochlorthiazide and metoprolol succinate in tablet and bulk dosage form. world research journal of organic chemistry. 2012; 1(1):1-5. 35 indian pharmacopoeia, govt of india, ministry of health and family welfare, 8th edition, the indian pharmacopoeia commission ghaziabad, 2018, iii: pp. 1616, 2583. 36 british pharmacopoeia, medicines and healthcare products regulatory agency london, 2019, ii: 1361, 1762. 37 ich expert working group. ich harmonized tripartite guideline-pharmaceutical development q 8 r2. in current step 4 version. 2009; p. 1-28. 38 ich expert working group. ich harmonized tripartite guideline-quality risk management q 9. in current step 4 version.. 2005; p. 1-23. 39 ich expert working group. ich harmonized tripartite guideline-pharmaceutical quality system q 10. in current step 4 version. 2008; p. 1-21. 40 ich expert working group. ich harmonized tripartite guideline-validation of analytical procedures: text and methodology q 2 r1. in current step 4 version.2005; p. 1-17 41 schweitzer m, pohl m, hanna-brown m, et al. implications and opportunities of applying qbd principles to analytical measurements. pharm technology. 2010; 34: 12-29. 42 vogt f g, kord a s. development of quality-by-design analytical methods. j of pharm science. 2011; 100:797-812. 43 bhatt d a, rane s i. qbd approach to analytical rp-hplc method development and its validation, int j of pharma science. 2011; (3):179-187. how to cite this article: dyade gk, jadhav rb, repal s, uparwad s, suryawanshi p, taware a, khapne s, kokare s, dnyaneshwar a. qbd approach and chemo metric method development for the estimation of metoprolol succinate and cilnidipine in combined solid dosage form. indian j pharm drug studies. 2024; online first. funding: none; conflicts of interest: none stated keywords: metoprolol succinate, cilnidipine, qbd, ich, absorbance correction method, amplitude modulation method saralaya & hiriyalu synthesis of lamotrigine and a glimpse of its closely related compounds: an overview vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 8 review article an overview of prior art disclosures about the synthesis of lamotrigine and a glimpse of its closely related compounds sanjay sukumar saralaya 1, shashikumar somashekar hiriyalu2 from, 1assistant professor, department of chemistry, sri dharmasthala manjunatheshwara institute of technology, [affiliated to visvesvaraya technological university (vtu), belagavi], ujire, belthangady taluk, dakshina kannada, karnataka, 2independent researcher, 436, b-block, sreenagar, jp nagar post, mysuru, karnataka, india. abstract in this review contribution, we have gathered the specific details in brief from the prior art disclosures regarding the synthesis of 1 and its closely related compounds. the prior art disclosures were in the form of patent publications and academic journal articles. during the review process it was noticed that, most of the disclosures on the synthesis of 1 were in the form of patent publications. with an emphasis to enhance the physico-chemical properties of 1, a few crystal/co-crystal/ionic salts of 1 are reported mainly in the academic journal articles. the glimpses of some closely related compounds to 1 are also included in this review contribution. this initiative can provide a platform for the global researchers to get an insight into the disclosures towards the synthesis of 1 and a glimpse on its closely related scaffolds. additionally, the researchers can design new routes to commercialize 1 in future with higher atom economy by adhering to the green chemistry principles. key words: lamotrigine, cyanation, condensation, cyclization, recrystallization. amotrigine 1 is a popular antiepileptic drug, belongs to the class of phenyl-triazines and to the sub-class of dichloro-benzenes or halo-benzenes. it has the cas number; 84057-84-1, trade name; lamictal, molecular weight; 256.09 g/mol, molecular formula; c9h7cl2n5, and iupac name; 6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5diamine. it is used in the treatment of both epilepsy and as a mood stabilizer in bipolar disorder [1]. it is even prescribed for the process of conversion to drug monotherapy for those patients with at least 16 years of age or older with partial seizures and currently they are treated with carbamazepine, phenytoin, phenobarbital, primidone, or valproate as the single anti-epileptic drug. additionally, it is also used for the maintenance treatment of bipolar-i disorder in adults with at least 18 years or older, delaying the time to mood episodes (which may include mania, hypomania, depression, mixed episodes) [2]. the mode of drug actions would resemble to those of phenytoin and carbamazepine, inhibiting voltage-sensitive sodium channels, stabilizing neuronal membranes, thereby access this article online received – 22nd feb 2024 initial review – 27th feb 2024 accepted – 04th mar 2024 quick response code modulating the release of presynaptic excitatory neurotransmitters [3]. it exhibits the characteristic binding capability to several different receptors during the mode of its pharmacological actions [4]. it is rapidly and completely absorbed with least first-pass metabolism effects and records a reasonably high bioavailability of around 98% [5, 6]. pharmacologically 1 undergoes routine metabolism and results in the generation of inactive metabolite 2-nglucuronide conjugate. moreover, about 94% of drug and its metabolites are eliminated through urine and around 2% would eliminate through feces [7, 8]. objectives this review was focused primarily towards the exfoliation of details on the synthesis of 1 in academic journals. additionally, salts, impurities and closely related scaffolds of 1 were also included. interestingly, most of the synthetic routes disclosed are in the form of patent publications. in this contribution as an overview, synthesis of 1 and the glimpses of some of its closely related compounds were considered to frame the article by referring the prior publications. __________________________________________________ correspondence to: sanjay sukumar saralaya, assistant professor, department of chemistry, sdm it, opposite to siddhavana gurukula, dharmasthala main road, ujire, belthangady taluk, dakshina kannada, karnataka, india 574 240. email: sanjayss@sdmit.in. l mailto:sanjayss@sdmit.in saralaya & hiriyalu synthesis of lamotrigine and a glimpse of its closely related compounds: an overview vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 9 1. synthesis of 1 (as per the disclosures in patent publications) numerous patents were published in various patent trade mark offices around the globe regarding the synthesis of 1 through different synthetic pathways. among those, majority of the synthetic pathways were reported with the use of starting material 2,3dichlorobenzoic acid 2 to prepare 1 through the isolation of an intermediate 2,3-dichlorobenzoyl cyanide 3. in some other illustrations, 3 was directly used as the starting material to obtain 1. in some other examples, the penultimate intermediate of the process (2e,z)-2-[cyano(2,3dichlorophenyl)methylidene]hydrazinecarboximidamide 4 itself was cyclized under various feasible conditions to isolate 1 in high yields and good purity. in a few instances, 1,2dichloro-3-methylbenzene 5 was used as the starting material to synthesize 1 via multi-step process. in one of the synthetic pathway, 1,2-dichloro-3-nitrobenzene 6 was employed as the starting material to isolate 1. in addition to these synthetic procedures, a few innovative multi-step strategies were also employed to prepare 1. to support the synthesis of 3, a noncatalytic approach was disclosed involving halogen displacement reactions to impart an intended cyanation. the above tabulated patent disclosures (figure 1) are very well elaborated with reaction schemes regarding the synthetic flourish of 1 by us in a review article [9]. furthermore, a few catalytic approaches were also disclosed for the synthesis of 3 in good yields. some of the close resemblance 1,2,4 triazines were reported much earlier for their synthesis and characterization [10]. a scalable process for the manufacture of 1 and other 1,2,4-triazines and their pharmaceutical applications were demonstrated by baxter mg, et al [11]. a few fluoro-substituted-triazines were reported by rees rw & russel pb, as effective anti-malarial chemotherapeutic agents [12]. cl cl n n nnh2 nh2 1 cl cl c oh o 2 cl cl o n 3 cl cl n nh n nh nh2 4 cl cl ch3 5 cl cl n o o 6 figure 1: product, major starting materials and intermediates featured in past patent disclosures 2. synthesis of 1 and a glimpse of its closely related compounds (as per the disclosures in academic journal publications) hitchings gh, et al., had reported the use of 3,5-diamino-astriazines as inhibitors of lactic acid bacteria and plasmodia. under the context, 3,4-dichlorophenyl-triazines have shown significantly superior inhibition activity against p. berghei [13]. settepani ja, et al., had reported the condensation of acylnitriles with aminoguanidine in the presence of 2n nitric acid to isolate acylnitrile amidinohydrazones and its subsequent base (potassium hydroxide) mediated cyclization gave some 3,5-diamino-l,2,4-triazines [14]. this work was inspired by the past initiatives from numerous researchers towards the isolation of triazines in varied yields [15-21]. roth b, et al., had attempted in vain to synthesize 3,5diamino-6-benzyl-as-triazines and the reaction failure was attributed to enolization [22]. rees rwa, et al., had reported the synthesis and antimalarial activity of some chlorinated, flourinated, methoxylated, and trifluoromethylated-6-aryl-as-triazines 7r. as per the disclosure, the condensation of arylglyoxylonitriles with aminoguanidine was done under aqueous acidic medium and the intermediate amidinohydrazone salts were isolated. these salts were subjected to cyclization under simple reaction condition to isolate various triazines [23]. march lc, et al., had demonstrated the synthesis of many substituted 1,2,4-triazines as potential antimalarials [24]. a series of pharmacological studies of 1 was conducted by various researchers to confirm its anti-convulsant properties [25-29]. janes rw, et al., had reported the crystal structure of methanol solvate of 1 [30]. kerr dib, et al., had reported the synthesis and pharmacological impact of 3-methyl-6-[2(trifluoromethyl)phenyl][1,2,4]triazolo[4,3-b]pyridazine 8 [31]. moreau s, et al., had demonstrated the synthesis and anti-convulsant activity of benzyl imidazo and benzyltriazolopyridazines having structural resemblance to 1 [32]. messenheimer ja, had reported a detailed study chapter on 1 with regard to its pharmacological properties, mechanism of action, pharmacokinetic behavior etc [33]. with the solidity of clinical efficacy of 1 as an anticonvulsant drug, a few researchers including dickins m, et al., had reported the synthesis of 1 in bulk for its commercialization [34]. janes aw, et al., had reported the crystal growth and study of 6-(2-fluorophenyl)-1,2,4-triazine3,5-diamine methanol solvate 9, 6-(2-methylphenyl)-1,2,4triazine-3,5-diamine monohydrate 10 and 6-(2-bromophenyl)1,2,4-triazine-3,5-diamine dimethanol solvate 11 [35-37]. sawyer da, et al., had reported the synthesis and pharmacological activity of 1i (isethionate salt of 1), an important contribution in the form of patent application for the enhancement of solubility of 1 [38]. potter b, et al., had reported the crystal and molecular structure of 1i with an emphasis on the protonation site of n in the triazine ring to form an ionic salt of the drug [39]. willmore lj, had reported an article comprising the studies on clinical advancements and pharmacokinetics of 1 with a comprehensive prior art coverage [40]. kubicki m, et al., had disclosed the hydrogen bonding patterns in 1 (hydrate) saralaya & hiriyalu synthesis of lamotrigine and a glimpse of its closely related compounds: an overview vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 10 and 1 (mesylate, hydrate) and confirmed the role of cocrystallizing solvent on the resultant hydrogen bonds [41]. many derivatives of 1 are reported by varying the substituent atoms in the benzene ring but the triazine part was untouched. in this regard, hlavác j, et al., had demonstrated the synthesis of oxo-analogues 12 and 13 of 1 and other related indolederivatives [42]. shridhar b, et al., had demonstrated the crystal structure details of benzoate-dimethylformamide solvate, dimethylformamide-sesquisolvate and hydrogenphthalate-dimethylformamide solvate of 1 in distinct experiments [43-45]. ulomskii en, et al., had demonstrated the cleavage (using triphenylphosphine or cu powder in acetic acid) of pre-prepared fused 6-aryl/6-hetaryl-7aminotetrazolo[1,5-b][1,2,4]triazines 14r in a simple accessible method to isolate a numerous 6-substituted 3,5diamino-1,2,4-triazines. from this re-constructive disclosure, the obtained yields of isolated triazines are high and it was 76.0% for 1 [46]. r n n nnh2 nh2 7r ff f n n n n 8 n n nnh2 nh2 f . ch3oh 9 n n nnh2 nh2 . h2o 10 n n nnh2 nh2 br . 2 (ch3oh) 11 n nh + nnh2 nh2 cl cl 1i so o o oh n nh n h o o cl cl r 12 r=h, cl, br, i, oh 13 cl cl n n h nh oo n n n h o o n n n n n n n nh2r 14r r=2,3 cl2c6h3 f or 1 figure 2: major related compounds of 1 featuring in [23-46] reddy vv, et al., had reported the studies related to the synthesis, isolation and characterization of isomeric impurities of 3 (impurity a-e) and 1 (impurity f-j) [47]. palmer ra, et al., had disclosed a report on low temperature x-ray crystallographic structures of two derivatives of 1, 2-methyl,3amino,5-imino-6-(2,3-dichlorophenyl)-1,2,4-triazine as water solvate 15 and 2-methyl,3,5-diamino-6-(2,3-dichlorophenyl)1,2,4-triazine as isethionate, hemi-hydrate 16 [48]. palmer ra, et al., had reported the x-ray crystal structures of two derivatives of 1, 3,5-diamino-6-(2-chlorophenyl)-1,2,4triazine as hydrate 17 and 3,5-diamino-6-(3,6dichlorophenyl)-1,2,4-triazine as methanol solvate 18 [49]. 15 n n n cl cl nh nh2 .h2o 16 n n n cl cl nh2 nh2 isethionate-hemihydrate 17 n n n cl nh2 nh2 .h2o 18 n n n nh2 nh2 cl cl .ch3oh figure 3: related compounds of 1 featuring in [47-49] qian y. et al., had demonstrated the synthesis, characterization and anti-microbial activity of 1 and its thirteen novel ammonium salt complexes. the work discloses an improved process for the synthesis of 1 as per scheme 1. 2,3-dichlorobenzoyl chloride 19 was cyanated through dehalogenation using cucn in mono-chlorobenzene to isolate 3. the condensation of 3 with hydrazinecarboximidamide hydrochloride 20 was achieved in the presence of methanesulfonic acid (meso3h) in dmf to obtain n''-[(e,z)cyano(2,3-dichlorophenyl)methylidene]carbonohydrazonic diamide 4a. an alkali driven cyclization had resulted in the formation of 1. the ammonium salts 21r were prepared by treating the solution of 1 in methanol with various acids in distinct experiments. the different acids used for the salt preparation are adipic acid, propanedionic acid, chloroacetic acid, bromoacetic acid, cis-butenedionic acid, formic acid, fumaric acid, nitric acid, acetic acid, trifluoroacetic acid, hydrochloric acid, oxalic acid and phosphoric acid (total: thirteen acids). as per the pharmacological studies, most of the novel salt complexes of 1 had exhibited good antibacterial activity against gram-positive bacterial strains. meanwhile, they were mild and even inactive against gram-negative bacterial strains [50]. sridhar b, et al., had reported the crystal studies and hydrogen bonding data of 1, its chloride and nitrate salts [51]. cheney ml, et al., had reported the studies related to the dissolution rate, solubility and pharmacokinetic behavior of ten novel forms of 1. those compounds are, 1 methylparaben co-crystal form-i, 1 methylparaben co-crystal form-ii, 1 nicotinamide co-crystal , 1 nicotinamide co-crystal monohydrate, 1 saccharin salt, 1 adipate salt, 1 malate salt, 1 nicotinate dimethanol solvate, 1 dimethanol solvate and 1 ethanol monohydrate [52]. razzaq sn, et al., had reported the saralaya & hiriyalu synthesis of lamotrigine and a glimpse of its closely related compounds: an overview vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 11 crystal structure and hydrogen bonding possibilities of novel 3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazin-2-iumdihydrogen-phosphate–4-(dimethylamino)-benzaldehyde 22 [53]. sridhar b, et al., had reported the crystal forms of 1 with fluorobenzoic acid, nicotinic acid, 2-thiobarbituric acid, 3picoline. additionally, the work reports the crystal nature of butyl alcohol solvate of 1 [54]. chadha r, et al., had reported the crystal structure, characterization and pharmaceutical properties of different multi-component crystalline forms of 1 with renowned coformers like nicotinamide, acetamide, acetic acid, 4-hydroxy-benzoic acid and saccharin [55]. rao sn, et al., had demonstrated the synthesis, isolation, characterization and formation basis of five impurities of 1. these reported impurities are 2-(2,3-dichlorophenyl)-2-(guanidinylimino) acetonitrile 4, n-guanidinyl-2,3-dichlorobenzamide 23, 3amino-6-(2,3-dichlorophenyl)4h-1,2,4-triazin-5-one 24, n[5-amino-6-(2,3-dichloro-phenyl)-1,2,4-triazin-3-yl]-2,3dichloro benzamide 25 and 3,5-bis-(2,3-dichloro-benzamido)6-(2,3-dichloro-phenyl)-1,2,4-triazine 26. among these impurities, 23 and 26 are the novel disclosures [56]. cl cl n n nnh2 nh2 1 cl cl cl o cucn, ki mono-chlorobenzene 70% cl cl o n dmf, meso3h 39.1% nh2 nh nh nh2 .hcl cl cl n n nh2 nh2 n 19 3 20 4a i-proh, koh, h2o80% cl cl n n nnh2 nh3 + 21r rh, meoh r scheme 1 (compound number –position to be changed, since numbers are getting cut in the view screen) clcl n o h n h nh nh2 clcl n n h nh nh2 n 4 23 cl cl n n n h nh2o 24 cl cl n n n n nh2 h o cl cl 25 cl cl n n n nn h o cl clh o cl cl 26 cl cl n n h n nh2 nh2 + o p oho oh o h n h h h h h h 22 figure 4: list of related compounds and the impurities of 1 featuring in [53-56] lekšić e, et al., had reported the synthesis, characterization and crystal nature studies of four novel cocrystals of 1 with phthalimide, pyromellitic-diimide: dmf, caffeine: 3-pentanone and isophthaldehyde [57]. with an intention to find an industrially feasible route of synthesis to manufacture 1 in high atom economy, venkanna g, et al., had disclosed a high yield and an improved process than the past referred routes scheme 2. the reported work emphasizes the importance of avoiding the alkali use along with alcohol for the intended cyclization of 4a. this adopted modification in the process will prevent the formation of 3-amino-6-(2,3dichlorophenyl)-1,2,4-triazin-5(4h)-one 27, thus enhancing the process yield. additionally, work reports the synthesis, characterization and in-process control measures about the novel 6-(2,3-dichlorophenyl)-n5-methyl-1,2,4-triazine-3,5diamine (n-methyl impurity) 28 [58]. saralaya & hiriyalu synthesis of lamotrigine and a glimpse of its closely related compounds: an overview vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 12 cl cl n n nh nh2 o 27 4a 1 cl cl n n n nh nh2 28 a) methanol, activ ated c, ref lux b) methanol, activ ated c, ref lux c) methanol, 0.1n naoh (3 drops), activ ated c, ref lux d) methanol, h2so4 (3 drops), activ ated c, ref lux a) 94% b) 84% c) 86% d) 80% scheme 2: synthesis of 1 and 28 as per venkanna g, et al. chadha r, et al., had reported the crystal structure studies and the physicochemical properties of four multicomponent forms of 1 with a few carboxylic acids like acetic acid, propionic acid, sorbic acid and glutaric acid [59]. young rb, et al., had disclosed a work regarding the direct photodegradation of 1 under the impact of simulated sunlight. the rate of degradation and the by-products formed are influenced by the medium ph [60]. leitch dc, et al., had demonstrated some different catalytic systems to prepare 3 from its chloride precursor. as per the conclusion of process optimization, the best cyanation (77% yield) was occurred upon the use of cucn, cetyltrimethylammonium bromide (ctab) in toluene [61]. du s, et al., had reported the synthesis and characterization of two novel co-crystals of 1 with 4,4′bipyridine and 2,2′-bipyridine [62]. kaur r, et al., had presented the drug-drug co-crystal of two renowned anticonvulsants, 1 and phenobarbital. the work disclosed the crystal nature characterization, intrinsic dissolution rate (idr) and solubility of the novel cocrystal [63]. makki m, et al., had demonstrated the synthesis, characterization and antiinflammatory properties of some fluorine substituted analogues of 1 [64]. kitson pj, et al., had illustrated the synthesis of 1 in a single cartridge (self-contained plastic reactionware device) through a platform-independent digital code. as per the example, 250 mg of 3 was converted 1 in a two-step process to get 112 mg of product (overall yield: 46%). this approach would pave the way for the local manufacture of drugs even in the absence of specialist facilities [65]. matias m, had reported the synthesis and characterization of many related compounds of 1 in his thesis [66]. kuang wj, et al., had reported the synthesis, characterization and crystal nature details of two novel co-crystals of 1. they are phthalimide co-crystal of 1 and succinimide co-crystal monohydrate of 1. the co-crystals have enhanced solubility and dissolution rate than the pure crystalline form of 1 [67]. kuang w, et al., had demonstrated the synthesis, characterization and construction of ternary phase diagrams of novel co-crystal of 1 with 1,2,3,6-4-hydro-phthalimide [68]. heravi mm, et al., had contributed a review article covering the pharmacological properties, medical applications and the adopted synthetic pathways of prescribed drugs containing nitrogen heterocyclics [69]. satapathy bs, et al., had illustrated the synthesis and characterization of crystalline form of 1 with citric acid. it was estimated to improve the release of 1 in gastric region and contribute to increased oral absorption [70]. samineni r, et al., had illustrated the synthesis, crystal studies and physicochemical parameter estimations of co-crystals of 1 with saccharin sodium, 4hydroxy benzoic acid, and methyl paraben. these co-crystals had exhibited better flow properties and higher dissolution rate than 1 [71]. li j, et al., had reported the synthesis, crystal morphology and thermal behavior studies of two polymorphs (form-i & ii) and two solvates (ethanolate & methanolate) of 1-tolfenamic acid salt [72]. a few review articles were also published with an aim to provide a broad spectrum view on the synthetic routes, characterization and biological significance of 1,2,4-triazine derivatives but not exclusively specific to 1 [73-75]. summary during the process to exfoliate the details of 1, we found that the disclosures on its synthesis were mainly dominated by numerous patent publications [9]. meanwhile, academic journals had prioritized to synthesize salts and structurally close resemblance derivatives of 1. this strategy was employed by many researchers to enhance the physicochemical properties of 1 (ex: solubility, stability etc) and the possible efforts to introduce the parallel drug having better clinical efficacy than 1. this review work could serve for the repurposing studies of 1 and also to venture new possibilities of synthesis. conclusion we have retrieved the prior art details on the synthesis of 1 and its related compounds from the various web search tools/databases like google scholar, google patents, global dossier, free patents online, uspto assignment, uspto (united states patent and trademark office) and espacenet (european patent office and trademark office). this initiative is an exfoliation of 1 with regard to its synthesis by various routes. additionally, this initiative provides the glimpses of some its very closely related compounds. this work provides an insight to the status of 1 and some of its closely related moieties in the global publications platform. in this context, this contribution is a good resource for the global researchers to plan new routes for the preparation of 1 and other associated initiatives. saralaya & hiriyalu synthesis of lamotrigine and a glimpse of its closely related compounds: an overview vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 13 references 1. prabhavalkar ks, poovanpallil nb, bhatt lk. management of bipolar depression with lamotrigine: an antiepileptic mood stabilizer. front pharmacol. 2015;6. available from: http://dx.doi.org/10.3389/fphar.2015.00242. 2. warshavsky a, eilam a, gilad r. lamotrigine as monotherapy in clinical practice: efficacy of various dosages in epilepsy. brain behav. 2016; 6(3). available from: http://dx.doi.org/10.1002/brb3.419. 3. goa kl, ross sr, chrisp p. lamotrigine: a review of its pharmacological properties and clinical efficacy in epilepsy. drugs. 1993; 46(1):152–76. 4. dibué-adjei m, kamp ma, alpdogan s, et al. cav2.3 (r-type) calcium channels are critical for mediating anticonvulsive and neuroprotective properties of lamotrigine in vivo. cell physiol biochem. 2017; 44(3):935–47. 5. rambeck b, wolf p. lamotrigine clinical pharmacokinetics. clin 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morphological differences. int j pharm. 2022; 628(122310):122310. 73. kumar r, kumar n, roy r, et al. triazines – a comprehensive review of their synthesis and diverse biological importance. curr med drug res. 2017. available from: https://rasayanjournal.co.in/vol-1/issue-2/17.pdf https://web.archive.org/web/20180413073436id_/http:/ijppsjournal.com/vol4issue1/2904.pdf https://web.archive.org/web/20180413073436id_/http:/ijppsjournal.com/vol4issue1/2904.pdf https://oa.mg/work/2805517906 saralaya & hiriyalu synthesis of lamotrigine and a glimpse of its closely related compounds: an overview vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 15 https://globalscitechocean.com/reportfile/3b9d1f5b088c44f8869 9e0432f29917b.pdf. 74. majid a, lawal am, ashid m, et al. synthetic routes, characterization and biological significance of 1, 2, 4-triazine derivatives: comprehensive review. journal of indian research. 2020; 8(1):37-52. 75. yaduwanshi ps, agrawal o, mishra mk. a review on recent approach in synthetic methods, chemical characteristics and biological potential of triazine and quinazolinone derivatives. nat volatiles essent oils. 2021; 5257–74. how to cite this article: saralaya ss, hiriyalu ss. an overview of prior art disclosures about the synthesis of lamotrigine and a glimpse of its closely related compounds. indian j pharm drug studies. 2024; 3(1):8-15. funding: none; conflicts of interest: none stated https://globalscitechocean.com/reportfile/3b9d1f5b088c44f88699e0432f29917b.pdf https://globalscitechocean.com/reportfile/3b9d1f5b088c44f88699e0432f29917b.pdf sikdar et al. advances in epilepsy research: from mechanisms to therapeutics vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 148 review article epilepsy unveiled: advances in understanding, diagnosis, and management shrijita sikdar1, udita dutta2 , ayan ghosh3, suparna mondal4, krisnendu das5 from, 1pg student, s.n pradhan centre for neuroscience, ballygunge science college, calcutta university, kolkata, 2ug student, eminent college of pharmaceutical technology, moshpukur, barbaria, paschim khilkapur, barasat, jagannathpur, 3ug student, department of pharmacy, gupta college of technological sciences, asansol, 4nursing tutor and coordinator, department of nursing, m.r institute of nursing, bira, 5assistant professor, school of pharmaceutical sciences, the neotia university, diamond harbour, west bengal, india abstract in addition to the numerous stress that characterize individuals today's fast-paced lives, the majority of people on the planet encounter various neurological problems. epilepsy, one of the most common neurological disorders of the brain, affects over 50 million people worldwide. 90% of whom are from developing nations. genetic factors, brain infections, strokes, tumors, and high body temperatures are all contributors to epilepsy. along with the social stigma that brings discrimination against patients and even their families in the community, it also, creates a significant financial burden on the healthcare systems of different countries. people with epilepsy frequently experience extreme mental suffering, behavioral problems, and little to no social engagement. numerous seizure types exist, as are numerous mechanisms by which the brain can trigger seizures. neuronal hyperexcitability and neural circuit hypersynchrony are the two fundamental features of seizure genesis. numerous mechanisms may disturb the equilibrium between excitation and inhibition, predisposing a specific or generalized region of the brain to hyperexcitability and hypersynchrony. the review will include the classification, background, epidemiology, etiology, pathophysiology, symptoms, diagnosis, and management of epilepsy. key words: epilepsy, seizure, anti-epileptic drug, pathophysiology pilepsy is a widespread chronic neurological illness that affects up to 1% of the population, making it the second most prevalent serious neurologic disorder after stroke [1]. it affects around 50 million individuals worldwide, with 90% of those affected living in underdeveloped nations [2]. epilepsy is defined by recurrent unprovoked 3-5 seizures, with substantial changes in the biology and consequences of seizures between the immature and mature brain [3]. it refers to a variety of seizures that vary in severity, appearance, cause, effect, and management. epilepsy frequently causes brief impairments in consciousness, putting people in danger and interfering with schooling and jobs. access this article online received – 30th oct 2023 initial review – 16th nov 2023 accepted – 07th dec 2023 quick response code it has no age, gender, geographical, social class, or racial boundaries. epilepsy is more common in young children and those over the age of 65, but it can develop at any age [4]. seizure onset can be focal, generalized, or unknown, and can be classified based on awareness or motor or nonmotor aspects. active epilepsy is defined by regular treatment with antiepileptic medications or the most recent seizure occurring within the last 5 years [5]. a condition known as status epilepticus (se) is a prolonged or repeated seizure that can lead to long-term consequences, including neuronal injury or death. a new diagnostic classification for se has been proposed [6]. sudden unexpected death in epilepsy (sudep) is defined as a sudden, unexpected, witnessed or unwitnessed death in epileptic patients, with or without seizure evidence, and excluding established se. _______________________________________________ correspondence to: krishnenedu das, 36a, mahanirban road, kolkata-700029, west bengal, india. email: krishnendas96@gmail.com, tel.: +91 9874377408 e mailto:krishnendas96@gmail.com sikdar et al. advances in epilepsy research: from mechanisms to therapeutics vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 149 seizures cause sudep, which may include seizureinduced cardiorespiratory changes. the frequency of epilepsy is determined by its incidence, prevalence, and mortality, whereas the burden is determined by disabilityadjusted life-years (dalys), years of life lost, and years of living with disability [7]. conventional treatment generally consists of anticonvulsant drugs, however even with the finest current treatments; more than 30% of persons with epilepsy do not have seizure control [8]. therapy is symptomatic because existing medications reduce seizures, but there is no effective preventive or cure. medication adherence is a big issue due to the longterm negative effects of many medicines [9]. history of epilepsy: epilepsy, originating from the greek term "epilepsia," which means "to seize," has long been tied with religion and demon possession. epilepsy was once thought to be a sacred sickness, with many believing that it affected those who were abducted by demons or that their visions were sent by the gods. epilepsy was considered a demonic spirit attack in hmong generations, although affected persons could become honored as shamans as a result of their explicit experiences [10]. epileptic people were scorned and even imprisoned in most societies. jean-martin charcot noted that epileptic persons were mentally retarded and criminally insane in the salpetriere, the birthplace of contemporary neuroscience. people in tanzania believed epilepsy was caused by bad spirits, witchcraft, poisoning, or was communicable. epilepsy was considered a god-given curse in rome, known as morbus comitialis [11]. stigma continues to this day, but it is gradually decreasing in developed countries. hippocrates predicted that it would not take much time to eradicate epilepsy as it is not divine. epidemiology: epilepsy is a common neurological illness, affecting an estimated 55 lakhs individuals in india, 20 lakhs in the united states, and three lacks in the united kingdom. in the united states, 120 persons out of every 100,000 have a seizure each year, with a recurrence rate ranging from 23% to 80%. the annual age-adjusted incidence of epilepsy is 44 per 100,000 persons. every year, around 125,000 new cases are diagnosed, with 30% of those diagnosed being under the age of 18. the elderly have a high prevalence of epilepsy, which is now acknowledged. at least 10% of patients in long-term care facilities are on at least six antiepileptic medications. the national sentinel audit of epilepsy-related fatalities highlights the problem, indicating that 1,000 people die in the uk each year as a result of epilepsy, with 42% of these fatalities being avoidable [12]. causes of epilepsy: epilepsy is a disorder in which the cause or severity of seizures is not directly connected. some cases are genetic, while others are the result of brain traumas, strokes, infections, high fever, or malignancies [13]. many incidences of epilepsy in young children are caused by heredity, but it can affect people of any age. specific precipitants or triggers, such as reading, flashing lights, mental stress, sleep deprivation, heat stress, alcohol, and febrile sickness, are required for reflex epilepsy disorders [14]. the impact of these precipitants varies depending on the epileptic syndrome. in epileptic women, the menstrual cycle can also alter seizure recurrence patterns. the most prevalent causes of epilepsy in infants and early infancy are hypoxic-ischemic encephalopathy, cns infections, trauma, congenital cns abnormalities, and metabolic problems. cns infections and trauma can cause febrile seizures in late infancy and early childhood. cerebrovascular disease is the most common cause of death in the elderly, followed by cns malignancies, head trauma, and degenerative disorders such as dementia [15]. pathophysiology of epilepsy: seizures are paroxysmal manifestations of the cerebral cortex caused by a transient imbalance in excitatory and inhibitory forces in the cortical neuron network. the seizure event is recognized in an unstable cell membrane or its surrounding cells, arising from either cortical or subcortical area's gray matter. a small number of neurons fire inappropriately at first, and normal membrane conductance and inhibitory synaptic current breakdown, as well as excess excitability, spread either locally to cause a focal seizure or more broadly to produce a generalized seizure. this onset spreads through physiologic pathways to include nearby and remote locations [16]. a failure in potassium conductance, a defect in voltage-activated ion channels, or a shortage in membrane atpases involved in ion transport can all lead to neuronal membrane instability and a seizure. certain neurotransmitters, such as glutamate, aspartate, acetylcholine, norepinephrine, histamine, corticotropinreleasing factor, purines, peptides, cytokines, and steroid hormones, increase neuronal excitability and propagation, whereas -amino butyric acid (gaba) and dopamine decrease neuronal excitability and propagation [18]. during a seizure, the demand for blood flow to the brain increases in order to remove co and bring substrate for neuronal metabolic activity. some types of epilepsy may be connected to mutations in many genes. sikdar et al. advances in epilepsy research: from mechanisms to therapeutics vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 150 figure 1: pathophysiology of epilepsy and understanding seizure mechanisms [17] diagnosis: a number of different tests have been developed to determine the epilepsy in an individual and its type. this may include a neurological exam that examines behavior, emotions, and mental function to diagnose and classify epilepsy. blood tests look for evidence of infections, genetic disorders, or other seizurerelated illnesses. genetic testing, which is commonly performed in children but can also benefit some adults with epilepsy, can provide more information about the condition and therapy [19]. some brain imaging tests and scans that detect changes are electroencephalogram (eeg) monitoring: electron encephalograms are extremely helpful in the identification of many seizure disorders. even if the eeg is normal in some people, they stillhave the clinical diagnosis of epilepsy. many people who do not have epilepsy have atypical brain activity. video monitoring is frequently used in conjunction with eeg to establish the type of seizures a person has [20]. high-density eeg:. in this test, electrodes are put closer together than in a traditional eeg. high-density eeg may aid in pinpointing which parts of the brain are affected by seizures [21]. computerized tomography (ct) scan: ct scans use xrays to create cross-sectional images of the brain, which can help detect tumors, bleeding & cysts that cause epilepsy [22]. magnetic resonance imaging (mri): an mri, like a ct scan, employs powerful magnets and radio waves to provide a precise image of the brain in order to detect future seizures, but it provides a more detailed view than a ct scan [23]. functional mri (fmri): a functional mri measures blood flow changes in brain parts, aiding in identifying critical functions like speech & movement before surgery, enabling surgeons to avoid these areas during the procedure [24]. positron emission tomography (pet): pet scans use low-dose radioactive material injected into veins to visualize brain metabolic activity and detect changes, potentially identifying low metabolism areas as seizures [25]. single-photon emission computerized tomography (spect): a spect test uses low-dose radioactive material to create a 3d map of blood flow during seizures, indicating seizure locations. siscom, or subtraction ictal spect connected to mri, overlaps spect results with brain mri results for more detailed results [26]. other techniques, such as statistical parametric mapping (spm), electrical source imaging (esi), and magneto encephalography (meg), can be used to detect seizures. spm contrasts locations with increased blood flow during seizures versus those without seizures. for a more thorough picture of seizures, esi projects eeg data onto an mri. meg detects magnetic fields generated by brain activity, resulting in more accurate results due to reduced sikdar et al. advances in epilepsy research: from mechanisms to therapeutics vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 151 interference from the skull and surrounding tissue. these approaches, when combined, provide images of areas impacted and not affected by seizures, allowing for a more complete knowledge of the brain's origins [27]. figure 2: a) types of seizure b) symptoms of seizure [28]. table 1: classification of drugs use in therapy of epilepsy [29] aed class of drug mechanism of action uses advantages disadvantages pregabalin anticonv ulsants inhibiting calcium influx and subsequent release of excitatory neurotransmitters. treat epilepsy, anxiety and neuropathic pain. add on efficacy: partial onset seizures, no drug interactions. no hypersensitivity skin reactions. work also in neuropathic pain and generalized anxiety disorders. weight gain. increased side effect risk in patients with low glomerular filtration rate requires a lower dose. seizure aggravation in idiopathic generalized epilepsy (absence or myoclonic seizures). phenobarbital/ primidone barbiturat e prolonged and frequency of gaba mediated chloride channel opening. blockade of ampa receptors. anti-seizure, anti-epileptic, treat insomnia, treat anxiety. efficacy: partial-onset seizures and generalized myoclonic seizures. rash is uncommon. phenobarbital is widely available and inexpensive parenteral formulation. drug interactions(may lower the efficacy of concomitant medications metabolized by the p450 hepatic enzyme system). sedation, cognitive slowing, arthralgia phenytoin anticonv ulsants voltage gated sodium channel blocker, keep maintain the sodium channel’s inactive condition and extending the neuronal refractory period. anti-epileptic. efficacy: partial-onset seizures. long accumulated experience. can be loaded orally or intravenously (intravenous phenytoin or the prodrug fosphenytion). drug interaction (through p450 enzyme induction and extensive protein binding). nonlinear pharmacokinetics (small changes in dose or bioavailability may produce large fluctuations in level). ataxia, rash. seizures aggravation in idiopathic generalized epilepsy (absence or myoclonic seizures). sikdar et al. advances in epilepsy research: from mechanisms to therapeutics vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 152 rufinamide anticonv ulsants it is a triazole derivative antiepileptic which prolong the inactive state of voltage gated sodium channel. control seizure. efficacy: lennoxgastaut syndrome. drug interactions (clearance decreased by valproate and increased by enzyme inducers, may reduce efficacy of oral contraceptives). tiagabine anticonv ulsants inhibit the reuptake of gaba into presynaptic neurons and increase the amount of gaba to postsynaptic neurons. treat partial seizure in epilepsy. efficacy: partial-onset seizures. does not affect other aeds. relatively favorable cognitive profile only indicated as adjunctive therapy. requires a show titration, given three or four times daily. can cause nonconvulsive status epilepticus or encephalopathy that resembles nonconvulsive status epilepticus, even in the absence of prior epilepsy. topiramate second generatio n antiepilep tic drug. it blocks voltage gated sodium channels. increasing gaba activity and inhibit glutamate activity. inhibition of kinateevoked currents. manage and treat epilepsy and migraine. efficacy: partial-onset seizures and generalized seizures. rash is uncommon. efficacy against migraine. weight loss. weight loss: aphasia and cognitive impairement, nephrolithiasis, metabolic acidosis, hypohidrosis. requires slow titration rate because of adverse cognitive effects valproate anticonv ulsants blocks voltage gated ion channel and increase the inhibitory neurotransmission. treat epilepsy and bipolar disorder. wide spectrum of efficacy against partialonset seizures and generalized seizures. no hypersensitivity skin reactions. work for bipolar disorder and migraine. intravenous preparation. weight gain: encephalopathy, tremor, parkinsonian syndrome. teratogenicity and permanent adverse cognitive outcomes in fetus. drug interaction (due to inhibition of p450 enzymes and extensive protein binding). vigabatrin antiepileptic/ anticonv ulsants inhibit gaba degraded enzyme gabatransaminase, increase gaba concentration in the brain treat refractory complex partial seizure. add-on efficacy for partial-onset seizures and west syndrome. no interactions. concentric visual field defects, irreversible. zonisamide sulfonam ide antiepileptic drug/ anticonv ulsants block voltage sensitive sodium channel and ttype calcium channel. enhancement of gabaergic transmission and inhibition of glutamatergic transmiss. treat partial onset seizure in the treatment of epilepsy. efficacy: partial-onset seizures; generalized onset seizures(evidence not rigorous). long half-life (allows once daily dosing). weight loss weight loss, aphasia and cognitive impairement, nephrolithiasis, metabolic acidosis; anhidrosis in children (fever). blood biomarkers in epilepsy: biochemical marker advancements can identify brain pathology, increasing hope in epilepsy. although connectivity/resting state imaging and gadgets such as smartwatches and implantable eeg can detect epilepsy, their cost and impracticality limit their usage in larger patient populations. blood testing may be a more scalable method of diagnosing epilepsy and seizure burden. pilot studies have shown feasibility, and trials with acute brain illnesses with a high risk of epileptogenesis could be used as well. biomarkers of disease activity, such as nt-probnp or hba1c, may help in illness management and intervention [30]. neuronal/brain biomarkers: neuronal/brain biomarkers like s100b, nse, gfap, nfl, tau, uchl-1, and mmp-9 are crucial for identifying epileptic pathophysiological changes. s100b, primarily expressed in astrocytes, is linked to poststroke epilepsy. gfap, nse, nfl, and uchl-1 are linked to neurodegenerative illnesses. elevated serum concentrations in epilepsy patients are detected in plasma. further research is needed to determine their clinical use and role in epilepsy [31]. neuroinflammatory biomarkers: neuroinflammatory indicators, such as cytokines like il-1, il-2, and il-4, are potential biomarkers for epilepsy. elevated interleukins (il) and plasma il-6 levels are associated with long-term seizures. individuals with epilepsy have higher levels of ifns, tnfs, ccl17, and other neuroinflammatory mediators. these factors contribute to inflammation and seizure development [32]. sikdar et al. advances in epilepsy research: from mechanisms to therapeutics vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 153 epilepsy and oral contraceptives: antiepileptic medications such as carbamazepine, phenytoin, barbiturates, topiramate, and oxcarbazepine can result in hormonal contraception failure, requiring patients to use a higher estrogen-containing oral contraceptive (50 mg/mg/day) [33]. pregnancy and epilepsy: pregnancy epilepsy can lead to difficulties for both the mother and the fetus, increasing the chance of spontaneous abortion and stillbirth. to avoid anoxiosis and metabolic abnormalities, it is critical to manage the illness effectively and cure it before pregnancy. minor seizures should not be eliminated, and patients should take folic acid and vitamin k supplements orally. some antiepileptic medicines can interfere with folic acid metabolism, resulting in neural tube abnormalities. hepatic enzyme-inducing medications can also cause postpartum bleeding by lowering the mother's vitamin k levels [34]. breastfeeding: antiepileptics are normally present in low amounts in breast milk, making breastfeeding safe when administered in standard doses. however, benzodiazepines and barbiturates, which are found in high concentrations in breast milk, might cause newborn drowsiness, posing a risk to breastfeeding [35]. epilepsy in children: children with epilepsy who have fits are treated similarly to adults, but they may react differently and be unpleasant. if febrile convulsions occur, a significant epilepsy medicine may be administered consistently until the kid reaches the age of five. because prolonged therapy may interfere with cognitive development, the medicine is discontinued [36]. future directions in research and treatment: epilepsy research is evolving towards preventive and curative strategies, emphasizing a shift from symptom control. recent breakthroughs involve identifying mutated genes in inherited epilepsy, characterizing brain networks at the molecular level, enhancing seizure origin imaging, and advancing quantitative eeg analysis for seizure prediction. therapeutic advancements focus on new molecular targets, eeg-tailored drug delivery, gene/cell therapy, and innovative surgical/non-ablative approaches [37]. integrated methods, like combining imaging with electrophysiology, play a central role in localizing epilepsy development and improving prediction and treatment. speculative approaches explore preventing epileptogenesis through cellular phenotype modulation, investigating protective factors, and understanding the fate of endogenous neural stem cells in the mature cns. novel pharmacotherapies, biosensor-coupled delivery systems, gene/cell therapy, and progressive surgical methods represent emerging directions for epilepsy therapy. these advancements collectively contribute to a comprehensive understanding and treatment of epilepsy, providing a foundation for further research and breakthroughs [38]. challenges in new drug development: epilepsy research and treatment encounter multifaceted challenges, including the diverse origins of the condition, hindrances in identifying reliable biomarkers for early diagnosis, and the emergence of drug resistance in some patients. the complex interplay of epilepsy with comorbidities necessitates a holistic patient care approach, while the incomplete understanding of underlying mechanisms impedes the development of targeted therapies [39]. societal stigma surrounding epilepsy and its impact on individuals' quality of life constitute significant hurdles. furthermore, global discrepancies in accessibility to specialized epilepsy care underscore the need for more equitable healthcare distribution. emerging research pointing to neuroinflammation and immune system involvement adds another layer of complexity, calling for further exploration and potential anti-inflammatory strategies [40]. addressing these challenges requires collaborative efforts involving diverse stakeholders, an increase in funding to support comprehensive research endeavors, and a multidisciplinary approach to advance both the understanding and treatment of epilepsy. the integration of knowledge from various fields, coupled with innovative strategies, is pivotal for overcoming these challenges and improving outcomes for individuals affected by epilepsy. conclusion epilepsy is a complex condition that affects people of all ages, with a special preference for children and the elderly. its causes range from monogenic to cortical acquired diseases and its severity varies from patient to patient. despite different treatments, public health surveys reveal that many people have a low quality of life as a result of the impact of recurring seizures on everyday activities. thus, the selection of an anticonvulsant medication is mostly based on its efficacy for specific types of seizures and epilepsy. despite adequate seizure control, a considerable proportion of epilepsy patients have intractable or drug-resistant epilepsy, necessitating the development of novel medications with better side effects and tolerance profiles, even at the expense of efficacy, when compared to existing antiepileptic therapies. sikdar et al. advances in epilepsy research: from mechanisms to therapeutics vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 154 reference 1. joshi r, tripathi m, gupta p, et al. prescription pattern of antiepileptic drugs in a tertiary care center of india. indian journal of pharmacology. 2020; 52(4): 283–289. 2. tchalla, achilleedem, et al. newly diagnosed epileptic seizures: focus on an elderly population on the french island of reunion in the southern indian ocean. epilepsia. 2011;52(12):2203-2208. 3. heger, katrine, et al. changes in the use of antiseizure medications in children and adolescents in norway, 2009– 2018. epilepsy res. 2022;181:106872. 4. bhui u, sarkar s, das j, et al. unraveling the enigma of anti-tubercular drug-induced vitamin b6 deficiency and its role in provoking convulsive seizures: a revelatory case report. indian j of pharm drug studies. 2023;jul 1:125-7. 5. vitaliti, giovanna, et al. molecular mechanism involved in the pathogenesis of early-onset epileptic encephalopathy. frontiers in molecular neuroscience. 2019;12:118. 6. hsieh liang-po, chin-yin huang. prevalence of treated epilepsy in western medicine among the adult population in taiwan: a study conducted using antiepileptic drug prescription data. epilepsy res. 2008;80.2-3:114-118. 7. shanmugapriya s, et al. drug prescription pattern of outpatients in a tertiary care teaching hospital in tamil nadu. perspectives clini res. 2018;9(3):133. 8. patterson victor. the development of a smartphone application to help manage epilepsy in resource-limited settings. seizure. 2020;79:69-74. 9. liang chun-yu, et al. prescription patterns and dosages of antiepileptic drugs in prevalent patients with epilepsy in taiwan: a nationwide retrospective cross-sectional study. epilepsy behavior. 2022;126:108450. 10. religioni, urszula, teresa pakulska. rational drug use in hospital settings–areas that can be changed. j med 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1999;54(4):211-6. sikdar et al. advances in epilepsy research: from mechanisms to therapeutics vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 155 37. jacobs mp, fischbach gd, davis mr, et al. future directions for epilepsy research. neurology. 2001;57(9):1536-42. 38. linehan c, tellez-zenteno jf, burneo jg, et al. future directions for epidemiology in epilepsy. epilepsy & behavior. 2011;22(1):112-7. 39. wahab a. difficulties in treatment and management of epilepsy and challenges in new drug development. pharmaceuticals. 2010;3(7):2090-110. 40. holmes gl, noebels jl. the epilepsy spectrum: targeting future research challenges. cold spring harbor perspectives in medicine. 2016;6(7). how to cite this article: shrijita sikdar, udita dutta, ayan ghosh, suparna mondal, krisnendu das. epilepsy unveiled: advances in understanding, diagnosis, and management. indian j pharm drug studies. 2023; 2(4):148-155. funding: none conflict of interest: none stated singha et al. leptin: its crucial role in the fight against obesity online first indian j pharm drug studies | 1 review article decoding leptin: unraveling the role of leptin signaling in the battle against obesity soumyadip singha1, soumyadip nayak2, rahul bishayee2, pritam sarkar3, priyanka ghosh3, udita dutta4, poulomi mishra5 from, 1lecturer, pandaveswar school of pharmacy, pandaveswar, west bengal, india, 2pg student, department of pharmacology, school of pharmaceutical sciences, lovely professional university, phagwara, punjab, india, 3ug student, department of pharmacy, school of pharmacy, seacom skills university, bolpur, west bengal, india, 4ug student, department of pharmacy, eminent college of pharmaceutical technology, moshpukur, barbaria, paschim khilkapur, barasat, jagannathpur, west bengal, india. 5assistant professor, pandaveswar school of pharmacy, pandaveswar, west bengal, india abstract obesity represents a major global health crisis, linked to a spectrum of metabolic disorders, including diabetes, cardiovascular diseases, and hypertension. leptin, a hormone predominantly produced by adipose tissue, plays a critical role in regulating energy balance, appetite, and metabolism. historically, leptin was believed to be a cornerstone in the management of obesity due to its ability to suppress appetite. however, the effectiveness of leptin therapy has been limited by the phenomenon of leptin resistance in obese individuals. this review, titled "decoding leptin: unraveling the role of leptin signaling in the battle against obesity," aims to delve into the molecular intricacies of leptin signaling pathways and their implications in obesity management. we explore the mechanisms underlying leptin resistance, the impact of leptin on neuroendocrine pathways, and the potential for resetting leptin sensitivity as a therapeutic strategy. additionally, we discuss recent advances in therapeutic approaches aiming to enhance leptin sensitivity or mimic its effects, including novel pharmacological agents and lifestyle modifications that influence leptin signaling. by providing a comprehensive overview of current research and emerging therapies, this review seeks to illuminate the path forward in leveraging leptin signaling for more effective obesity interventions. key words: leptin resistance, obesity management, therapeutic strategies, neuroendocrine regulation besity can be defined as a condition which can be characterized by excessive accumulation of body fat which has the capability to show negative impact of health, in general a person could be said that he or she is suffering from obesity by checking the bmi over 30 kg/m3[1]. there are certain east asian countries they apply lower value for defining the obese condition. obesity can be linked with various other health risk factors, such as cardiovascular diseases, type ii diabetes, obstructive sleep apnea, certain cancers, osteoarthritis, obesity can also be influenced by a reciprocal relationship by depression. obese condition for children ranging between the age of 519, can be defined with the bmi with two standard deviations above the median their age group [2], and for the age group below the age of 5, the obese condition is considered to be the three-standard deviation above the median. the assessment of fat distribution and cardiovascular risk factorcould be done by subdividing bmi by the cdc [3]. when it comes to the etiological factors of obesity, excessive calorie intake and access this article online received – 24th may 2024 initial review – 30th september 2024 accepted – 28th october 2024 quick response code leading a sedentary life plays major role, while the genetics also have some role when it comes to obesity with certain genes showing influence towards susceptibility to obesity [6][9]. in addition, there are certain drug metabolism system which also contributes to the condition of obesity, particularly fat-soluble drugs, like anti-tuberculosis medications [10]. the increase risk of obesity is also accompanied with the treatment for certain physical and mental illness like psychiatric disorder [11]. finally, the contribution of the hormone leptins plays the vital role in obesity. leptin is a hormone with helps in regulating the intake of food and energy usage. obsess condition may occur when the hormone leptin have lost its ability to signal the brain about proper functioning of food intake and usage of energy. leptin and its receptors: leptin is a neurohormone that acts in the hypothalamus to regulate energy balance and food intake [12]. recessive mutations in the leptin (obese, ob) or its receptor obr gene result in profound obesity and type ii diabetes mellitus. further studies demonstrated that in __________________________________________________ correspondence to: poulomi mishra. pandaveswar school of pharmacy, pandaveswar, west bengal, india. email: mishrapoulomibeliatore@gmail.com o mailto:mishrapoulomibeliatore@gmail.com singha et al. leptin: its crucial role in the fight against obesity online first indian j pharm drug studies | 2 addition to its role as a neurohormone, leptin can modulate immune response, fertility, and hematopoiesis, acting as a mitogen, metabolic regulator, or pro angiogenic factor [13]. the central regulation of food intake and energy expenditure is mediated through the binding of leptin to its receptor obr, a type 1 cytokine receptor. several isoforms of obr have been described as a result of alternative mrna splicing leading to several short isoforms (obra, obrc, obrd, and obrf), one long isoform obrb with a long cytosolic c-terminus tail and one soluble isoform obre [14]. hence, obr isoforms differ in the length of their intracellular region but share identical extracellular domains. while short isoforms are ubiquitously expressed, obrb expression is more restricted with high levels in hypothalamic nuclei such as the arcuate nucleus (arc). the hypothalamic arc has an important role in the development of leptin resistance. accordingly, exposure of rodents to a high-fat diet rapidly decreases the phosphorylation of stat3 in the arc or the ventral tegmental area (vta), while leptin-sensitivity is simultaneously maintained in some other hypothalamic nuclei. while the biological function of the short isoforms is still elusive, it is well established that obftb is the main isoform responsible for the effect of leptin on body weight control. the weight lowering properties of leptin via obrh has been suggested to be centrally mediated. once activated after leptin binding, obrh is able to trigger various signal transduction pathways. activation of the janus tyrosine kinase 2 (jak2)/signal transducer and activator of transcription 3 (stat3) pathway leads to an increase of anorexigenic signals and a decrease of orexigenic signals [15]. leptin is also able to activate the insulin receptor substrate (irs)/phosphatidylinositide 3-kinase (pi3k) pathway, essential for the regulation of glucose homeostasis [16]. moreover, leptin inhibits the energy 10 sensor adenosine monophosphate activated protein kinase (ampk) in the brain, to decrease eating. the activation of extracellular signal regulated kinase (erk) is another pathway mediating the anorectic action of leptin in the hypothalamus. leptin expression in obesity: early onset obesities has the capability to form rare genetic mutations which can hamper leptin signaling which might lead to congenital leptin deficiency or leptin resistance. hyperleptinemia and resistance to reducing body mass is the general characteristics of leptin resistance [5][6][17][18]. a person is leptin resistant, can only be said with the presence of leptin in plasma at higher amount, high amount of leptin in blood plasma certainly corelates with body fat percentage. several weight loss studies have revealed that the level of leptin can decrease initially and then gradually rise with continued weight loss. matheny et al.’s research put light on leptin resistance can be induced in the arcuate nucleus (arc) and ventral tegmental are (vta) of the brain with high fat diets, while when it comes to medial basal hypothalamic region it retains sensitivity [20][21]. the critical role of arc in leptin can be indicated by targeting downregulation of leptin receptor expression in the arc promoting diet-induced obesity. activation of socs3 and stat3 resistance to leptin in neuron such as pro-opiomelanocortin (pomc) and agrp in rodents with high-fat content diet. while agrp neurons shows increased sensitivity towards leptin when the rodents are shifted to low-fat content diet. polymorphism in the crystalline structure of the leptin (ob) and leptin receptor (lep-r) genes which has the capability to disrupt leptin functions resulting in obesity [22][23][24]. the polymorphism of lep-2548 g/a are widely studied in humans for understanding obesity. a protein quantitative trait locus (pqtl) analysis study was performed by carayol et al. to understand the genetic influences on leptin levels and identified fam46a as the negative signaling regulator in adipose tissue [25][26]. in addition to that distant enhance sequences, le1 and leprei, modulate leptin gene expression through ppar gama/ rxra binding sites. noncoding rnas and epigenetic factors like dna methylation found in the leptin (ob) and lep-r genes, are expressions of obesity and leptin insensitivity [27][28]. mechanisms underpinning leptin resistance: it is worth noting that leptin resistance occurs when there is an impairment of the effectiveness of the obrb downstream signaling transduction, although in the presence of hyperleptinemia, a lack of anti obesity action of leptin appears. therefore, leptin resistance is one of the most frequent features in the onset and progression of obesity [29]. this condition is very common in obese humans and occurs after only few weeks of high fat diet (hfd) in rodents. since the hypothalamus mediates the anti-obesity actions of leptin, three mechanisms are currently accepted to mediate central leptin resistance, such as the reduction in leptin access to cns through the bbb, the impairment of leptin signaling in firstorder neurons expressing obrb, or in second-order leptintargeted neurons and neural circuits. it has been recently suggested that other mechanisms, such as the onset of hypothalamic inflammation, autophagy deficiency or er stress, can also mediate the obesityassociated central leptin resistance. indeed, since leptin exerts its biological effects not only in cns but also in peripheral tissues, parallel to central leptin resistance a peripheral dampening in leptin sensitivity can occur. reduction in leptin access to cns: as it is well-known, obra, which is highly expressed by capillary cells of the choroid plexus, actively transports leptin across the bbb to reach the majority of obrb expressing neurons in the cns. this transport system is saturable and recently it has been shown to be flanked by another transport mechanism involving the endocytic receptor megalin, as demonstrated by a decrease in leptin cerebrospinal fluid (csf) levels inmegalin deficiency [30]. in obese individuals the saturation of leptin singha et al. leptin: its crucial role in the fight against obesity online first indian j pharm drug studies | 3 transport can occur due to the hyperleptinemia, resulting in an only slightly increase in the csf leptin levels [31]. oh-i et al. showed that the impairment of leptin transport across bbb can also be caused by the higher plasmatic levels of cytokines figure the pathophysiology of leptin signaling on its crucial role in regulating appetite and maintaining energy balance [52] and fatty acids in obese individuals relative to lean subjects. to date, it is still unclear the extent to which the impairment of leptin transport to cns can contribute to the leptin resistance. indeed, leptin can reach the cns through the median eminence, which lacks the bbb. therefore, arc firstorder neurons can sense leptin trough their projections into the median eminence [32]. impairment of leptin signaling in hypothalamic neurons: leptin resistance can occur by alterations in each component of the ob-rb downstream signaling cascade. in particular, previous findings have highlighted three potential mechanisms: a reduced expression of ob-rb at plasma membrane level, an upregulation of negative regulators of leptin signaling, and a downregulation of its positive regulators. diano et al. have demonstrated that ob-rb has a predominant localization in the golgi apparatus in hypothalamic neuronal and glial cells, thus its trafficking to the plasma membrane is necessary to obtain a physiological response to leptin stimulation. the ob-rb expression pattern at cell surface depends on a delicate balance between the activity of the bardet-biedl syndrome (bbs) proteins, which mediate its transport to the plasma membrane, and the rate of the ligand independent endocytosis, a process that promotes the ob-rb internalization. regarding the second molecular mechanism underpinning leptin resistance, the ob-rb physiological signaling is under control of two negative regulators: socs-3 and phosphor tyrosine phosphatase (ptp) 1b [33]. socs3 is involved in the onset of leptin resistance at central level, as well as at peripheral level. it has been demonstrated that an increase in socs3 mrna expression is involved in the development of leptin resistance in skeletal muscle from rats on high-fat diet (hfd). moreover, socs3 inhibits ampk activation in peripheral metabolically active tissues, such the liver, white adipose tissue (wat) and skeletal muscle, contributing to abnormalities of fatty acid metabolism. ampk is a fuel-sensing enzyme, whose activity is finely regulated by leptin: in peripheral tissues, i.e. wat, leptin increases its enzyme activity, promoting the catabolic pathways toward the fatty acid oxidation and glucose internalization, exceeding the anabolism rate. on the contrary, leptin inhibits ampk activity at central level, where this enzyme is involved in food intake regulation, singha et al. leptin: its crucial role in the fight against obesity online first indian j pharm drug studies | 4 since it controls the release of hypothalamic neuropeptides. in addition to socs3, ptp1b is a negative regulator of both leptin and insulin signaling. leptin resistance is also associated with a downregulation of positive regulators of obrb signal transduction. in particular, the leptin-induced stat3 phosphorylation is essential to obtain a clear activation of hypothalamic neurons, releasing thus the anorexigenic neuropeptides. impairment of mc4r downstream signal transduction in neural circuits: the melanocortin system controls the energy balance, especially trough the mc4r, which is mostly expressed in the brain. therefore, alterations in the mcardependent brain-derived neurotrophic factor (bdnf)/tropomyosin receptor kinase (trk) b signalling pathway, in the ventromedial nucleus (vmn), can affect leptin resistance. as demonstrated by liao et al. [34], mice harbouring a truncated long bdnf 3 utr develop a severe hyperphagia and a clear obese phenotype. in this genetic model, leptin is unable to activate hypothalamic neurons and reduce food intake. role of hypothalamic er stress in central leptin resistance: the er is responsible for folding nascent proteins and this process is possible until there is a perfect balance between the er capability to fold these macromolecules and the amount of loaded proteins. as soon as an imbalance between these folding and loading processes occurs, er stress appears, leading to the activation of several pathways (ie. unfolded protein response (upr), inositol-requiring protein (ire)-1 and protein kinase rna (pkr)-like kinase (perk) pathways), which collectively attempt to counteract the er stress itself, restoring the er homeostasis. to date, a growing body of evidence has demonstrated the involvement of the hypothalamic er stress in central leptin resistance and obesity. consistently, pharmacological approaches, consisting in the central administration of er stress inducers or chemical er chaperons, are able to modulate leptin responsiveness in an opposite manner, identifying a role of er stress in leptin resistance [35]. defective autophagy as a contributor of leptin resistance: it has been recently emphasized a key role for the autophagy in regulating the overall energy balance, since the inhibition of this process, by a neuronspecific deletion of autophagy related protein (atg) 7, can alter the phenotype in mice. in particular, mice show an obese phenotype when this wellknown autophagy component is selectively knocked down in pomc neurons, probably because this deletion is associated also with a reduction of leptininduced stat3 phosphorylation; in agreement with these results, the deletion of this gene in agrp neurons causes a reduction in fat mass collectively, these data highlight that the hypothalamic autophagy deficiency is involved in leptin resistance and obesity [36]. strategies to overcome leptin resistance: caloric restriction and exercise: caloric restriction is the first approach for the treatment of obesity able to reduce circulating leptin levels, as an alternative to pharmacologic reversal of leptin resistance. it has been reported that long term exercise, not only decreased leptin levels, but also increases the activation of stat3 and ampk signalling pathways in the hypothalamic arcuate nucleus. prevention of leptin resistance by exercise was also demonstrated by zhou [37], who showed a reduction in hypothalamic socs3 mrna expression and jak2/stat3 signalling pathway in rats fed a high fat diet by exercise. however, when exercise was combined with caloric restriction the effect was more evident compared to those obtained by exercise or diet approach alone [38]. reversal of the inhibition of socs3 and ptp1b: as already depicted, socs3 and ptp1b are negative regulator proteins of leptin receptor signalling. therefore their downregulation can be considered a useful approach to revert leptin resistance [39]. the inhibition of socs3 expression and/or activity could possibly lead to an interruption of the negative feedback loop related to leptin resistance and restore leptin activity. accordingly, obrb mutation in transgenic mice, disabling socs3 binding [40]. reduced food intake, increases leptin sensitivity and reduced weight gain. besides socs3, also ptp1b inhibition seems to be an attractive target to overcome lentin resistance [41]. in fact deletion of ptp1b in mice increases lentin sensitivity, reduces body weight and increases energy expenditure. these animals also showed an improvement in glucose metabolism and uptake, and they were protected by dio. the selective inhibition of ptp1b resulted in dampening of stat3 activation by leptin in hek cells. to date, inhibitors of ptp1b based on capability to bind ptpb1 active site (without hydrolysis) have been designed. in particular, thiazolidinedione compounds have been shown to exert anti obesity effects as ptp1b inhibitors and ppar-a activators, ameliorating blood lipid profile in mice on high fat diet [42]. pomc neuron activation:last molecular targets of leptin effect on energy balance are pom neurons, therefore their activation seems another attractive strategy to overcome leptin receptor signal to induce a-msh-mediated suppression of food intake and weight gain together with an increase in energy expenditure [43]. increase in leptin receptor expression and cell surface localization:an increase in obr expression and its localization at the cell surface are key determinants for cell sensitivity to leptin [44]. we demonstrated that in leptin resistant ovariectomized obese rats, estradiol replacement therapy or long term raloxifene treatment, reduced leptin levels and body weight and restored leptin receptor expression both in adipose tissue and hypothalamus [45]. unfortunately, singha et al. leptin: its crucial role in the fight against obesity online first indian j pharm drug studies | 5 differently from rodent studies, a recent review of the literature regarding estrogen effect on leptin levels in postmenopausal women, did not show evident beneficial effects by hormonal intervention in modulating leptin levels and attenuating weight gain. therefore, the authors discourage the hormonal intervention in relation to cardiovascular and neoplastic risk associated with the replacement therapy [46]. previously, clinical studies showed an increase in leptin levels and body fat content in other studies reported an increase in leptin levels in treated woman, not related to change in fat mass. it has also been shown that metformin, acting at obrb hypothalamic gene level, is able to increase receptor expression and leptin sensitivity, and exert an anorectic effect. moreover, the inverse agonist of cannabinoid receptor 1, jd5037,can overcome leptin resistance and reduce weight gain [47]. treatment of obesity based on leptin: leptin analogous: metreleptin is a once daily subcutaneously administered leptin analogue approved by the fda in 2014 for use in people with leptin deficiency or congenital/acquired lipodystrophy, with good clinical results in these conditions. whilst metreleptin monotherapy supports weight loss in obese individuals, it is not clinically meaningful with a mean 1.5 kg additional weight loss over 24 weeks noted in a previous trial [48]. to enhance the effect of leptin analogues, amylin mimetics such as pramlintide have been used in combination with metreleptin. one study found that use of the pramlintide/metreleptin combination resulted in 11.5 kg weight loss over 20 weeks compared with 7.4 kg and 7.9 kg weight loss respectively in participants receiving either metreleptin or pramlintide monotherapy. unfortunately, the development of the combination therapy was discontinued in 2011 following commercial reassessment [49]. phentermine and topiramate combination therapy: phentermine plus topiramate food intake may be due to an increase in hypothalamic crh, which is an anorexigenic neuropeptide [50]. furthermore, tpm seems to reduce energy deposition even in the absence of changes in food intake, suggesting a role for tpm in increasing energy expenditure. nonetheless, (tpm) in combination markedly decreased body weight in overweight and obese patients and the us. food and drug administration just recommended this drug to be approved to treat obesity tpm treatment has been shown to reduce adiposity in humans and rodents. this reduction in adiposity is related to decreased food intake and reduced body fat gain. in rodents, the tpm treatment induced reduction in the molecular mechanisms by which tpminduced weight loss occurs are contradictory and remain to be clarified [51]. conclusion obesity is a global health concern till date, it having such strong epidemiological evidences which link it to various other health issues. genetic factors like mutation in leptin signaling plays an important role in obesity. this influences both somatic and hereditary genomic events. activation of specific pathways due to metabolic shift which is associated with obesity and tumor progression is contributed by these genetic factors along with the gut microbiome. comprehensive approaches are required to examine leptin’s role in obesity can be done by metagenomics, metatranscriptomics, metaproteomics and metabolomic studies. further research should be targeted on the biology of the leptin focusing more on the acquired genetic variations and understand more about there interaction with the gut microbiome. longitudinal studies focusing on unveiling these factors from early life to adulthood may contribute to future research in building critical preventive strategies, potentially leading to innovative treatments and managing obesity in a better way. reference 1. wauters m, considine rv, van gaal lf. human leptin: from an adipocyte hormone to an endocrine mediator. 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s, bishayee r, sarkar p, ghosh p, dutta u, mishra p. decoding leptin: unraveling the role of leptin signaling in the battle against obesity. indian j pharm drug studies. 2024; online first. funding: none; conflicts of interest: none stated https://doi.org/10.1210/mend.16.4.0800 https://doi.org/10.1152/ajpregu.1998.274.1.r204 https://doi.org/10.1210/jcem.86.2.7245 https://doi.org/10.3389/fendo.2021.585887 tamboli et al. natural immunomodulators vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 163 review article natural immunomodulators: promising therapy for disease management muskan altaf tamboli, rutuja ramdas bhosale, naziya ashpak tamboli from, professor & amp, department of pharmacology, sahydri college of pharmacy, methwade, maharashtra, india abstract the immune system comprises a complex group of processes that provide defense against diverse pathogens. these defenses can be divided into innate and adaptive immunity, in which specific immune components converge to limit infections. we reviewed currently available experimental and clinical evidence to prove the efficiency, safety, and feasibility of immunomodulation in-vitro and in-vivo. we also reviewed the advantages and limitations of the described techniques. despite its limitations, immunomodulation is considered as the therapy itself or as an adjunct with promising results and developing potential. many plants and some phytoconstituents responsible for immunomodulation have been explained. the study also discusses biological screening methods for various plant drugs that focus on revealing the mechanism involved in immunomodulation. nutraceuticals are essential food constituents that provide nutritional benefits as well as medicinal effects. the benefits of these foods are due to the presence of active compounds such as carotenoids, collagen hydrolysate, and dietary fibers. key words: immune system, immunomodulation, nutraceutical plants, anti-inflammatory utraceuticals which have also been called medical foods, designer foods, phytochemicals, functional foods and nutritional supplements, include such everyday products as “bio” yoghurts and fortified breakfast cereals, as well as vitamins, herbal remedies, and even genetically modified foods and supplements. nutraceuticals may be used to improve health, delay the aging process, prevent chronic diseases, increase life expectancy, or support the structure or function of the body [1]. immunomodulation is an alteration of the immune system and interfering with its functions; if it results in an enhancement of immune reaction, it is named an immune stimulation which primarily implies stimulation of non-specific system, that is, granulocytes, macrophages, complement, certain tlymphocytes and different effector substances. immunosuppression implies mainly to reduce resistance access this article online received – 08th nov 2023 initial review – 27th nov 2023 accepted – 14th dec 2023 quick response code against infections, and stress and may occur on account of environmental or chemotherapeutic factors. immunostimulation and immunosuppression both need to be tackled to regulate normal immunological functioning. hence, both immunostimulating and immunosuppressing agents have their standing, and the search for better agents exerting these activities is becoming a field of major interest all over the world [2]. many immunomodulators in clinical use are cytotoxic drugs with significant adverse effects. to overcome toxicity and existing available cytotoxic drugs there is a need for new immunomodulatory medications. traditional medicines are the oldest approach for treating and managing any illness without causing severe or minor adverse effects. further, the search for chemicals of plant origin as fresh lead for the creation of potent and safe immunomodulators is receiving a lot of attention [3]. _________________________________________________ correspondence to: muskan altaf tamboli, department of pharmacology, sahydri college of pharmacy, methwade, maharashtra, india. email: muskanatamboli17@gmail.com tel.: +91 7387963031 n mailto:muskanatamboli17@gmail.com tamboli et al. natural immunomodulators vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 164 immunity the resistance offered by the host to the harmful effect of pathogenic microbial infection is called immunity [4]. types of immunity: broadly speaking, immunity or body defense mechanism is divided into two types, each with humoral and cellular components [5]. natural or innate immunity is non-specific and is considered the first line of defense without antigenic specificity. it has two major components: a) humoral: comprised by complement. b) cellular: consists of neutrophils, macrophages, and natural killer (nk) cells. specific or adaptive immunity is specific and is characterized by antigenic specificity. it too has two main components: a) humoral: consisting of antibodies formed by b cells. b) cellular: mediated by t cells. the various components of both types of immunity are interdependent and interlinked for their function. the mechanisms of innate and adaptive immunity are demonstrated in (figure 1) [6]. figure 1 mechanism of innate immunity and adaptive immunity structure of immune system [5] organs of immune system: although functioning as a system, the organs of the immune system are distributed at different places in the body. these are as under: a) primary lymphoid organs: i) thymus ii) bone marrow b) secondary lymphoid organs: i) lymph nodes ii) spleen iii) malt (mucosa-associated lymphoid tissue) located in the respiratory tract and git. cells of immune system [5] the cells comprising the immune system are as follows: lymphocytes: lymphocyte is the master of the human immune system. morphologically, lymphocytes appear as a homogeneous group but functionally two major lymphocyte populations, t and b lymphocytes are identified; while a third type, nk cells, comprises a small percentage of circulating lymphocytes having the distinct appearance of large granular lymphocytes [5]. monocytes and macrophage: the role of macrophages in inflammation consists of circulating monocytes, organspecific macrophages, and histiocytic. circulating monocytes are immature macrophages and constitute about 5% of peripheral leucocytes. they remain in circulation for about 3 days before they enter tissues to become macrophages. the macrophage subpopulations like the dendritic cells found in the lymphoid tissue and langerhans cells seen in the epidermis, are characterized by the presence of dendritic cytoplasmic processes and are active in the immune system [5]. mast cells and basophil: basophils are a type of circulating granulocytes (0-1%) while mast cells are their counterparts seen in tissues, especially in connective tissue around blood tamboli et al. natural immunomodulators vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 165 vessels and in submucosal locations. basophils and mast cells have ige surface receptors; thus, on coming in contact with antigen binding to ige (e.g. allergic reaction to parasites), these cells get activated and release granules i.e. degranulate. these granules contain substances such as histamine platelet-activating factor, heparin, and certain chemical mediators (e.g. prostaglandins, leukotrienes). mast cells and basophils are thus involved in mediating inflammation in allergic reactions and have a role in wound healing [5]. neutrophils: polymorphonuclear neutrophils (pmns) are normally the most numerous of the circulating leucocytes (40-75%). the cytoplasm of pmns contains lysosomal granules of three types: primary (azurophilic), secondary, and tertiary. pmns have similar functions to those of macrophages and are therefore appropriately referred to as ‘macrophages’ owing to their role as the first line of defense against an invading foreign organism in the body. however, these cells have limitations in size and type of organisms to be engulfed e.g. while they are capable of acting against bacteria and small foreign particulate material but not against viruses and large particles [5]. eosinophils: eosinophils are also circulating granulocytes (1-6%). these cells play a role in allergic reactions and intestinal helminthiasis. the granules of eosinophils contain lysosomal enzymes, peroxidases, and chemical mediators of inflammation (e.g. prostaglandins, leukotrienes). on coming in contact with ige opsonized antigen (e.g. helminths), eosinophils degranulate and release the chemicals stored in granules and incite inflammation [5]. diseases of immunity the diseases of the immune system are broadly classified into the following four groups: immunodeficiency disorder: failure or deficiency of the immune system, which normally plays a protective role against infections, manifests in the occurrence of repeated infections in an individual having immunodeficiency disease. traditionally, immunodeficiency diseases are classified into two types: a. primary immunodeficiencies: are usually the result of genetic or developmental abnormality of the immune system. b. secondary immunodeficiencies: arise from acquired suppression of the immune system. since the first description of primary immunodeficiency by bruton in 1952, an increasing number of primary and secondary immunodeficiency syndromes have been added to the list, the latest addition being the acquired immunodeficiency syndrome (aids) in 1981 [5]. hypersensitivity reactions: hypersensitivity is defined as an exaggerated or inappropriate state of normal immune response with the onset of adverse effects on the body. the lesions of hypersensitivity are a form of antigen-antibody reaction. these lesions are termed hypersensitivity reactions or immunologic tissue injury, of which 4 types i, ii, iii, and iv. depending upon the rapidity, duration, and type of the immune response, these 4 types of hypersensitivity reactions are grouped into immediate and delayed types [5]. a. immediate type in which on the administration of antigen, the reaction occurs immediately (within seconds to minutes). immune response in this type is mediated largely by humoral antibodies (b cell-mediated). immediate type of hypersensitivity reactions includes type i, ii and iii. b. delayed type in which the reaction is slower in onset and develops within 24-48 hours and the effect is prolonged. it is mediated by the cellular response (t cell-mediated) and it includes type iv reaction [5]. mechanisms of hypersensitivity reaction: humans live in an environment teeming with substances capable of producing immunologic responses. contact with antigens leads not only to the induction of a protective immune response, but also to reaction that can be damaging to tissue exogenous antigen occur in the dust, pollens, foods, drugs microbiologic agent, chemicals and many blood product used in clinical practice the immune responses that may result from such exogenous antigen take a variety of from ranging from annoying but trivial discomforts, such as itching of skin, to potentially fatal diseases, such as bronchial asthma. the various reactions produced are called hypersensitivity reactions, a tissue injury in the reaction may be caused by humoral or cell-mediated immune mechanisms. injurious immune reactions may be evoked not only by exogenous environmental antigens, but also by endogenous environmental antigens, but also by endogenous tissue antigens. some of these immune reactions are triggered by homologous antigens that differ among individuals with different genetic backgrounds. transfusion reactions and graft rejection are examples of immunologic disorders evoked by homologous antigens. another category of disorders, those incited by self -, or autologous, antigens, tamboli et al. natural immunomodulators vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 166 constitutes the important group of autoimmune diseases (discussed later). these diseases arise because of the emergence of immune responses against self–antigens [6]. table 1 comparative features of 4 types of hypersensitivity reactions [5] feature type i [anaphylactic, atopic] type ii [cytotoxic] type iii [immune-complex, arthus reaction] type iv [delayed hypersensitivity] definition rapidly developing immune response in a previously sensitized person reaction of humoral antibodies that attack cell surface antigen and cause cell lysis result from deposition of antigen-antibody complexes on tissues cell-mediated slow and prolonged response peak action time 15-30 minutes 15-30 minutes within 6 hours after 24 hours mediated by ige antibodies igg or igm antibodies igg or igm antibodies cell-mediated examples i. systemic anaphylaxis. ii. local anaphylaxis [hay fever, bronchial asthma, food allergy]. i. cytotoxic antibodies to blood cells [autoimmune haemolytic anaemia, transfusion reactions]. ii. cytotoxic antibodies to tissue components [graves’ disease, myasthenia gravis]. i. immune complex glomerulonephritis. ii. goodpastures syndrome. iii. collagen diseases [sle, rheumataoid arthritis]. iv. pan. v. drug-induced vasculitis. i. reaction against microbacterial antigen [tuberculin reaction, tuberculosis]. ii. reaction against virus-infected cells. iii. reaction against tumour cells. autoimmune diseases: autoimmunity is a state in which the body’s immune system fails to distinguish between ‘self’ and ‘non-self’ and reacts by formation of auto-antibodies against one’s tissue antigens. in other words, there is a loss of tolerance to one’s tissues; autoimmunity is the opposite of immune tolerance [5]. depending upon the type of autoantibody formation, autoimmune diseases are broadly classified into two groups: a. organ-specific diseases: in these, the autoantibodies formed react specifically against an organ or target tissue component and cause its chronic inflammatory destruction. the tissues affected are endocrine glands (e.g. thyroid, pancreatic islets of langerhans, and adrenal cortex), alimentary tract, blood cells, and various other tissues and organs. b. organ non-specific (systemic) diseases: these are diseases in which several auto-antibodies are formed that react with antigens in many tissues and thus cause systemic lesions e.g. various systemic collagen diseases [5]. possible immune disorders: these are the disorders in which the immunologic mechanisms are suspected in their etiopathogenesis. a classic example of this group is amyloidosis. immunomodulators immunomodulators: are natural or synthetic components that regulate the immune system and induce innate and adaptive defense mechanisms. immunomodulators are drugs or components that suppress the immune system (immunosuppressants) or stimulate the immune system (immunostimulants) [4]. classification of immunomodulators immunostimulants: immunostimulants are substances that stimulate the immune response or enhance body resistance against various infections by increasing the basal level of immune responses. immunostimulants are used for the treatment of autoimmune diseases, chronic infections, viral infection, and cancer-like diseases. there are two types of immunostimulants a. specific immunostimulant: it provides antigenic specificity in immune response e.g. antigen, vaccines. b. non-specific immunostimulants: it act irrespective of antigenic specificity to augments immune response of other antigen or stimulate components of the immune system without antigenic specificity e.g. adjuvants. figure 2 examples of immunostimulants tamboli et al. natural immunomodulators vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 167 immunosuppression: immunosuppression is a reduction of the activation or efficacy of the immune system. it is a phenomenon wherein the ability of an organism to form antibodies in response to an antigenic stimulus is reduced or suppressed. this suppression may be the result of a disease that targets the immune system, such as hiv infection or a consequence of pharmaceutical agents used to fight cancer. in some cases, immunosuppression may be deliberately induced. induction may be required for therapeutic interventions of tissue or organ transplantation to reduce the risk of organ rejection. it is also used for treating graftversus-host disease after a bone marrow transplant or for treatment of auto-immune diseases such as systemic lupus erythematosus, rheumatoid arthritis, sjogrens syndrome or crohn’s disease [4]. two types of immunosuppression 1. non-specific immunosuppression: this immunosuppression invariably takes places, particularly in the natural instances related to immune deficiency disorders, or may even be induced by the gradual depletion of lymphoid tissue or by the administration of immunosuppressive drugs. it has been observed adequately that undue radiation exposure gives rise to significant depletion of lymphocytes. 2. specific immunosuppression: specific immunosuppression is usually induced either by antigen or antibody. azathiopurine and corticosteroid combination therapy is commonly used in tissue transplantation to inhibit cell-mediated immunity [cmi]. cyclosporine is commonly used in immunosuppressive therapy. the monoclonal antibody [okt3] is also used as an immunosuppressive agent after organ transplantation in humans [4]. nutraceuticals: nutraceuticals is defined as substances that can be considered food or its part which, in addition to their normal nutritional value provide health benefits including prevention of disease or promotion of health. the knowledge about the nutritive value of various food items and their basic chemical roles has increased [4]. classification of nutraceuticals 1. nutraceuticals from food source: nutraceuticals are obtained from plants, animals and microbial resources and are restricted to them only. linolenic acid is found in animal flesh and is also synthesized in plants. choline and aphosphatidyl choline are available in plants, animals, and also in microbes. 2. nutraceuticals indicating their action of mechanism: nutraceuticals indicate their pharmacological activities i.e. anti-oxidants, anti-inflammatory, anti-cancer, antibacterial, and bone protectives. 3. nutraceuticals according to their chemical nature: the chemical nature of nutraceuticals is phenolic compounds, protein-based isoprenoids, carbohydrate derivatives, fatty acids and structural lipids, microbial, minerals, etc. 4. nutraceuticals according to their higher contents in specific food items: the food substances used as nutraceuticals contain antioxidants, prebiotics, probiotics omega-3-fatty acids, certains phytochemicals and dietary fibers. expect probiotics, all these components are present in fruits, vegetables and different types of foods. [4] advantages of nutraceuticals a. they are substances like food and not considered drugs so have fewer side effects. b. they are mainly used as preventive are prophylactic to improve medical conditions. c. they have natural ingredients and dietary supplements to balance diet discrepancies. d. they enhance overall health and well-being. e. they are available without prescription means accessible and affordable [9]. disadvantages of nutraceuticals a. quality and regulation issues: as compared to pharmaceuticals, nutraceuticals are not regulated very strictly and directly. some of the health claims made for nutraceuticals may not be scientifically validated. there is a lack of proper regulation for nutraceuticals. b. bioavailability: nutraceuticals may have poor bioavailability and data related to pharmacokinetics and pharmacodynamics may not be available in some cases. c. placebo effect: the body has a mechanism to recover on its own. in such cases, consumers may not use nutraceuticals to treat ailments and have a placebo effect. d. safety and interactions: rarely, nutraceuticals may have some side effects. as there is a lack of appropriate regulations, there may be possible side effects, interaction with other drugs, or maybe in effectiveness in treating disorders [9]. tamboli et al. natural immunomodulators vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 168 table 2 a brief description of immunomodulators derived from nutraceuticals common name botanical name (family) part used chemical constituents other biological activity ref. no. tea camellia sinensis l. (theaceae) leaves epigallocatechin gallate, quercetin, gallic acid anti-oxidant 25 tulsi ocimum sanctum l.(labiateae) entire plant essential oil such as eugenol,cavacrol immunomodulator 8 (14.14) ginger zingiber officinale roscoe (zingiberaceae) dried rhizome camphene, citral, borneol, cineolc anti-oxidant 9 (4.394.42) garlic allium sativum (liliaceae) bulbs diallyl disulfide, diallyl trisulfide, zinc, vit-a anti-oxidant immunomodulator 9 (4.454.46) turmeric curcuma langa (zingiberaceae) rhizome curcumin hydrophobic anti-oxidants antiinflammatory 9 (7.6) amla emblica officinalis gaetan (euphorbiaceae) fruit tannins, punigluconin, pedunculagin anti-oxidant 9 (4.504.51) pepper piper nigrum (piperaceae) fruit volatile oil, guineensine, limonenecamphene, eugenol anti-oxidant 9 (5.21) tomato solanum lycopersicum (solanaceae) fruit lycopen, lipophilic anti-oxidant 9 (6.15) thyme thymus vulgaris l. (labiateae) leaves flavonoids, caffeic acid, labiatic acid anti-oxidant 8 (14.6614.67) spirulina spirulina plantensis (oscillatoriceae) algae proteinous nitrogen, lipid, protein, oleic immunostimulant 8 (18.918.10) echinacea echinacea purpurea (compositae) leaf, flower arabinogalactan immunostimulant 8 (8.5) alfalfa medicago sativa linn. (leguminosae) herb vit.k, vit.c, thiamin, riboflavin immunomodulator 9 (4.344.35) chicory cichorium intybus l. (asteraceae) root sucrose, cellulose, protein, volatile oil, chicoric acid anti-oxidant, immunomodulator 9 (4.364.39) fenugreek (methi) trigonella foenumgraceum l (fabaceae) herb ascorbic acid, b carotene, fibers, graicunins immunomodulator 9 (4.424.44) amra (mango) mangifera indica linn. (anacardiaceae) bark tannin, catechin, mangiferin antioxidant 8 (10.3) ashoka saraca indica linn. (leguminosae) dried stem bark tannin, haematoxylin, ketosterol, saponin immunostimulant 8 (10.1410.16) ginseng panax ginseng (araliaceae) root ginsenosides, ginsenine, βsitosterol anti-oxidant, immnomodulator 8 (9.509.52) tamboli et al. natural immunomodulators vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 169 ashvagandha withania somnifera dunal (solanceae) root withanolides, withaferin-a, amino acids immunomodulator 9 (4.564.58) ginkgo biloba ginkgo biloba l. (ginkgoaceae) leaves quercitrin, rutin, kaempferol, ginkgolide-a anti-oxidant 9 (5.135.14) shatavari aspargus racemosus wild (liliaceae) root shathavarin i-iv, quercetin, rutin anti-oxidant 8 (9.629.63) milk-thistle milk thistle silybum marrianum gaerth (compositae) seed silymarin, silybin, silycrystin anti-oxidant 8 (9.779.78) guduchi tinospora cordifolia , miers (menispermaceae) leave and stem tinosporine, tinosporic acid, berberine immunostimulant 8 (9.1019.103) rice bran oil oryza sativa (gramineae) seed .fatty acid, palmitic acid anti-oxidant 8 (11.3311.34) teel (sesame) sesamum indicum linn. (pedaliaceae) seed mucilage, hcl, fixed oil. anti-oxidant, antiinflammatory 7 (220) wormwood artemisia annua linn. (compositea) herb artemisinin immunosuppressive 10 beggar-tricks bidens pilosa l. (asteraceae) flower, leave polyacetylenes anti-inflammatory , immunosuppressive 11 japanese summer grape fruit citrus nastudaidai hayata (rutaceae) fruit auraptene, flavonoids antioxidant 12 fig marigold carpobrotus edulis l. (aizoaceae) flower, fruit alkaloids immunomodulator 13 cone flower echinacea angustifolia (asteraceae) flower polysaccharides immunomodulator 14 bringraja eclipta alba l. (compositae) leaves triterpenoids, glucoside antioxidant 15 sahijan moringa oleifera l. (moringaceae) leaves vit.a, carotenoids, saponins antioxidant 16 paarijaata anti nyctanthes arbortr tristis l. (oleaceae) leaf, seed iridoid glicoside inflammatory, antispasmodic 17 kutki picrorhiza scrophulariiflora benth (scrophulariaceae) root iridoid glycoside, amphicoside antioxidant 18 roseroot rhodiola imbricate gray (crassulaceae) rhizomes phenolics immunostimulating property 19 glasswort salicornia herbacea (chenopodiaceae) herb polysaccharides immunomodulator 20 white cedar thuja occidentalis l. (arborvitae) leaves polysaccharide immunomodulator 21 haussknechtia haussknechtia elymatica (apioideae) herb phenolics immunomodulator 22 tamboli et al. natural immunomodulators vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 170 cispanche cistanche desertisola (orobalachaceae) herb polysaccharide immunomodulator 23 brahmi centella asiatica linn. (umbelliferae) herb triterpenoids, saponins immunomodulator 24 dragon head dracocephalum kotschyi (lamiaceae) herb essential oil immunomodulator 25 bay leaves (tejpata) cinnomomum tamala (lauraceae) leaves eucalyptol, terpineol, eugenol immunosuppressant 27 cumin (jira) cuminum cyminum l. (apiaceae) seeds cymol, cuminol immunostimulant, immunosuppressant 28 tamarind ( imali) tamarindus indica l. (leguminosae) fruits compesterol, seven hydrocarbon, acetic acid, tartaric acid antioxidant, immunomodulator 29, 30 black cumin nigella sativa l. (ranunculaceae) seeds thymoquinone, dithymoquinone immunomodulator 31 bitter melon (karela) momordica charantia l. (cucurbitaceae) fruits , seeds triterpene, proteid, steroid antioxidant, immunostimunt 32 key lime (lemon) citrus aurantiifolia swingle (rutaceae) fruits, leaves limonene, linalool, citronellal antioxidant 33 red spiderling boerhaavia diffusa l. (nyctaginaceae) roots boerhavia acid , boeravinone, palmitic acid antioxidant 34 sea buckthorn hippophae rhamnoides l. (elaeagnaceae) leaves, fruits quercetin derivatives, hydrocinnamic acid antioxidant 35 physic nut jatropha curcas l. (euphorbiaceae) leaves phenolic acid, lignans, coumarins antioxidant, immunomodulator 36 sweet flag acorus calamus l. (araceae) rhizome asarone, monoterpene, acorenone antioxidant, immunosuppressive 37 chemistry of plant-derived immunomodulators 1. glycosides: these organic compounds from plant and animal sources, upon enzymatic or acid hydrolysis, yield one or more sugar moieties. chemically, they are the acetals or sugar ethers, formed by the interaction of the hydroxyl groups of the sugar and non-sugar moieties, with the loss of a water molecule. numerous glycosides have been shown to exert the desired immunomodulatory action [38]. 2. flavonoids: chemically, flavonoids have a fifteencarbon skeleton (c6-c3-c6) which consists of two phenyl rings connected by a three-carbon bridge. several types of flavonoids exert immunomodulatory activities, including apigenin (3), oligomeric proanthocyanidins (4), isoflavonoids, flavones, and anthocyanidins. such flavonoids are found in terminalia arjuna [38]. 3. coumarins: these glycosides are derivatives of benzoa-pyrone (5); the furanocoumarins (6) are formed by fusion of furan ring to a coumarin at either the 6 and 7 position or the 7 and 8 position. these glycosides also exert immunomodulatory activities. these glycosides also exert immunomodulatory activites [39]. 4. alkaloids: these organic compounds are of natural or synthetic origin, basic in nature, containing one or more nitrogen atoms, normally heterocyclic, of limited distribution and have specific physiological actions on the human or animal body [39]. figure 3 flavonoids showing immunomodulatory activity tamboli et al. natural immunomodulators vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 171 figure 4 – coumarin figure 5 plant alkaloids with immunomodulatory activity concept of rasayana the word rasayana, a combination of two words (rasa and ayana), refers to nutrition and its transportation throughout the body. rasayana therapy enhances the qualities of rasa, enriching it with nutrients so one can attain longevity, improved memory and intelligence, freedom from disorder, youthfulness, excellence of hair, complexion and voice, optimum development of physique and sense organs, mastery over phonetics and brilliance. as a dedicated stream of medication for immune promotion, anti-degenerative and rejuvenating health care, the rasayana therapy of ayurveda is known to prevent the effects of ageing and improve the quality of life for healthy as well as diseased individuals. rasayana is helpful to improve immunity and is normally advised during the degenerative phase of life, which starts from around 45 years in both male and female pharmacology of immunomodulatory activities from putative medicinal plants mechanism of action of the rasayanas/ immunomodulators it has been reported that the “rasayanas” are rejuvenators, and nutritional supplements and possess strong antioxidant activities. they also exert antagonistic action on oxidative stressors, giving rise to the formation of different free radicals. they are used mainly to combat the effects of aging, atherosclerosis, cancer, diabetes, rheumatoid arthritis, autoimmune disease and parkinson’s disease. the rasayana herbs seem to operate through immunostimulant, immunoadjuvant, and immunosuppressant activities or by affecting the effector arm of the immune response. [40] modulation of the immune responses through the stimulatory or suppressive activity of a phyto-extract may help maintain a disease-free state in normal or unhealthy people. agents that activate host defense mechanisms in the presence of an impaired immune response can provide supportive therapy to conventional chemotherapy. [41] a high degree of cell proliferation renders bone marrow a sensitive target, especially to various cytotoxic drugs. bone marrow is the organ most affected during any immunosuppression therapy with this class of drugs. loss of stem cells and the inability of the bone marrow to regenerate new blood cells results in thrombocytopenia and leucopenia. [42] many studies have reported the identification of immunomodulatory compounds with pharmacological activity and limited toxicity. in this context, ethnopharmacology represents the most important way possible to uncover interesting and therapeutically helpful molecules. the phytochemical analysis of rasayana plants has revealed a large number of compounds including tannic acid, flavonoids, tocopherol, curcumin, ascorbate, carotenoids, polyphenols, etc., which have been shown to have potent immunomodulatory properties. the herbal mixture preparations of indian traditional medicine may stimulate immunomodulation due to the content of plants with immunomodulatory properties that probably act synergistically. this hypothesis along with the lack of toxicity can be important to understand their use in the past as well as currently. [43] conclusion the immune system is a complex organ with highly specialized cells and even a circulatory separate from blood vessels. immunodeficiencies occur when one or more of the components of the immune system are inactive. immunomodulation is the ruling of immune responses by stimulating them to prevent transmittable diseases or by suppressing them in undesired circumstances. many proteins, amino acids, and natural compounds have shown a significant ability to regulate immune responses, including interferon-γ (ifn-γ), steroids, and dmg. several medicinal tamboli et al. natural immunomodulators vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 172 plants exhibit not only immunomodulatory activity but also a wide range of antioxidant, anti-inflammatory, and other medicinal activities. new immunomodulatory plants are important for the discovery of drugs with fewer side effects, less costly, more potent, and effective treatments developed for immune and related diseases. acknowledgements i would like to thank dr. tamboli n.a, dr. tamboli a.m sir, for providing the necessary facilities during my entire work. i would also like to thank principal dr. m.s patil sir, for continuous support and encouragement. references 1. harshad patel. a focus on hepatoprotective and immunomodulatory nutraceuticals.2007; 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167,231 40. chulet r, pradhan p. a review on rasayana. phcog rev 2010; 3(6): 229-34. 41. wagner h. in: hikino h, farnsworth nr, editors. economic and medicinal plant research, vol. 1. london: academic press; 1984; 113e53. 42. bafna ar, mishra sh. immunostimulatory effect of methanol extract of curculigo orchioides on immunosuppressed mice. j ethnopharmacol. 2006; 104:1e4. 43. blasdell ks, sharma hm, tomlinson jpf, et al. (1991) subjective survey, blood chemistry and complete blood profile of subjects taking maharishi amrit kalash (mak). fed american soc exp biol 5(5):a1317(2014). how to cite this article: muskan altaf tamboli, rutuja ramdas bhosale, naziya ashpak tamboli. natural immunomodulators: promising therapy for disease management. indian j pharm drug studies. 2023; 2(4):163-173. funding: none conflict of interest: none stated sanjay s s patents disclosed synthetic overview of rasagiline vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 132 review article an overview of prior patents for the sequential progress in the synthetic approaches of rasagiline, its salts, crystallographic forms and impurities sanjay sukumar saralaya from, assistant professor, department of chemistry, sri dharmasthala manjunatheshwara, institute of technology, [affiliated to visvesvaraya technological university, belagavi], ujire, belthangady taluk, dakshina kannada, karnataka, india. pin code 574 240. abstract this review work was intended to provide the essential details disclosed in prior patents on the synthesis of rasagiline, its salts (as crystalline or amorphous forms) and a few impurities. quite a high number of patents were published in various patent trademark offices around the world regarding the synthesis of rasagiline. among them, the patents which fall under the similar family are excluded to prevent the possible duplication of the information. the remaining distinct patents were carefully reviewed and the particulars are grouped in chronological order. moreover, this initiative can provide an essential backyard for the global researchers and the organizations to get the details on the methodological flourish of rasagiline. more importantly, the work forms a firm basis for to invent/innovate a few more new strategies to commercialize rasagiline in its pharmaceutically suitable and stable forms. additionally, researchers can avail the information about the organizations which are behind the continuous process improvement on various aspects of rasagiline. key words: rasagiline base, rasagiline mesylate, propargylation, n-alkylation, racemization. he popular drug, rasagiline is an irreversible inhibitor of monoamine oxidase. it is used widely as a monotherapy during the initial stages of parkinson's disease or as an adjunct therapy in more advanced scenarios [1, 2]. nh h s o o oh1 rasagiline mesylate (c13h17no3s) 1 has the iupac name: [(1r)-n-(prop-2-yn-1-yl)-2,3-dihydro-1h-inden-1-amine methanesulfonate] with a cas registry number: 16173579-1 and a molecular weight: 267.34 g/mol. it is commercially marketed under the brand/trade name azilect®. rasagiline is a renowned propargylamine access this article online received – 24th august 2023 initial review – 09th september 2023 accepted – 10th october 2023 quick response code derivative with a good clinical efficacy. interestingly, r(+)-enantiomer is therapeutically active and hence gained a wide clinical importance to treat parkinson's disease, memory disorders and dementia of the alzheimer type (dat), depression, and hyperactive syndrome in children [3]. the s-(-)-enantiomer of rasagiline has a bit of neuroprotective properties but the potency of r-(+)enantiomer over the monoamineoxidase enzyme (mao-b) is around 1000-fold higher. however, the racemic rasagiline hydrochloride was discovered in 1970s and was effectively used to treat hypertension [4]. after achieving the resolution of enantiomers, it was found that r-(+)enantiomer was an active mao-b inhibitor with a reasonably high degree of selectivity. meanwhile, s-(-)enantiomer had showed relatively very low mao-b inhibitory activity [5]. _______________________________________________ correspondence to: sanjay sukumar saralaya, assistant professor, department of chemistry, sri dharmasthala manjunatheshwara, institute of technology, [affiliated to visvesvaraya technological university, belagavi], ujire, belthangady taluk, dakshina kannada, karnataka, india. pincode 574 240. email: sanjay.saralaya@gmail.com. t mailto:sanjay.saralaya@gmail.com sanjay s s patents disclosed synthetic overview of rasagiline vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 133 literature review a chronological flow was maintained in this review initiative to tabulate all the essential process centric details disclosed in the prior patents regarding the synthesis, racemization, purification, impurity profiling, various stable salts of rasagiline and its crystallograhic forms. nh2 2 br 3 n4 cl 5 nh2 6 cl 7 oh13 s o o s o o o 14 s o oo 8 o nh9 11 cl12 nh2 10 huebner cf., in 1966, had reported the condensation of 1amino-indane 2 with propargyl bromide 3 in the presence of sodium carbonate and acetone to isolate 1-(n,ndipropargyl-amino)-indane hydrochloride 4 with a melting point of 160-163oc (yield: 11.82%, recrystallized from ethyl alcohol). furthermore, the condensation of 1-chloroindane 5 with propargylamine 6 was achieved in isopropyl alcohol to isolate the racemic rasagiline hydrochloride with a melting point of 178-179oc (yield: 22.27%, recrystallized from ethyl alcohol) [6]. maurice wg, et al., in 1968 & 1970, had demonstrated synthesis and applications of n-substituted 1aminoindanes. the condensation of 5 with 6 was executed in the presence of sodium iodide in ethyl alcohol to isolate the racemic rasagiline hydrochloride with a melting point of 185-187oc (recrystallized from isopropyl alcohol). it was believed that, n,n-di(1-indanyl)-propargyl amine hydrochloride (dimer) had formed as an intermediate [4,7]. youdim mbh, et al., in 1991, 1995 & 1996, had illustrated the synthesis and effective resolution of the racemic rasagiline base. rasagiline base was prepared by treating 2 with propargyl chloride 7 in the presence of potassium carbonate and acetonitrile. the isolated rasagiline base was treated with ethereal hydrochloride (hcl gas purged to diethyl ether) to isolate the rasagiline hydrochloride with a melting point of 182-184oc (yield: 46.82%, recrystallized from isopropyl alcohol).the racemic mixture of rasagiline base was resolved in a preparative hplc (high performance liquid chromatography) column and converted to s-(-)enantiomer hydrochloride with a melting point of 182184oc (isolated from diethyl ether). similarly, r-(+)enantiomer hydrochloride was isolated with a melting point of 179-181oc (isolated from diethyl ether). moreover, the work extends to report the reaction of r-(-)enantiomer of 2 with 7 in the presence of potassium carbonate and acetonitrile to isolate r-(+)-enantiomer of rasagiline hydrochloride with a melting point of 183185oc (yield: 35.18%, recrystallized from isopropyl alcohol). under the similar context, s-(+)-enantiomer of 2 was reacted with 7 to get s-(-)-enantiomer of rasagiline hydrochloride with a melting point of 183-185oc (recrystallized from isopropyl alcohol). furthermore, r(+)-rasagiline base was treated with l-tartaric acid to isolate r-(+)-di-rasagiline tartarate with a melting point of 175-177oc (yield: 46.59%, isolated from methyl alcohol). in an illustration, propargyl benzenesulfonate 8 was reacted with racemic 2 in the presence of aqueous sodium hydroxide in toluene to isolate r-(+)-di-rasagiline tartarate. it was treated with methanesulfonic acid in isopropyl alcohol to isolate 1 with a melting point of 157oc. [5,8-11]. gutman al, et al., in 2002, had reported the condensation of 2,3-dihydro-1h-1-indanone 9 with benzylamine 10 in the presence of acetic acid and benzene to form the important imine intermediate. it was reduced using sodium borohydride in ethyl alcohol and treated with sodium hydroxide solution to isolate the racemic n-benzyl-2,3dihydro-1h-inden-1-amine 11 with a boiling point of 125135oc (yield: 82%, purity: 95.9% by gas chromatographyhttps://patents.google.com/?inventor=huebner+charles+ferdinand sanjay s s patents disclosed synthetic overview of rasagiline vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 134 gc). in an alternate pathway, that involved the condensation of 1-chloro-2,3-dihydro-1h-indene 12 with 10 in acetonitrile to isolate racemic 11 with a boiling point of 125-135oc (yield: 64%) [12]. it was resolved under the influence of r,r-tartaric acid in water to isolate the crude salt. it was recrystallized from water to isolate the r,rtartarate salt of 11 with a melting point of 135-144oc (yield: 32%). s-isomer of 11 was recovered and resolved using potassium-t-butoxide in dimethylsulfoxide to isolate 11 (yield: 80%). r,r-tartarate salt of 11 was reduced using palladium-carbon in water to isolate r-isomer of 2 with a boiling point of 130-140oc (yield: 72%). this can also be treated with 3 or 7 to isolate rasagiline base as per the past disclosures [12]. lee tb, et al., in 2006, had demonstrated the condensation of s-1-indanol 13 with 6 in the presence of methanesulfonyl anhydride 14 and triethylamine in dichloromethane to isolate the r-rasagiline base (yield: 68%, as brown liquid) [13]. anton f, et al., in 2007 & 2009, had illustrated the reaction of 2 with 8 in the presence of aqueous sodium hydroxide in toluene to isolate rasagiline base through the extraction method (yield: 66.71%, as brown oil). it was treated with l-tartaric acid in isopropyl alcohol to isolate r-rasagiline tartarate with a melting point of 176.3176.8oc (yield: 28.8%, s-isomer: approx. 4%). with the use of similar key reagents, an additional two illustrations were reported. they are, direct and prolonged precipitation methods to isolate rasagiline tartarate with the melting points in the range of 160.8-163.2oc having the s-isomer content to about 4-16%. the work had even disclosed a few other precipitation methods such as, rasagiline sulfate isolation, recrystallization of rasagiline salts from solvents/water and salt inter-conversions etc [14, 15]. n oh 15 n16 nh nh17 18 nh o 19 luo jh., in 2007, had reported the reduction of 2,3dihydro-1h-1-indanone oxime 15 using alumino-nickel catalyst in the presence of sodium hydroxide solution and ethyl alcohol to isolate 2 (yield: 80.11%, as oily mass). in another experiment, 9 was treated with oxammonium hydrochloride in the presence of sodium hydroxide solution and ethyl alcohol. later to the reaction mixture added alumino-nickel catalyst and worked up to isolate the hydrochloride of 2 with a melting point of 208.4-209.5oc (yield: 76.68%, purity: 98.64% by hplc, as white crystals). it was treated with 8 in the presence of sodium hydroxide solution and toluene to isolate the racemic rasagiline base (yield: 79.07%, purity: 93.08% by hplc, as brown oily mass). in another instance, 15 was dissolved in ethyl alcohol and treated with sodium hydroxide solution. to the reaction mixture added alumino-nickel catalyst for the reduction and later added 8 to isolate the racemic rasagiline base (yield: 64.89%, purity: 90.45% by hplc, as brown oily mass). in another example, 9 was treated with oxammonium hydrochloride in the presence of sodium hydroxide solution and ethyl alcohol. to it, added alumino-nickel catalyst for the reduction and then added 8 to isolate racemic rasagiline base (yield: 62.3%, purity: 92.03% by hplc, as pale brown oily mass). in a few separate experiments, racemic rasagiline base was treated with l-tartarate in isopropyl alcohol to get the crude solid, which was recrystallized in isopropyl alcohol to isolate r-di-(rasagiline) tartarate with the melting points in the range of 174.2-177oc (yield range: 20-26%, purity range: 97.46-98.54% by hplc, as white puffy crystals). it was treated with methanesulfonic acid in isopropyl alcohol to isolate 1 with a melting point of 155155.8oc (purity: 99.85% by hplc, as white crystals) [16]. feng q., in 2007, had demonstrated a simple process to isolate the reacemic rasagiline base by reacting 9 in ethyl alcohol with 6 in the presence of sodium borohydride/palladium-carbon. around six illustrations were reported with a slight modulated reaction conditions and isolation procedures with good yield (60.19-73.10%) [17]. n21 nh22 nh2 20 sanjay s s patents disclosed synthetic overview of rasagiline vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 135 zongxuan s, et al., in 2008, had illustrated the condensation of 9 in isopropyl acetate with s-(-)-1phenylethylamine 20 in the presence of p-toluene-sulfonic acid monohydrate to form (1z)-n-phenyl-2,3-dihydro-1hinden-1-imine 21 (82.5%). it was reduced under the impact of raney-nickel in isopropyl acetate to isolate n-phenyl2,3-dihydro-1h-inden-1-amine 22 (yield: 88%). it was dissolved in tetrahydrofuran and treated with phosphorus pentachloride in the presence of triethylamine and then treated with oxammonium hydrochloride to isolate risomer of 2 (as free base or its hydrochloride salt, yield: 70-90%). hydrochloride salt of 2 in acetonitrile was treated with 3 in the presence of potassium carbonate followed by the addition of methanesulfonic acid in diethyl ether to obtain 1 with a melting point of 156-158oc [18]. bosch ilj, et al., in 2009, had reported a few reactions of r-isomer of 2 in toluene with 8 in the presence of sodium hydroxide solution to get r-rasagiline base (yield: 67.8272.49%). it was treated with methanesulfonic acid in toluene or acetonitrile to isolate the crude 1 (yield: 85.5497.39%, purity: 79.65-93.32% by hplc). furthermore, a double recrystallization of crude 1 was done from acetonitrile and then the solid obtained was suspended in acetonitrile/water mixture to get 1 (overall yield: 22-33%, purity: 99-100% by hplc). they had reported the recrystallization of 1 in isopropyl alcohol, but the final product and the filtrate had isopropyl mesylate traces as an impurity (by gc analysis) [19]. frenkel a, et al., in 2009, had demonstrated the isolation of crystalline solid r-rasagiline base (yield: 70-90%) from its mesylate or tartarate by a few methods like splitting, extraction, water crystallization, melt crystallization, quenching to water, reverse quenching, seeding crystallization etc. under the usual conditions, rrasagiline base would be used in its crude form as an oily liquid. the work had reported the isolation of the solid form of r-rasagiline base by various crystallization approaches with a melting point of 15-20oc (isolated from toluene), 38.2-38.4oc (isolated from water), 39.0-39.2oc (isolated from isopropyl alcohol), 40.8oc (isolated from isopropyl alcohol/water by seeded emulsion) and 41.3oc (isolated from isopropyl alcohol/water by seeding crystallization) [20-22]. gore v, et al., in 2009, had disclosed a process to get enantiomerically pure r-isomer of 2 by treating racemic 2 with 2,3,4,6-di-o-isopropylidene-2-keto-l-gulonic acid monohydrate in methyl alcohol to isolate the crude salt. it was dissolved in aqueous methyl alcohol and crystallized to isolate the salt. pure r-isomer of 2 (yield: 40-42%, chiral purity: 96-97% by hplc, as light green oily liquid) was isolated by salt breaking method in the presence of sodium carbonate solution and extracting the free base to dichloromethane [23]. the reported work was much superior to the past disclosed resolving techniques [12,24]. frenkel a & koltai t., in 2009, had illustrated a process to manufacture of rasagiline tannate. rasagiline base was treated with tannic acid solution to obtain the first mixture. a partial removal of the liquid from the first mixture and the addition of a polar water soluble solvent (ethyl alcohol) had resulted in the formation of second mixture. furthermore, liquid/solvent was removed completely at ambient temperature to isolate the tannate salt. the isolated rasagiline tannate had the water content of below 10% and the rasagiline content was got varied around in the range of 3-64%. the work had disclosed the cleavage of 1 to isolate rasagiline base as an oily mass and its crystallization to isolate in the solid form with a melting point of 39-39.3oc. it was then treated with tannic acid solution in various methods to isolate the tannate salt. an improved rasagiline tannate salt formation was observed in polar solvents than in non-polar solvents (ethyl acetate and hexane) [25]. caigu h & huimin h., in 2009, had reported the process to isolate the crystal form-i of 1. it was prepared by taking 1 in (ethyl acetate/ethyl alcohol) or (acetone/ethyl alcohol) or acetonitrile or isopropyl alcohol. the isolated solid had exhibited a prominent dsc endotherm at 157-157.5oc [26]. stephen bdw., in 2009, had reported the synthetic pathway to isolate the crystal form-i of 1. the work had employed various solvents to dissolve 1 and isolate the crystal form-i at 25-70oc [27]. patil ns, et al., in 2009, had demonstrated a process to prepare 1 with 90 volumepercent of the particles (d-90) with a size of about (6001500 microns) and (255-1500 microns). it was prepared from 1 or rasagiline base using the suitable solvents [28]. s o o cl n o o 23 nh so o n o o 24 n so o n o o 25 sanjay s s patents disclosed synthetic overview of rasagiline vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 136 qiandong l, et al., in 2009, had demonstrated the condensation of r-isomer of 2 in dichloromethane with onitro-benzene-sulfonyl chloride 23 in the presence of triethylamine to isolate r-n-(2-nitro) benzenesulfonyl-1indenamine 24 (yield: 95%, as white solid). it was dissolved in toluene and treated with 3 in the presence of sodium hydroxide and catalytic amount of tetrabutylammonium bromide to get r-n-propargyl-n-(2nitro)-benzenesulfonyl-1-indenamine 25 (yield: 81%, as pale yellow solid). n,n-dimethylformamide, lithium hydroxide monohydrate and mercapto-propionic acid were added to 25 to isolate r-rasagiline base (yield: 96%, as yellow oily liquid). the work had also reported the use of racemic 2 for the above steps to get racemic rasagiline base, which was treated with l-tartaric acid in isopropyl alcohol to isolate the salt. it was dissociated further to isolate the r-rasagiline base (as yellow brown oil) [29]. huang c & he h, in 2009, had disclosed the preparation of form-i of 1 from various solvents like isopropyl alcohol, ethyl alcohol, acetone, ethyl acetate and acetonitrile. the crystal form-i had showed the characteristic endothermic peak at about 157.07oc [30]. cherukupally p, et al., in 2010, had illustrated the reaction of 9 in methyl alcohol with 6 to isolate hydrochloride salt of (1z)-n-(prop-2-yn-1-yl)-2,3-dihydro-1h-inden-1-imine 16. it was then reduced using sodium borohydride in methyl alcohol to get the crude racemic rasagiline base. it was treated with isopropyl alcohol/hcl (18%) to get the racemic rasagiline hydrochloride. moreover, the salt cleavage had resulted in the formation of rasagiline base. the work also had reported the use of a few suitable reducing agents like raney nickel, palladium on carbon, and platinum dioxide; lithium aluminium hydride; sodium borohydride; sodium cyanoborohydride; sodium borohydride in acidic conditions; and sodium bis(2methoxyethoxy)-aluminum hydride (vitride®) to isolate 16 in reasonably high yields. a few suitable chiral resolving agents were used like l-(+)-tartaric acid and (-)-di-ptoluoyltartaric acid (dptta) to isolate the required risomer. with the use of these reagents, enantiomerically pure salts of rasagiline (hydrochloride/tartarate/mesylate) were prepared with good yields. furthermore, the work provides a process for the preparation of 1 having d90 below 6 μm. it was done by adding the solution of 1 in isopropyl alcohol to chilled methyl-t-butyl ether. the work involved the isolation of rasagiline & its salts which are significantly free from the critical impurities like n,ndi(prop-2-yn-1-yl)-2,3-dihydro-1h-inden-1-amine 4, n(prop-2-en-1-yl)-2,3-dihydro-1h-inden-1-amine 17, npropyl-2,3-dihydro-1h-inden-1-amine 18 & 3-(prop-2-yn1-ylamino)-2,3-dihydro-1h-inden-1-one 19 [31]. o oh 26 s o nh2 30 nh s o 31 n s o 32 br 27 nh28 oh oh o o nh br br 29 br nh nh br 33 34 marras g, et al., in 2010, had disclosed a multi-step process starting from (±)-2, 3-dihydro-lh-indene-lcarboxylic acid 26 to isolate r-rasagiline base (as light green oily mass). the process had involved a series of reaction steps like curtius rearrangement, resolution, hydrolysis, de-protection etc to isolate the intended product [32]. allegrini p, et al., in 2010, had illustrated the condensation of 6 and 9 in the presence of sodium borohydride in tetrahydrofuran to isolate the racemic rasagiline base (yield: 91%). it was treated with l-(+)tartaric acid in ethyl alcohol to get r-rasagiline tartarate. it was cleaved under nitrogen atmosphere using sodium bicarbonate solution and ethyl acetate to isolate rsanjay s s patents disclosed synthetic overview of rasagiline vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 137 rasagiline base (as an oily liquid). it was taken in isopropyl alcohol and treated with methanesulfonic acid to isolate 1 (yield: 89% from its tartarate salt). the work was extended further to provide the procedure to isolate the crystalline form of r-rasagiline base with a melting of 4041oc [33]. phull ms, et al., in 2010, had reported a multi-step process to isolate 1. in an example, 9 was reacted with allyl bromide 27 in the presence of potassium carbonate in acetonitrile and then treated with oxalic acid to obtain r-()-n-allyl-1-aminoindan oxalate 28. it was de-oxalated and then brominated (by bromine) in dichloromethane to isolate r-(-)-n-(2,3-dibromo-propyl)-1-aminoindan 29. it was hydrolyzed using potassium hydroxide solution in ethyl alcohol to isolate r-rasagiline oxalate. under the similar conditions, racemic 29 was synthesized and hydrolyzed. it was then treated with l-tartaric acid to isolate the crude solid. it was then de-tartarated by treating with sodium hydroxide solution and mesylated by the addition of methanesulfonic acid in isopropyl alcohol to obtain 1 (purity: 99.8%, chiral purity: 99.5%). the resolving process can be adapted to racemic 28 using ltartaric acid or it can be effectively implemented to racemic 29 using l-tartaric acid. the work had provided an improved process by eliminating the drawbacks of past disclosures [34]. stahl hp., in 2010, had demonstrated the preparation, solubility profile, and hygroscopicity details of two new salts of rasagiline. rasagiline base was taken in isopropyl alcohol and ethanedisulfonic acid was added to isolate rasagiline edisilate with a melting point of 201oc (yield: 56.3%). under the similar conditions, rasagiline base was treated with oxalic acid in isopropyl alcohol to obtain rasagiline oxalate with a melting point of 204oc (yield: 93.1%) [35]. frenkel a, et al., in 2010, had reported the isolation of mono-rasagiline citrate, di-rasagiline citrate or trirasagiline citrate or a mixture of all them. these salts or salt mixtures were prepared by treating rasagiline base (solid) with citric acid in ethyl alcohol or a few other solvents/water [36]. stephen bdw, et al., in 2010, had illustrated a few more rasagiline salts such as tartrate with a melting point of 176.2-177.3oc, maleate with a melting point of 87.287.8oc, sulphate with a melting point of 159.4-161.1oc, hydrochloride with a melting point of 177.0-180.0oc, tosylate with a melting point of 129.3-129.9oc, fumarate with a melting point of 125.4-126.2oc, phosphate with a melting point of 109.5-110.4oc, acetate with a melting point of 69.2-69.7oc, besylate, tannate, benzoate, galactarate, gluconate, glucuronate, succinate, hetartarate etc from rasagiline base using various suitable solvents/water. some of those salts were isolated in their crystalline forms (form i/ii) and a few salts in their amorphous form [37]. thomas t, et al., in 2011, had reported the preparation and advantages of a few salts of rasagiline such as gluconate, l-aspartate, citrate, dl-lactate, saccharinate, docusate, lauryl sulphate, 4-dodecylbenzenesulfonate, linoleate, pentanoate, propanoate, acetate, decanoate, octanoate, hexanoate and oleate. these salts were prepared from rasagiline base using appropriate solvents [38]. patil ns, et al., in 2011, had demonstrated the isolation of a few rasagiline salts. the salts reported are maleate (form-ii), mandelate (form-i) and salicylate (form-i), surprisingly these salts had good purity and had exhibited an adequate stability, good flowability and a good dissolution properties [39]. dongwei c, et al., in 2011, had illustrated a multi-step process to isolate 1. the reaction of 9 with t-butylsulfinamide 30 was carried out under the catalytic impact of isopropyl titanate in tetrahydrofuran to get n-(2,3dihydro-1h-inden-1-yl)-2-methylpropane-2-sulfinamide 31 (yield: 59%). it was dissolved in dimethyl formamide and reacted with sodium-t-butoxide. to the reaction mass, 7 was added to isolate n-(2,3-dihydro-1h-inden-1-yl)-2methyl-n-(prop-2-yn-1-yl)propane-2-sulfinamide 32 (yield: 51% from 9). the solution of methanesulfonic acid in diethyl ether was added to the solution of 32 in methyl alcohol to isolate the enantiomerically pure 1 with a melting point of 156-158oc (yield: 46% from 9, as white crystals, isolated from methyl-t-butyl ether) [40]. chi-hsiang y & tsung-ting c., in 2011, had reported the condensation of 9 with 6 in methyl-t-butyl ether using ptoluene sulfonic acid to form the intermediate 16. it was effectively reduced using 20% di-isobutyl-aluminumhydride (dibal-h) in hexane to isolate the racemic rasagiline base (yield: 81%). it was treated with s-(+)mandelic acid in methyl-t-butyl ether to isolate rrasagiline mandelate (yield: 45%). de-salting it with 2% sodium hydroxide solution gave r-rasagiline base (yield: 90%). it was treated with methanesulfonic acid in isopropyl alcohol to isolate 1 (yield: 80%) [41]. gore v, et al., in 2011, had illustrated the condensation of r-isomer of 2 with 8 under the influence of 1,8diazabicyclo-[5,4,0]-undec-7-ene (dbu) in tetrahydrofuran to get r-rasagiline base (yield: 80-82%, sanjay s s patents disclosed synthetic overview of rasagiline vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 138 purity: 64.33% by hplc, as yellow oily mass). it was treated with methanesulfonic acid in isopropyl alcohol to isolate 1 (yield: 47%, purity: 99.84%, chiral purity: 100% by hplc) [42]. thanedar aa, et al., in 2011, had demonstrated a comparative example to condense racemic 2 in acetonitrile with 7 in the presence of potassium carbonate to isolate racemic rasagiline base (yield: 101%, purity: 72.33% by hplc, as oily residue). it was done as per the previous disclosure [10]. in another example, 9 in ethyl alcohol was reacted with 6 in the presence of titanium (iv) isopropoxide to form the respective titanium complex. it was reduced by sodium borohydride and performed acid-base isolation to get racemic rasagiline base (yield: 22.57%, purity: 98% by hplc). it was resolved using l-(+)-tartaric acid in isopropyl alcohol to isolate the crude salt. it was recrystallized from methyl alcohol to isolate r-rasagiline tartarate (recovery: 80%, purity: 99.89% by hplc). it was de-salted and treated with methanesulfonic acid in acetone to isolate 1 (yield: 72.12%, purity: 99.97% by hplc). a direct conversion process of tartarate salt to 1 (yield: 88.14%, purity: 99.5%, chiral purity: 99.9% by hplc) was also reported [43]. selic l., in 2011, had disclosed the preparation of a few salts of rasagiline and those are exclusively used to resolve racemic rasagiline base. the major salts reported are rasagiline-l-mandelate, rasagiline-d-mandelate, rasagiline-r-mandelate (yield: 33%, with a melting point of 107-111oc), r-rasagiline-(+)-camphor-10-sulfonate (yield: 27%, with a melting point of 167-170oc), rasagiline orotate, rasagiline cinnamate, rasagiline-1hydroxy-2-naftoate, rasagiline fumarate, rasagiline benzoate and rasagiline-(-)-camphor-10-sulfonate. the work also had disclosed the recrystallization methods of those salts along with an efficient analysis method [44]. zope ss, et al., in 2011, had demonstrated the synthesis of some rasagiline salts like, r-rasagiline phosphate (purity: 99.96%), r-rasagiline benzoate (yield: 78%, purity: 99.94%), r-rasagiline mandelate (yield: 61%, purity: 99.89%) and r-rasagiline oxalate (purity: 99.9%) from rrasagiline free base in isopropyl alcohol [45]. sun j, et al., in 2011, had reported a method to prepare rrasagiline base starting from racemic 2 as the raw material through enzyme-catalyzed asymmetric acylation reaction, hydrolysis and n-propargylation reactions. the r-isomer of 2 in isopropyl ether was reacted with 8 in the presence of sodium carbonate solution to isolate the desired propargylated product (yield: 82.5%, as yellow oily liquid) [46]. dwivedi sd, et al., in 2011, had illustrated the synthesis of many salts of rasagiline from its base using the suitable solvents. the salts reported are r-rasagiline hydrobromide form-i, amorphous rasagiline hydrobromide, r-rasagiline hydrogen phosphate, 1 from r-rasagiline hydrobromide form-i, form-i of 1, rrasagiline 1,2-edisylate form-ii, crystalline r-rasagiline2-napsylate, r-rasagiline-1,5-dinapsylate form-i, rrasagiline 1,5-dinapsylate form-ii, crystalline rrasagiline-l-napsylatate, r-rasagiline ascorbate, amorphous rasagiline ascorbate [47]. sathe dg, et al., in 2011, had demonstrated the reaction of r-(-)-isomer of 2 hydrochloride with 8 in the presence of sodium hydroxide solution and tetra-butyl-ammoniumbromide to get the crude r-rasagiline base (yield: 60%, as an oily mass). it was subjected to column chromatography to isolate r-rasagiline base (recovery: 90%). the unreacted r-(-)-isomer of 2 was recovered by the basification and extraction processes. r-rasagiline base was treated with methanesulfonic acid in isopropyl alcohol to isolate 1 (yield: 83%). the work was extended further to provide the preparation methods of rasagiline hydrochloride, form-i and form-ii, rasagiline hydrobromide, rasagiline palmitate. furthermore, the work reported the route to synthesize and isolate the impurities like 33 (impurity a) and 34 (impurity b) [48]. sanjay s s patents disclosed synthetic overview of rasagiline vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 139 elffrink wwj, in 2011, had disclosed the techniques to synthesize a few crystallographic forms of rasagiline hydrochloride like form-i (as per the past disclosures), form-ii and form-iii (by the use of an inert solvent for a sufficient time to attain the conversion) [49]. bohumil d., in 2012, had illustrated the resolution of racemic 2 in methanol using l-(+)-aspartic acid, l-(-)malic acid and (2r, 3r)-tartaric acid. the work had reported the conversion of s-isomer of 2 to its r-isomer (yield: 94%, purity: 96% by hplc) in the presence of potassium-tert-butoxide in dimethyl sulfoxide. it also extends to report the condensation of r-isomer of 2 hydrochloride with 3 in the presence of sodium hydroxide solution under the toluene medium to form rasagiline base (not isolated). to the rasagiline base dissolved in toluene, added methanesulfonic acid in isopropyl alcohol to isolate 1 (purity: 99.86%, chiral purity: 100%, by hplc) [50]. liu g, et al, in 2012, had reported the condensation of risomer of 2 with methyl trifluoroacetate 35 in methyl alcohol to isolate n-[(1r)-2,3-dihydro-1h-inden-1-yl]2,2,2-trifluoroacetamide 38 (yield: 96%, purity: 99.5% by hplc). similarly, ethyl trifluoroacetate 36 in ethyl alcohol was used to isolate 38 (yield: 98%, purity: 99.7% by hplc). additionally, butyl-trifluoroacetate 37 in pentyl alcohol or tetrahydrofuran was used to get 38 (yield: 94%, purity: 99.2% by hplc). in the next step, 38 was treated with 7 in the presence of n,n-dimethylformamide and sodium hydroxide solution to obtain n-[(1r)-2,3-dihydro1h-inden-1-yl]-2,2,2-trifluoro-n-(prop-2-yn-1yl)acetamide 39 (yield: 86%, purity: 99.1% by hplc). a slight process modification was done to condense 3 to with 38 to obtain 39 (yield: 90%, purity: 99.7% by hplc). a few different reagents and solvents were used to condense 3 or 7 with 38 to isolate 39 with good optical purity. hydrolysis of 39 under the suitable alkaline conditions had resulted in the formation of r-rasagiline base (yield: 8696%, purity: 99.8-99.3%). it was treated with methanesulfonic acid in diethyl ether to get 1 (yield: 82%, purity: 99.3%) [51]. oemer r, et al, in 2012, had demonstrated a high yield method for the synthesis of 1 by the alkylation of 39. the trifluoroacetyl protection had enabled to carry out an alkylation of 38 with a high yield and purity under very mild conditions with a wide range of reaction conditions and reagent selection. r-isomer of 2 hydrochloride was treated with trifluoroacetic anhydride 40 in the presence of pyridine and dichloromethane to isolate 38 (yield: 95%, purity: 99.5% by hplc). it was alkylated with 3 in the presence of cesium carbonate in acetonitrile to isolate 39 (purity: 97.8% by hplc) and then hydrolyzed in the presence of potassium hydroxide solution and methyl alcohol to get r-rasagiline base (purity: 99.5% by hplc). it was taken in isopropyl alcohol and added methanesulfonic acid to get 1 (yield: 88%, purity: 100% by hplc, as white crystals) [52]. yao q & chen z, in 2012, had illustrated the condensation of 9 with 6 in the presence of p-toluenesulfonic acid in methyl-t-butyl ether to form 16. it was dissolved in-situ in toluene and added 20% di-isobutylaluminum hydride solution (dibal-h dissolved in n-hexane) and worked up to isolate racemic rasagiline base (yield: 81%). it was resolved by converting to its mandelate salt (yield: 45%) to isolate the required r-isomer. it was subjected to desaltation (yield: 90%) using sodium hydroxide solution and then treated with methanesulfonic acid in isopropyl alcohol to obtain 1 (yield: 80%) [53]. tang l, et al., in 2012, had reported the treatment of 15 in aqueous ethyl alcohol with aluminium-amalgum to isolate racemic 2 (purity: 98.9-99.9%). it was condensed with 8 in the presence of sodium hydroxide solution and dichloromethane to form racemic rasagiline base. it was resolved by the formation of tartarate salt in isopropyl alcohol to isolate r-rasagiline tartarate with a melting sanjay s s patents disclosed synthetic overview of rasagiline vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 140 point of 174.9-176.3oc (yield: 41.05%). it was dissolved in isopropyl alcohol and treated with methanesulfonic acid to obtain 1 with a melting point of 150.1-151oc (yield: 81%) [54]. nagarajan k, et al., in 2012, had demonstrated a process to isolate 1 with a particle size of about 255-590 microns. it was achieved by the crystallization from isopropyl alcohol and the disclosed method was devoid of routine comminution techniques to control the particle size of 1 [55]. dwivedi sd, et al., in 2011, had illustrated the process to isolate r-rasagiline besylate form-i with the larger particle size by treating rasagiline base with benzene sulfonic acid solution in ethyl acetate. similarly, a few other salts were also prepared like r-rasagiline hydrobromide form-i and its larger particle size, amorphous rasagiline hydrobromide, r-rasagiline hydrogen phosphate form-i and form-i of 1. the reaction of 9 in methyl alcohol with hydroxylamine hydrochloride was carried out in the presence of sodium hydroxide solution to obtain 15. it was reduced under the catalytic impact of raney-nickel (with 5 kg of hydrogen pressure) in the presence of ammoniacal methyl alcohol to get racemic 2. it was treated with 7 in the presence of potassium carbonate and sodium hydroxide using the solvent dimethyl formamide to isolate the racemic rasagiline base (as yellow oil). the isolated base was resolved using l-(+)-tartaric acid and desalted to isolate r-rasagiline base (as oil) and then converted to required salt forms [56]. zhang r, et al., in 2012, had disclosed the condensation of r-isomer of 2 in acetonitrile with propargyl methanesulfonate 44 to isolate 1 with a melting point of 156-158oc (yield: 97.8%). the isolation of 1 in different scales as above was reported via one-pot processes (yield: 85-92%) along with a few comparative examples as per the past disclosures [57]. bahar e, et al., 2012, had demonstrated the synthesis and applications of deuterated rasagiline, its salts. the work was primarily focused on the varied metabolic profile of deuterated forms of rasagiline than the protonated forms. to substantiate it, phase-i bio-trasformations of deuterated rasagiline was conducted with encouraging results [58]. ulanenko k, et al., in 2013, had reported the synthesis of 2-(2-((2, 3-dihydro-li/-inden-l-yl)(prop-2-ynyl)amino)-2oxoethyl)-2-hydroxysucckiic acid 42 from citric acid 41. in the first step, 41 was esterified to form trimethyl citrate. it was then converted to 1, 2-dimethyl citrate by a selective stearically controlled saponification. in the next step, an amidation reaction was conducted between r-rasagiline base and 1, 2-dimethyl citramide and finally the esters were hydrolyzed to isolate 42 (yield: 3.5%) [59]. santosh vp, et al., in 2013, had reported the condensation of r-(-)-isomer of 2 hydrochloride in acetonitrile with 7 in the presence of potassium carbonate to isolate crude base. it was purified by the selective ph adjustment and selective extraction to suitable solvents to isolate rrasagiline base (purity: 99.82% by hplc, as oily mass). it was treated with methanesulfonic acid in isopropyl alcohol to obtain 1 (purity: 99.99% by hplc). an optional isolation of free base was reported by converting in-situ to 1 with good purity. this work avoids the tedious and timeconsuming column chromatographic purification method & the solvent recrystallization techniques to get pure free base, instead had incorporated a simple workup techniques to get the r-rasagiline base with high purity. [60]. gade sr, et al., in 2013, had demonstrated the treatment of rasagiline base with l-(+)-tartaric acid in methyl alcohol to isolate rasagiline hemi-tartarate (yield: 70-75%, purity: 99.91-99.98% by hplc) [61]. sanjay s s patents disclosed synthetic overview of rasagiline vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 141 sun y, et al., in 2014, had demonstrated a one-pot synthetic process to condense r-(-)-isomer of 2 in acetonitrile or n,n-dimethyl formamide with propargyl-ptoluenesulfonate 43 or 44 in the presence of sodium carbonate or potassium carbonate or triethylamine to form an intermediate. it was not isolated, but immediately treated with methanesulfonic acid to isolate the crude solid. it was recrystallized in isopropyl alcohol to isolate 1 with a melting point of 156-157oc (yield: approximately 40-50%, purity: more than 99.5% by hplc). the work was extended further to isolate a few impurities like prop2-yn-1-yl (1r)-2,3-dihydro-1h-inden-1-ylcarbamate 45 and prop-2-yn-1-yl-(1r)-2,3-dihydro-1h-inden-1-yl-(prop2-yn-1-yl)carbamate 46 from the filtrate by the column chromatography technique [62]. sun y, et al., in 2014, had illustrated the isolation and analysis methods of 45, 46 and r-mesylate of 4. the lcms data of isolated impurities are [m+h]+ 216.1 for 45, [m+h]+ 254.1 for 46 and [m+h]+ 210.1 for mesylate of 4 [63]. prudic d, et al., in 2015 & 2016, had reported a process for the preparation of optically pure r-isomer of 2 by a diastereomeric resolution of racemic 2 using n-acetyl-lglutamic acid 47 as an effective resolving agent. the formation of diastereomeric salts of r-isomer of 2 with 47 and their use in the synthesis of optically enriched rasagiline base was also reported. rasagiline base was treated with methanesulfonic acid in isopropyl alcohol to obtain 1 (yield: 76.6%, purity: 100% by hplc). the reaction of 9 with hydroxylamine hydrochloride was carried out in ethyl alcohol to get 15 (yield: 91.6%). it was reduced by raney-nickel in the presence of ammonia enriched methyl alcohol to isolate racemic 2 (yield: 100%) [64, 65]. frenkel a, et al., in 2015, had reported the synthesis of 19 from n-(2,3-dihydro-1h-inden-1-yl)acetamide 48. impurity 19 was formed during the production of 1 under certain specific conditions. the work was extended further to contribute a commercial process to get 1 with very low content of 19. racemic 2 was treated with 8 in the presence of sodium hydroxide solution to isolate racemic rasagiline base. it was treated with l-tartaric acid in isopropyl alcohol to isolate r-rasagiline tartarate, it was then de-salted and treated further with methane sulfonic acid to isolate 1 (with around 0.01-0.02% of 19 content) [66]. chen j, et al., in 2019, had demonstrated the reaction of r-(-)-isomer of 2 with propynoic acid 49 in dichloromethane under the presence of suitable acid amine coupling agents like dicyclohexylcarbazone/ 4dimethylaminopyridine to get n-[(1r)-2,3-dihydro-1hinden-1-yl]but-3-ynamide 50 (yield: 97.7%, purity: 96.76% by hplc). similarly, the use of 1hydroxybenzotriazole/ 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride in tetrahydrofuran gave 50 (yield: 95.2%, purity: 96.59% by hplc). the use of (azabenzotriazol-1-yl)-n,n,n',n'-tetramethyluroniumhexafluorophosphate/ di-isopropylethylamine in 2methyltetrahydrofuran had resulted in the formation of 50 (yield: 96.8%, purity: 96.84% by hplc). it was dissolved in toluene/xylene/benzene and treated with diphenylsilane/triphenylsilane/diphenylmethylsilane and di-o-chlorophenylboronic acid/ bis-fluorophenylboronic acid to isolate rasagiline base (yield: 59.4-60.7%, purity: 99.72-99.77% by hplc). it was diluted in isopropyl alcohol and treated with methanesulfonic acid to obtain 1 (yield: 58.5%, purity: 99.89% by hplc) [67]. li j, et al., in 2020, had reported a one-pot synthetic procedure to isolate rasagiline base by the condensation of sanjay s s patents disclosed synthetic overview of rasagiline vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 142 9 with 6 in the presence of a suitable dehydrating agent/s (sodium borohydride, glacial acetic acid and tetraisopropyl titanate) in tetrahydrofuran to isolate racemic rasagiline base (yield: 93.6%, purity: 95.67% by hplc, as red-brown oily liquid). it was resolved by the use of l(+)-tartaric acid to get r-rasagiline tartarate (yield: 39%, purity: 99.46% by hplc, as off-while solid). it was desalted and treated with methanesulfonic acid in isopropyl alcohol to get 1 (yield: 87.6%, purity: 99.80% by hplc) [68]. ma y, et al., in 2020, had demonstrated the condensation of r-(-)-isomer of 2 with 3 in the presence of n,ndiisopropylethylamine in acetonitrile to isolate rrasagiline base (yield: 45.3-58%, purity: 91.4-97.54% by hplc). it was dissolved in isopropyl alcohol and added methanesulfonic acid to isolate 1 (yield: 83.1%, purity: 99.3% by hplc). the work had even covered the process to recover the un-reacted 2 from the filtrate (recovery: 32%, purity: 81% by hplc) [69]. ma y, et al., in 2021, had illustrated the synthesis of a few critical genotoxic impurities like (1r)-n-(2-chloroprop-2en-1-yl)-2,3-dihydro-1h-inden-1-amine 51 (purity: 84.6% by hplc, as light yellow oil), (1r)-n-[(2e)-3-chloroprop2-en-1-yl]-2,3-dihydro-1h-inden-1-amine 52 (yield: 89.3% by hplc, as yellow oil), (1r)-n-[(2z)-3chloroprop-2-en-1-yl]-2,3-dihydro-1h-inden-1-amine 53 (purity: 88.4% by hplc, as yellow oil), (1r)-n-nitroso-n(prop-2-yn-1-yl)-2,3-dihydro-1h-inden-1-amine 54 (purity: 88-8.9% by hplc, red-brown oil) and n-[(1r)2,3-dihydro-1h-inden-1-yl]-n-nitroso-2,3-dihydro-1hinden-1-amine 55 (purity: 75-79.8% by hplc, as brownblack oil). these are the process related impurities of 1, and are synthesized by the suitable reagents under the favorable reaction conditions. the work had even extended to provide the characterization details of impurities and their detection methods in the finished product [70,71]. hu a, et al., in 2022, had demonstrated an electroreduction synthetic pathway (a green chemistry approach) to isolate racemic 2 hydrochloride. the reduction of 15 under electrolytic conditions (alkaline) was performed in the presence of an organic solvent to obtain racemic 2 hydrochloride with a melting point of 210-212oc (yield: 90.6%, as a white solid) [72]. tang h, et al., in 2023, had reported the cyclization of 3phenylpropanoic acid 56 in the presence of trifluoromethanesulfonic acid and trifluoromethanesulfonic anhydride to isolate 15 (yield: 86.84%, purity: 99.37% by hplc). it was dissolved in tetrahydrofuran and treated with sodium triacetoxyborohydride and added the solution of 6 in tetrahydrofuran to obtain racemic rasagiline hydrochloride (yield: 79%, purity: 99.67% by hplc). it was de-salted and resolved using l-tartaric acid in methyl alcohol to isolate r-rasagiline tartarate (yield: 34.38%, purity: 98.83%). it was treated with methanesulfonic acid in isopropyl alcohol to isolate 1 (yield: 94.97%, purity: 100%, chiral purity: 100% by hplc). the disclosed initiative was proved to be an industrially feasible process involving cyclization, reductive amination, resolution and salification using the readily available starting materials, simple reagents and solvents [73]. 57 s o o nh n o o s o o n no o 58 wu g, et al., in 2023, had illustrated the use of 9 as the starting material to obtain s-isomer of 13 (yield: 75.6-82%, purity: 98.3-98.6% by hplc) in high purity through the asymmetric reduction of a chiral auxiliary and borane intermediate. it was dissolved in tetrahydrofuran and treated with 2-nitro-n-(prop-2-yn-1yl)benzenesulfonamide 57, triphenylphosphine and diisopropyl azodicarboxylate under the nitrogen atmosphere to isolate n-[(1r)-2,3-dihydro-1h-inden-1-yl]-2-nitro-n(prop-2-yn-1-yl)benzenesulfonamide 58 (yield: 81.5-85%, purity: 98-98.8% by hplc). in this step, a chiral amine was constructed in a simple pathway and the amino group was protected by the nitro sulfonyl group. the reported work successfully avoids the formation of unwanted polysubstituted byproducts. intermediate 58 was dissolved in dimethylformamide and treated with lithium hydroxide solution. to the mixture, mercaptopropionic acid in dimethyl formamide was added drop-wise and worked-up by ph adjustment to obtain r-rasagiline base (yield: 92%, 97.5% by hplc). upon the use of lithium hydroxide solution in dimethyl acetamide and mercapto-acetic acid, 1 was isolated with relatively good purity (yield: 84.4%, purity: 97.1% by hplc). in another experiment, lithiumbistrimethylsilylamide in n-methyl pyrrolidone and thioglycolic acid was used to isolate 1 in reasonably good purity (yield: 77%, purity: 95.8% by hplc) [74]. summary numerous researchers (inventors) associated to various global organizations (assignees) had contributed to commercialize rasagiline, its salts and crystallographic forms. sanjay s s patents disclosed synthetic overview of rasagiline vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 143 the information regarding the assignee of all the disclosures till date was revealed in table 1. to the context, “teva pharma” leads under the category of publication of patents on various aspects of rasagiline and its close related compounds. table 1. list of organizations/institutions behind the disclosed work (patent/s applied by/assignee) ref. no. patent no. applicant/s or assignee organization/s [4], [7]. us3513244a de1443403a1 aspro nicholas ltd [5], [8], [9], [10]. us5453446a, ep0436492a2, wo9511016a1, us5532415a teva pharma [il]; technion res & dev foundation [il] [6]. us3253037a ciba geigy corp [11]. cn1031995c orvet bv [nl] [12]. wo02068376a1 isp finetech ltd [il] [13], [14], [15], [20], [25], [36], [66]. us 2006/0199974a1, us2007/112217a1, us7491847b2, ep2101569b1, us 7547806b2, us7855233b2 ep2939669a1 teva pharma [il] [16]. cn101062897a chongqing pharm res inst co [cn] [17]. cn1990455a beijing d venture pharm tech [cn] [18]. cn101260048a suzhou chireach biomedical tec [cn] [19], [27], [37]. us2009292141a1, wo2009118657a2 us20100041920a1 medichem sa [es] [21], [22], [53], [58], [59]. wo2009154777a, wo2009154782a1, wo2012058219a2, wo2012058219a2 wo2013055684a1 teva pharma [il]; teva pharma [us] [23]. ca2723869a1 generics uk ltd [gb] [24]. ep0235590a2 warner lambert co [us] [26]. cn101486655a meide jiangxi biotechnology co [cn] [28], [39]. wo2009122301a2 wo2011080589a2 actavis group ptc ehf [is] [29]. cn101381314a chengdu healthcare pharmaceuticals [cn] [30]. cn101486655a meide jiangxi biotechnology co [cn] [31]. wo2010059913a2 dr. reddys lab ltd [in]; dr. reddys lab inc [us] [32]. wo2010049379a1 chemo iberica sa [es] [33]. us2010029987a1 dipharma francis s r i [it] [34]. ep2231582a1 cipla ltd [in] [35], [38]. us2010234636a1 wo2011003938a1 ratiopharm gmbh [de] [40]. cn102010353a winchem science and technology co ltd [41]. us2011218361a1 everlight usa inc [us] [42]. cn102203053a generics uk ltd [43], [45]. wo2011048612a2, wo2011095985a2 glenmark generics ltd [in] [44]. wo2011064216a1 lek pharmaceuticals [si] [46]. cn102154432a bengbu bbca medicine science dev co ltd sanjay s s patents disclosed synthetic overview of rasagiline vol 2 | issue 4 | oct – dec 2023 indian j pharm drug studies | 144 [47], [51]. wo2011121607a2, wo2012153349a2 cadila healthcare ltd [in] [48]. ep2364967a2 usv ltd [in] [49], [50], [56]. wo2011012140a2 wo2012116752a1 wo2012153349a2 synthon bv [nl] [51]. cn102464589a chiral quest suzhou co ltd [52]. wo2012096635a1 fargem farmasoetik arastirma gelistirme merkezi sanayi ve ticaret a s [tr] [53]. cn102476998a taiwan everlight chemical ind corp [57]. cn102675122a dongguan daxin biolog technology co ltd [55], [50]. us20120321896a1, wo2013054346a2 alkem lab ltd [in] [52]. cn102786422a topharman shanghai co ltd; shanghai inst materia medica; shandong topharman medical raw material co ltd [61]. ep2610239a1 dr. reddys lab ltd [in] [62], [63]. cn103804200a, cn103864646a changzhou no 4 pharmaceutical factory co ltd [64], [65]. wo2015070995a, wo2016116607a1 farma grs d o o [si] [67]. cn109180499a shanghai bocimed pharmaceutical co ltd [68]. cn110776429a qilu pharmaceutical co ltd [69], [70], [71]. cn111333517a, cn113030283a, cn113045456a shanghai aobo pharmtech inc ltd; zhejiang huahai pharm co ltd [72]. cn114438531a univ hunan [73]. cn115838333a jiangsu szyy pharmaceutical res institute co ltd [74]. cn115947675a boji medical technology co ltd conclusion this work was primarily aimed to cover the disclosures in prior patents for the synthetic approaches on rasagiline, its salts (in crystalline or amorphous forms) and a few impurities. numerous patents were published at various patent trademark offices over the years covering many aspects of the drug, rasagiline. to the context, the patents with attempts/explorings on the synthesis, impurity profiling, salt formation (in crystalline or amorphous form) of rasagiline were considered, as retrieved from the web search tools/databases like google patents, uspto (united states patent and trademark office) and espacenet (european patent office). this review work provides the essential information regarding the key starting material/s, reagents and solvents employed to obtain rasagiline and its salts. the present review initiative can assist global researchers to venture further on the synthetic aspects and reaction optimization studies to isolate rasagiline and its clinically acceptable salts. racemization, recovery of s-isomer and its effective reuse are the key process bottle-necks either at the initial phase or at the end. an optimized process with the use of commercially viable starting material/s and the use of green solvents/ reagents under mild reaction conditions would favor the large scale manufacturing of rasagiline and its stable salts. references 1. akao y, maruyama w, yi h, et al. an anti-parkinson’s disease drug, n-propargyl-1(r)-aminoindan 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mesylate. 2023. available from: https://worldwide. espacenet.com/patent/search/family/085574928/publication/c n115838333a?q=cn115838333 74. wu g, wang t, zuo l, et al. rasagiline intermediate and preparation method and application thereof. 2023. available from: https://worldwide.espacenet.com/patent/search/family/ 087288402/publication/cn115947675a?q=pn%3dcn11594 7675a how to cite this article: sanjay sukumar saralaya. an overview of prior patents for the sequential progress in the synthetic approaches of rasagiline, its salts, crystallographic forms and impurities. indian j pharm drug studies. 2023; 2(4):132-147. funding: none conflict of interest: none stated https://worldwide.espacenet.com/patent/search/family/046939731/publication/wo2012153349a2?q=pn%3dwo2012153349a2 https://worldwide.espacenet.com/patent/search/family/046939731/publication/wo2012153349a2?q=pn%3dwo2012153349a2 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https://worldwide.espacenet.com/patent/%20search/family/081362416/publication/cn114438531a?q=pn%3dcn114438531 https://worldwide.espacenet.com/patent/%20search/family/081362416/publication/cn114438531a?q=pn%3dcn114438531 https://worldwide.espacenet.com/patent/%20search/family/081362416/publication/cn114438531a?q=pn%3dcn114438531 https://worldwide.espacenet.com/patent/search/family/%20087288402/publication/cn115947675a?q=pn%3dcn115947675a https://worldwide.espacenet.com/patent/search/family/%20087288402/publication/cn115947675a?q=pn%3dcn115947675a https://worldwide.espacenet.com/patent/search/family/%20087288402/publication/cn115947675a?q=pn%3dcn115947675a umarkar et al. alum-based herbal handwash online first indian j pharm drug studies | 1 original article alum-based herbal handwash: formulation and evaluation study mohit j. umarkar1, pawan v. jibhkate1, payal g. thakare1, nilesh b. banarase2, koshish b. gabhane2, vikrant l. salode2 from, 1b. pharmacy student, 2professor, department of pharmacy, p.r. patil institute of pharmacy, talegaon (s.p.), wardha, india abstract hand washing is one of the most important strategies for preventing the spread of surface-borne diseases. a variety of hand washes are currently available on the market, the majority of which are alcohol-based and synthetic. since synthetic substances can cause various allergic reactions among people, as well as roughness of the skin, society's interest has shifted towards the use of herbal hand washes. however, in addition to the herbal extracts, we investigated alum as one of the most ancient and effective antimicrobial ingredients in hand wash formulations. in this study, f-1 to f-5 formulation batches of hand washes were prepared using a trial and error method by varying the quantity of ingredients used. in these formulations, hydro-alcoholic extracts of lemon peels and reetha, as well as lemon grass oil as a volatile oil and alum as a mineral, were used as the main constituents, which were analysed by phytochemical screening. these formulations were then evaluated using a variety of methods, such as ph, foam height, foam retention, skin irritation, stability study, and an antimicrobial assay. according to our findings, the alum-based f-3 formulation performed significantly better than the other formulations. with the success of this study, we can conclude that, in the future, as herbal drug technology advances, various hygienic and life-saving products will firmly hold their place in human life. key words: herbal extracts, alum, soap base, physical evaluation, antimicrobial and hygiene, or washing your hands, is a crucial, practical, easy, and reasonable way to stop the spread of disease [1]. in the past, the earliest and best sources of pharmacologically active compounds were plant species. for centuries, bioactive compounds and plant extracts have been employed in the preparation of traditional and ayurvedic medicines, foods, natural dyes, and cosmetics for the treatment of various illnesses. it has been discovered that herbal drugs, which contain a diverse range of bioactive compounds like flavonoids, terpenoids, volatile oils, tannins, and alkaloids, have the potential to exhibit effective antimicrobial properties in vitro against a broad range of microorganisms with relative safety [2-5]. nowadays, people are very much aware of the adverse effects of the synthetic materials used in various health products. as a result, the demand for herbal products worldwide is on the rise. although on the market, various hand wash formulations are available, most of them contain synthetic antimicrobials [6-7]. so, in this study, the formulation and evaluation of herbal hand wash is proposed using various herbal drug extracts, volatile oils, and minerals such as lemon peel, reetha, lemon access this article online received – 21st june 2024 initial review – 04th october 2024 accepted – 08th october 2024 quick response code grass oil, and potassium alum, as they are easily available, less expensive, and more efficient with fewer side effects. plants and mineral profile lemon peel (limonis cortex) is obtained from the fruit of citrus limon (l.) burm. belonging to the family rutaceae. it’s a small tree, 3–5 m high, cultivated in the countries bordering the mediterranean countries. dried lemon peel is official in the bp and ep. the major chemical constituents of dried lemon peels are 2.5% volatile oil, vitamin c, hesperidin, flavanone glycosides, and mucilage. in this work, the lemon peel extract was used as an antimicrobial agent and as a perfuming agent [8]. reetha consists of the fruits of sapindus mukorossi gaertn., also known as soapnut or washnut, a member of the sapindaceae family. the plant is a deciduous tree that grows in tropical and subtropical regions of asia, including india, china, japan, and pakistan. saponins, sugars, sesquiterpene oligoglycosides, and mucilage are among the most important chemical constituents of reetha. it has been used since antiquity as a foaming and cleansing agent. several studies have demonstrated its potential for antibacterial, anticancer, and hepatoprotective activity. in this work, the reetha extract was used as an antimicrobial, foaming, and cleansing agent [9]. ________________________________________________ correspondence to: nilesh b. banarase, department of pharmacy, p.r. patil institute of pharmacy, talegaon (s.p.), wardha, india email: nbanarase7@gmail.com h mailto:nbanarase7@gmail.com umarkar et al. alum-based herbal handwash online first indian j pharm drug studies | 2 lemongrass oil is distilled from the cymbopogon flexuosus plant, which belongs to the gramineae family. lemongrass oil is either reddish yellow or brown. it has an odor similar to lemon oil. lemongrass oil primarily contains citral and citronellal (75–85%). other terpenes found include geraniol, nerol, linalool, methyl heptenol, and limonene. it is primarily used as a flavoring and perfume agent in soaps and cosmetics. in this work, lemongrass oil was used as a perfuming and antimicrobial agent [10]. potassium alum, also known as alum, was first mentioned in ancient indian texts such as the charaka samhita and sushruta samhita. in ayurveda, alum in the form of bhasma, known as ‘sphatika bhasma’, is used to treat whooping cough. it is widely used in the purification of water, leather tanning, aftershave lotion, and deodorant. various studies have demonstrated that alum can be effectively used in various formulations to kill and control the growth of microorganisms. so, in this work, the alum was used as an antimicrobial agent [11–12]. materials and methods plant materials, chemicals, and microbes for this formulation, lemon and reetha fruits were purchased from the local market of talegaon, district-wardha (india). lemon grass was obtained from the farm of talegaon. all these plant materials were thoroughly cleaned with distilled water after collection and dried in the shed. in the case of lemons, after removing their juice, the peels were sundried and used. further, these plant materials were subjected to pulverization and extraction to obtain extract and volatile oil. potassium alum (aluminium potassium sulfate purified dodecahydrate), hydroxyl propyl methyl cellulose (hpmc), and sodium lauryl sulfate (sls) were purchased from cdh fine chemical, india. ethanol (purity ≥ 99%) was purchased from alsucrose corporation, india. a soap base was prepared in the laboratory. throughout the experiment, the doubledistilled water prepared in the laboratory was used. for the evaluation of antimicrobial activity, the soil sample was used as a source of microbes. preparation of extracts and isolation of volatile oils the plant materials, namely lemon peels and reetha, were pulverized to a coarse powder after drying. it was then extracted with solvent (ethanol: water 50:50) using a soxhlet apparatus. after the extraction, the solvent recovery was carried out using a distillation apparatus. further, the extracts were concentrated and dried in a water bath, and the yield was noted down. to obtain the lemon grass oil, the clevenger apparatus was used, and the yield was calculated. preparation of soap base take 6 g of cooking fat into an erlenmeyer flask. add the 12.5% naoh solution prepared in 40 ml of distilled water and ethanol (1:1) to the oil and mix thoroughly. heat the solution in a boiling water bath for 45 minutes. after 45 minutes, remove the mixture and place the flask in ice-cold water, followed by pouring into the 16.5% nacl solution for several minutes. filter the precipitated soap and wash twice with ice-cold water [13]. phytochemical screening the prepared extracts were further subjected to phytochemical screening for the presence of carbohydrates, proteins, alkaloids, saponin glycosides, steroids, flavonoids, terpenoids, tannins, and amino acids by various standard procedures [1416]. tests for carbohydrates molisch’s test (general test) to 2-3 ml of aqueous extract, add a few drops of alpha-napthol solution in alcohol, shake, and add conc. h2so4 from the sides of the test tube. a violet ring is formed at the junction of two liquids. fehling’s test (for reducing sugars): mix 1 ml of fehling’s a and 1 ml of fehling’s b solution; boil for one minute. add an equal volume of test solution. heat in a boiling water bath for 5-10 minutes. first yellow, then brick red precipitate is observed. barfoed’s test (for monosaccharides): mix equal volumes of barfoed’s reagent and test solution in the test tube. heat in boiling water bath for 5 minutes. the solution appears green, yellow, or red depending on the amount of reducing sugar present in the test solution. bial’s orcinol test (for pentose sugars): to boil bial’s reagent, add a few drops of test solution. green or purple coloration appears. selwinoff’s test (for hexose sugars): heat 3 ml of selwinoff’s reagent and 1 ml of test solution in a water bath for 1-2 minutes. a red color is formed. tollen’s phloroglucinol test (for hexose sugars): mix 2.5 ml conc. hcl and 4 ml 0.5% phloroglucinol. add 1-2 ml of test solution. heat the mixture. yellow to red color appears. tests for proteins biuret test (general test): to the 3 ml test solution, add 4% naoh and a few drops of 1% cuso4 solution. violet or pink color appears. million’s test: mix 3 ml of test solution with 5 ml of million’s reagent. white precipitate obtained. warm precipitate, turn brick red, or the precipitate dissolves, giving a red-colored solution. test for sulfur-containing proteins: mix 5 ml test solution with 2 ml of 40% naoh solution and 2-3 drops of 10% lead acetate solution. boil. solution turns black or brownish due to lead sulfide formation. umarkar et al. alum-based herbal handwash online first indian j pharm drug studies | 3 tests for alkaloids for the alkaloidal test, 2 ml of dilute hcl was added to 1 g of dry extracts, shaken well, filtered, and used for the following tests. mayer’s test: to 3 ml of the filtrates, add 1 ml of mayer’s reagent (potassium mercuric iodide). the creamy precipitate indicates the presence of alkaloids. wagner’s test: to 3 ml of the filtrates, add 1 ml of wagner’s reagent (iodine in potassium iodide). the reddish brown precipitate indicates the presence of alkaloids. hager’s test: to 3 ml of the filtrates, add 1 ml of hager’s reagent (saturated picric acid solution). the yellow precipitate indicates the presence of alkaloids. dragendroff’s test: to 3 ml of the filtrates, add 1 ml of dragendroff’s reagent (potassium bismuth iodide). the appearance of orange-brown precipitate indicates the presence of alkaloids. tests for saponin glycosides foam test shake a little quantity of extract with water. the persistent foam for 10 minutes confirms the presence of saponins. haemolysis test: mix a small amount of extract with blood on the slide. the hemolytic zone represents the saponin glycosides. tests for steroids salkowski’s test: to the 2 ml test solution, add 2 ml chloroform and 2 ml conc. h2so4. shake well. the chloroform layer appears red, and the acid layer shows greenish-yellow fluorescence. legal’s test (for cardenolides): to the 1 ml of test solution, add 1 ml of pyridine and 1 ml of sodium nitroprusside solution. pink to red color appears. tests for flavonoids shinoda tests dissolve the extract in 5 ml of 95% v/v ethanol and add a few drops of conc. hcl and 0.5 g of magnesium turnings. the pink, crimson, or magenta color represents flavonoids. tests for terpenoids salkowaski’s test: to the extract, add 2 ml of chloroform and 2 ml of conc. sulfuric acid from the side of the test tube. shake it for few minutes. red color forms. liebermann-burchard’s test: dissolve the extract in chloroform, add a few ml of acetic anhydride, and heat it. cool it and add a few drops of conc. sulphuric acid from the side of the test tube. the blue colour forms. tests for tannins ferric chloride test: with the 5% ferric chloride solution, the extract gives a dark green or deep blue color. lead acetate test: add a 10% w/v solution of basic lead acetate in distilled water to extract. precipitate is obtained. potassium dichromate test: with the extract, potassium dichromate solution produces a dark precipitate. gelatin test: add a 1% w/v solution of gelatin in water containing 10% sodium chloride. white precipitate indicates presence of tannins. tests for amino acids ninhydrin test: heat 3 ml of test solution and 3 drops of 5% ninhydrin solution in a boiling water bath for 10 minutes. purple or bluish color appears. handwash formulation procedure the formulation of alum-based herbal handwash was carried out by trial and error method. in brief, a total five formulation batches with varying amounts of ingredients were used (table 1.1), and the best formulation was finally selected based on the various evaluation parameters to produce the bulk quantity. table1.1: formulation of handwash batches by trial and error method s. no. ingredients used formulation batches /quantity used (mg) f-1 f-2 f-3 f-4 f-5 1. potassium alum 400 450 500 550 600 2. lemon peel extract 500 650 750 800 900 3. reetha extract 500 650 750 800 900 4. lemon grass oil 500 600 400 300 250 5. soap based 1500 2000 2700 3000 3500 6. hpmc 200 400 700 800 900 7. sls 50 200 350 500 600 8. distilled water q.s. to 100 ml umarkar et al. alum-based herbal handwash online first indian j pharm drug studies | 4 figure 1.1: alum-based handwash formulation (batch f-3) in this process, the desired quantity of the soap base in the form of powder was allowed to dissolve in the warm water, followed by hpmc. the prepared extracts were further dissolved in the distilled water and added to the gel prepared in the previous step, followed by sls, potassium alum, and lemon grass oil, and the quantity was adjusted with distilled water. the formulations were made homogenous at room temperature and stored for further studies (figure 1.1). evaluation of prepared handwash physical evaluation the prepared handwash formulations were first evaluated for various physical parameters like colour, odour, appearance, texture, homogeneity, and grittiness by visual inspection. ph the digital ph meter was used for the determination of the ph of the various prepared formulations. for this process, a 1% handwash solution was used, and readings were recorded at room temperature. foam height for the determination of foam height, 1 g of handwash formulation was diluted with 50 ml of distilled water in a 500 ml stoppered measuring cylinder, followed by water up to 100 ml. further, the stopper measuring cylinder was shaken vigorously for 25 strokes and kept aside for some time, and the height of the foam was measured. foam retention to determine foam retention, 1 g of handwash formulation was diluted with distilled water in a 500-ml stoppered measuring cylinder up to 100 ml. the preparation was then shaken vigorously ten times, and the quantity of foam produced was measured for four minutes at one-minute intervals. skin irritation test to determine whether any skin irritation or redness was caused by the prepared handwash formulation, 15 students were voluntarily selected. the formulations were applied to their skin for 30 minutes, then washed off. during this time, any skin irritation or redness that occurred was noted down. stability study the stability of handwash was tested for one month at 5 ± 3°c, 30 ± 2°c, and 40 ± 2°c, following ich guidelines. after one month, the tested formulation was evaluated for physical changes as well as changes in other parameters [17]. selection of microbes and antimicrobial assay for the evaluation of the antimicrobial potential of the prepared handwash, a soil sample was used as a source of microbes. in this process, a soil sample was prepared by the serial dilution method and used. for the antimicrobial assay, the agar well diffusion method was used. in brief, the sterilized nutrient agar medium plates were spread with the diluted soil sample prepared in sterile water using a sterile glass spreader. further, the holes were punched using a sterile cork borer, and a volume of 50 μl (0.1%) of prepared handwash formulation, marketed handwash formulation, and sterile water was filled in each bore. here, sterile water was used as a control and marketed handwash as a standard. the plates were incubated at 35-37 °c for 24 hours. after 24 hours, the diameter of the inhibition zone in mm was measured [18]. result after being extracted using ethanol and water, the extractive values of the crude drugs, lemon peel, and reetha were found to be 3.7% and 4.3%, respectively, upon complete drying on the water bath. while the yield of volatile oil from the lemon grass using the clevenger apparatus was found to be 3.4%. in order to analyse the classes of chemical constituents present in these extracts, various phytochemical tests were carried out. the details of the phytochemical screening are tabulated below (table 1.2). the phytochemical screening indicated the presence of all the phytoconstituents for which the phytochemical screening has been conducted except amino acids. umarkar et al. alum-based herbal handwash online first indian j pharm drug studies | 5 table 1.2: phytochemical screening of extracts phytoconstituents tests lemon peel extract reetha extract carbohydrates molisch’s + + fehling’s + + barfoed’s + + bial’s orcinol + + selwinoff’s + + tollen’s phloroglucinol + + proteins biuret + + million’s + + sulphur containing + + alkaloids mayer’s + + wagner’s + + hager’s + + dragendroff’s + + saponin glycosides foam + + haemolysis + + steroids salkowski’s + + legal’s + + flavonoids shinoda + + terpenoids salkowski’s + + liebermannburchard’s + + tannins ferric chloride + + lead acetate + + potassium dichromate + + gelatin + + amino acids ninhydrin + present ; absent the evaluation results of the different parameters for the prepared formulation batches are as follows: physical evaluation each of the five formulations had a light buff colour and a pleasing scent of lemon peel extract and lemon grass. the texture was glossy, non-gritty, and had an opaque appearance. the preparations were uniform and simple to clean. ph the ph of all the tested formulations was found to be in the range of 6.7–7.4, which is within the permissible limit (table 1.3). table 1.3: evaluation of prepared formulation batches formulation batches ph foam height (cm) foam retention (min.) f-1 6.9 24 8 f-2 6.9 19 3 f-3 7.1 32 12 f-4 6.8 19 5 f-5 7.4 22 5 foam height the foam height of all the tested formulations of herbal handwash was given in the table (table 1.3). from the observation table, it was found that the f-3 formulation has good foam height as compared to other formulations. foam retention according to the observation table (table 1.3), the f-3 formulation has a better retention time than the others, which was almost stable for 12 minutes. so, the f-3 formulation can be considered good on this parameter as well. skin irritation test no irritation or redness to the skin was reported on the evaluation of all formulations of handwash. stability on testing prepared handwash formulations for one month at various conditions, the f-3 formulation was found to be more stable as compared to others. other than the f-3 formulation, slight changes in the colour as well as the odour have been observed. table 1.4: antimicrobial assay s. no. diameter of inhibition zone in (mm) formulation batches 50μl (0.1%) standard formulation 50μl (0.1%) 1. f-1 11 42 2. f-2 06 3. f-3 24 4. f-4 15 5. f-5 16 antimicrobial assay the antimicrobial assay revealed that the f-3 handwash formulation is effective at inhibiting the growth of microorganisms, similar to the marketed product (table 1.4). as umarkar et al. alum-based herbal handwash online first indian j pharm drug studies | 6 a result, we can conclude that the f-3 formulation is suitable for bulk preparation and subsequent application. discussion the herbal handwash formulations in this study were made with reetha and lemon peel hydro-alcoholic extracts. lemon grass and alum were also utilized as primary ingredients. all of the extracts had a similar chemical composition, with the exception of the presence of amino acids, as discovered during the phytochemical screening process. these phytochemical data suggested that the strong antimicrobial activity observed in the antimicrobial assay may be attributed to the presence of a few key constituents, primarily tannins, saponin glycosides, and alkaloids. additionally, the quality parameters for the aforementioned herbal handwash were found to be satisfactory. conclusion with covid-19, people are becoming more aware of the various diseases that spread through skin surfaces, such as hands. hand washing is one of the simplest ways to protect ourselves from infectious diseases. hand wash liquids are commonly used in society to provide protection. various handwash formulations with a variety of ingredients are available on the market, the majority of which are synthetic or alcohol-based. despite the fact that these formulations have passed various quality tests, people prefer formulations containing natural ingredients such as herbs, minerals, and volatile oils due to their relative non-toxic effects and to control the continuous buildup of microbe resistance. so, in this study, while keeping this fact in mind, alum-based herbal extracts and a volatile oil-containing herbal handwash were prepared. the prepared handwash formulation batches were tested for a variety of parameters, including antimicrobial activity. among them, we discovered that formulation batch f-3 performed exceptionally well in all aspects of the hand washing requirement. the antimicrobial assay revealed that f3 at 0.1% concentration effectively inhibited the growth of soil bacteria. so, to summarize the study, the alum-based herbal handwash was successfully developed and tested. acknowledgement we are grateful to the college management for providing laboratory and library facilities during our course of work. references 1. mathur p. hand hygiene: back to the basics of infection control. indian j med res. 2011; 134(5):611-20. doi: 10.4103/09715916.90985 2. samy rp, pushparaj pn, gopalakrishnakone p. a compilation of bioactive compounds from ayurveda. bioinformation. 2008; 3(3):100-10. 10.6026/97320630003100 3. veeresham c. natural products derived from plants as a source of drugs. j adv pharm technol res. 2012; 3(4):200-1. doi:10.4103/2231-4040.104709 4. kaushik p, ahlawat p, singh k, et al. chemical constituents, pharmacological activities, and uses of common ayurvedic medicinal plants: a future source of new drugs. adv tradit med. 2023; 23:673–714. doi: 10.1007/s13596-021-00621-3 5. parham s, kharazi az, bakhsheshi-rad hr, et al. antioxidant, antimicrobial and antiviral properties of herbal materials. antioxidants (basel). 2020; 9(12):1309. doi: 10.3390/antiox9121309 6. hassen gw, ghobadi f, kalantari h. synthetic drugs: a new trend and the hidden danger. am j emerg med. 2013; 31(9):1413-5. doi:10.1016/j.ajem.2013.05.047 7. chaachouay n, zidane l. plant-derived natural products: a source for drug discovery and development. drugs and drug candidates. 2024; 3(1):184-207. doi: 10.3390/ddc3010011 8. rqfiq s, kaul r, sofi sa, et al. citrus peel as a source of functional ingredient: a review. j saudi soc agri sci. 2018; 17(4): 351-8. doi: 10.1016/j.jssas.2016.07.006 9. sochacki m, vogt o. triterpenoid saponins from washnut (sapindus mukorossi gaertn.)-a source of natural surfactants and other active components. plants (basel). 2022; 11(18):2355. doi: 10.3390/plants11182355 10. shah g, shri r, panchal v, et al. scientific basis for the therapeutic use of cymbopogon citratus, stapf (lemon grass). j adv pharm technol res. 2011; 2(1):3-8. doi: 10.4103/22314040.79796 11. dutta s, de sp, bhattacharya sk. in vitro antimicrobial activity of potash alum. indian j med res. 1996; 104:157-9 12. sahoo i, more ss, jadhav v, et al. clinical appraisal on therapeutic efficacy of tankana & sphatika bhasma with madhu pratisarana in tundikeri. journal of drug delivery and therapeutics. 2019; 9(6): 130-4. doi: 10.22270/jddt.v9i6.3707 13. the royal society of chemistry. examples of interdisciplinary chemistry-biology laboratory experiments: synthesis and properties of soap [internet]. chemistry education research and practice. 2017. available from: https://www.rsc.org/suppdata/c7/rp/c7rp00133a/c7rp00133 a2.pdf 14. kokate ck. practical pharmacognosy. vallabh prakashan, 2014. 15. banarase n, khadabadi s, sawarkar h. pharmacognosy practicals (an illustrative guide). scholar’s press, 2018. 16. khandelwal kr, sethi v. practical pharmacongosy (techniques and experiments). nirali prakashan, 2016. 17. irfan z, giri s, khatun a, et al. development and detection of antimicrobial properties of polyherbal handwash. yyu j agr sci. 2023; 33(3):441-9. doi: 10.29133/yyutbd.1271260. 18. wadibhasme p, verma v, banarase n, et al. antimicrobial activity of caesalpinia pulcherrima (l.) leaves extracts against food borne pathogenic and spoilage microorganisms. research journal of pharmacognosy and phytochemistry 2024; 16(1):5-8. doi: 10.52711/0975-4385.2024.00002. how to cite this article: mohit j. umarkar, pawan v. jibhkate, payal g. thakare, nilesh b. banarase, koshish b. gabhane, vikrant l. salode. alum-based herbal handwash formulation and evaluation study. indian j pharm drug studies. 2024; online first. funding: none; conflicts of interest: none stated jacson role of clinical pharmacists in the precision medicine landscape online first indian j pharm drug studies | 1 review article precision medicine and clinical pharmacists janice jacson mandumpala pharm d intern, department of pharmacy practice, nirmala college of pharmacy, muvattupuzha, ernakulam, kerala, india abstract the term "bedside pharmacist" also applies to clinical pharmacists. pharmacists are in a position to take the lead in pharmacogenomic testing, clinical interpretation of data, and recommendations for individualized medication therapy because pharmacogenomic testing can offer patient-specific predictors for drug response. there are career paths for pharmacists in both inpatient and outpatient settings, including managing clinical pharmacogenomics consultation services and teaching families and patients about pharmacogenomic screening. therefore, clinical pharmacists play a crucial role in improving lives through proper medical management. the pharmacy curriculum includes coursework that offers opportunities to gain knowledge and skills in pharmacogenomics. these opportunities also extend to postgraduate education (such as residencies, fellowships, and continuing education). the clinical pharmacy advocacy group emphasizes the need for improved training for chemists in practice as well as students to take precision medicine into account. this review elaborates on the scope of precision medicine, its applications, and the role of a clinical pharmacist in advocating this practice. key words: clinical pharmacist, precision medicine, oncology, pharmacogenomics he concept of "precision medicine" has gained popularity in recent years, owing to scientific and political perspectives. despite its popularity, it is unclear what it means and how it differs from other popular concepts such as "stratified medicine," "targeted therapy," or "deep phenol-typing." commonly used definitions focus on patient stratification, also known as a novel taxonomy, and are created using large-scale data sets that include clinical, lifestyle, genetic, and additional biomarker information, thereby going beyond the traditional "signs-and-symptoms" approach. while these points are important, the description raises several problems. when, for example, does precision medicine begin? in what ways can patient classification translate into improved medical care? and, as implied, is precision medicine the end-point of a novel classification of patients, or is it part of a larger whole? furthermore, are our pharmacists' important members of the precision medicine clinical care team? [1]. personalized medicine is a more traditional concept that is frequently used interchangeably with precision medicine. it aims to use therapies or prevention techniques that are specific to a person's disease process or symptoms [2]. treatment procedures utilized a one-size-fits-all framework in which all access this article online received – 20th july 2024 initial review – 13th august 2024 accepted – 24th august 2024 quick response code individuals presenting with a comparable constellation of symptoms received the same treatment. this practice has led to a reasonable desire for more exact forms of diagnosis and treatment, allowing patients with specific symptoms to receive more individualized care. in many ways, communicable disease management has long been congruent with precision medicine goals, including identifying causal organisms and building data warehouses to drive particular treatments for infections. infectious illness management has integrated technology over time to acquire a better understanding of resistant organisms and to safeguard people [3]. personalized medicine then aims to incorporate technology with medicine to develop a data ecosystem capable of better identifying and treating a patient's ailment. this strategy intends to integrate clinical characteristics and biological details, from imaging to testing in laboratories (including omics data) and health records, in a seamless manner. the reasoning behind this is to create an entirely novel classification of human disease based on molecular biology. the national council report of 2011 has implied that this will lead to better diagnosis, treatment selection, and novel therapies [4]. precision medicine has also had an impact on ________________________________________________ correspondence to: janice jacson mandumpala, department of pharmacy practice, nirmala college of pharmacy, muvattupuzha, ernakulam, kerala, india email: janice.jacson@gmail.com t mailto:janice.jacson@gmail.com jacson role of clinical pharmacists in the precision medicine landscape online first indian j pharm drug studies | 2 non-communicable disease therapy. notionally, precision medicine approaches will help to inform and improve disease taxonomy, leading to greater specificity about the pathogenesis of complex conditions such as cancer, heart disease, and overweight and obesity, as well as a paradigm shift in potential therapeutic interventions that maximize disease treatment while minimizing adverse events [5]. although many consider precision medicine to be one of the most exciting advancements in medicine, it has also generated important problems at multiple levels. many of these concerns relate to what kimmelman and tannock refer to as the 'paradox of precision medicine'. interestingly, ambiguity appears to be a significant feature of precision medicine in practice [6]. with the advent of various treatment-related threats, it is important to identify the role of precision medicine in treatment practice [7]. clinical pharmacists play an important role in identifying various problems within the treatment regimens of a patient and could be pivotal in implementing precision medicine strategies [8]. with this backdrop, we must fully understand the potential of precision medicine and its effectiveness in treating various diseases using a customized approach. method and materials precision medicine and the epigenetic landscape of various diseases the metabolic condition with the fastest-rising incidence in the world, type 2 diabetes mellitus, is linked to epigenetics, according to groundbreaking studies conducted over the past few decades [9]. importantly, these investigations found epigenetic alterations in the pancreatic islets, adipose tissue, skeletal muscle, and liver of people with type 2 diabetes mellitus, including altered dna methylation [10]. since epigenetic changes have been linked to non-genetic factors that influence the risk of type 2 diabetes mellitus in healthy persons, including obesity, a poor diet, physical inactivity, aging, and the intrauterine environment, epigenetics is likely also a factor in the development of type 2 diabetes mellitus. additionally, the epigenome in human tissues is impacted by genetic variables linked to type 2 diabetes mellitus and obesity [11]. a notable finding of causal mediation analysis was the identification of dna methylation as a potential mediator of genetic correlations with metabolic characteristics and disease. translational studies conducted in the last few years have found blood-based epigenetic markers that may be further refined and used in precision medicine to enable patients with type 2 diabetes mellitus to obtain the best care possible and to identify people at risk of problems [12]. the etiology of lung disorders is influenced by both genetic predisposition and environmental risk factors. epigenetic mechanisms indicate possible biological pathways that could close this gap [13]. there is growing evidence of abnormal epigenetic marks, primarily dna methylation and histone modifications, which mediate reversible alterations to the dna without changing the genomic sequence, in individuals with copd, asthma, and pulmonary arterial hypertension. micrornas and post-translational processes may have a role in the development of diseases and be controlled epigenetically. thus, peripheral blood, sputum, nasal and buccal swabs, or lung tissue may be able to detect novel disease pathways and possible biomarkers [14]. additionally, environmental exposures may have an impact on dna methylation during the early stages of fetal development, which may then affect an individual's eventual susceptibility to copd, asthma, and pulmonary arterial hypertension [15]. modeling epigenetic variability in a network framework rather than as individual molecular abnormalities offers insights into potential molecular pathways underlying the pathogenesis of copd, asthma, and pulmonary arterial hypertension thanks to advancements in omics platforms and the use of computational biology methods. clinical applications for epigenetic alterations as non-invasive pulmonary disease indicators are possible [16]. to further improve the primary prevention of lung diseases and their subsequent clinical management, network analysis of epigenomic data combined with molecular tests may help to elucidate the multistage shift from a "pre-disease" to a "disease" state. throughout the natural course of tumor formation, epigenetic abnormalities such as aberrant covalent histone modifications and dna methylation deficiencies are selected for [17]. changes are detectable in early initiation, progression, and eventually recurrence and metastasis. it is becoming more clinically relevant to discover these markers and use them to categorize patient populations at risk, improve diagnostic standards, and provide prognostic and predictive indicators to help with treatment choices [18]. a great potential to change therapy paradigms and offer new therapeutic alternatives for patients whose cancers have these aberrant epigenetic alterations is also presented by the targetable nature of epigenetic modifications, opening the door to novel and personalized treatments [19]. due to its stability and comparatively simple testing, dna methylation has been demonstrated to be quite useful in therapeutic settings. therefore, both precision medicine and epigenetics go hand in hand while working on the treatment of cancers. precision medicine and cancer treatment the rapidly developing amount of information regarding the roles of genetics and the immune system in cancer has allowed for the creation of medicines that target specific molecular abnormalities or other biologic traits, such as those involved in immune suppression. however, genomics has revealed a convoluted truth about malignancies that necessitates a jacson role of clinical pharmacists in the precision medicine landscape online first indian j pharm drug studies | 3 significant shift in the therapy paradigm: away from tumortype-centered treatment and towards gene-directed, histologyagnostic treatment that is tailored to each patient based on biomarker analyses. the rise of precision medicine trials with creative design reflects this paradigm shift [20]. nextgeneration sequencing of advanced malignancies has revealed that genomic changes do not neatly fall into categories defined by the tumor organ of origin. furthermore, metastatic tumors have extraordinarily complicated and distinct genetic and immunological landscapes [21]. as a result, to target cancers with "precision," treatment must be personalized. although immunotherapy has a limited ability to treat ovarian cancer, it may be more effective if sensitive/resistant target treatment subpopulations are assessed based on tumor biomarker stratification. tumor mutation burden, pd-l1, tumor-infiltrating cells, homologous recombination deficit, and neoantigen intratumoral heterogeneity are among these markers. the use of these biomarkers to determine suitable candidates is one of the next directions in the treatment of ovarian cancer [22]. furthermore, precision medicine and targeted therapies have a long history of use in the treatment of breast cancer and continue to hold promise for more specialized and personalized care. targeted medicines and precision medicine continue to push the management of breast cancer towards more individualized care, from the discovery of endocrine and her2-targeted medications to multigene arrays in chemotherapy for more specific patient selection, to radionics and genetic subtyping [23]. precision medicine has emerged as a key concept in the treatment of biliary tract tumors (btcs). although the prognosis remains dismal, advances in molecular characterization, as well as the approval of numerous targeted medicines by the us food and drug administration, have altered the therapeutic landscape of advanced btc. chronic inflammation of the liver and biliary tract, independent of anatomical subtype, is a hallmark of btc oncogenesis [24]. btc subtypes correspond to various molecular properties, making btc a molecularly diverse group of tumors. up to 40% of btcs have a potentially targetable molecular aberration, according to the national comprehensive cancer network guidelines, and molecular profiling is recommended for all patients with advanced btc [25]. the use of circulating tumor dna, immunohistochemistry, and nextgeneration sequencing for biomarker-driven management and molecular surveillance of btc is expanding. improving outcomes for non-targetable tumors utilizing biomarkeragnostic treatment is also a focus, and combinational treatment techniques such as immune checkpoint blockade plus chemotherapy have promise for this patient group [26]. with the development of precision medical tools, research, and treatments, the diagnosis and treatment of diseases such as cancer are getting more accurate and specialized. diagnostic tests can determine particular, individual information from each patient and direct clinicians to a more accurate treatment plan by reaching down to the cellular and even sub-cellular level. with this increased knowledge, researchers and providers may better assess the efficacy of medications, radiation, and other therapy, resulting in a more accurate, if not more optimistic, prognosis. new methodologies, equipment, materials, and testing methods will be necessary as precision medicine becomes more entrenched (figure 1). figure 1 – evolving precision medicine techniques for cancer treatment ethical issues in precision medicine precision medicine bases disease treatment and prevention on a patient's unique gene, environmental, and lifestyle variations. it is a logical continuation of existing research that profiles and identifies therapeutically useful markers utilizing multi-omics-based laboratory tests. in essence, the objective is to gather genotypic and phenotypic data to guide precise and efficient patient therapy. typically, significant and difficult testing would be needed before any practical clinical applicability to patients. precision medicine's integration into healthcare will heavily rely on clinical laboratories. reflex testing based on algorithms or specific case judgments is frequently required for laboratory work. laboratory and clinical data will be integrated to provide the basis for interpretation [27]. the clinical laboratory must become a more active collaborator in clinical treatment as a result of both indirect and direct transmission of test data to patients. large databases of research and clinical data, as well as numerous multi-omics-based laboratory experiments, serve as the foundation for both research and the emerging clinical practice of precision medicine. this situation may lead to moral conundrums involving justice, autonomy, additional findings, consent, and privacy. precision medicine's requirements must be balanced with the idea that patients' interests come first, which leads to conflicts that frequently can go unresolved. jacson role of clinical pharmacists in the precision medicine landscape online first indian j pharm drug studies | 4 in precision medicine, data collection and analysis go further than is required to look into specific clinical conditions [28]. over time, the data gathered and research objectives frequently change. this makes it difficult to gain informed consent based on proper counseling. some of the issues might be resolved with flexible permission that can alter over time. in other circumstances, assumed consent will be regarded as sufficient [29]. data profiles can be used to identify people in anonymous databanks, which raises serious privacy concerns in databanks used for precision medicine. how can data access be restricted to preserve privacy without jeopardizing support for precision medicine, and under what circumstances should this be permitted? in this area, we are still in the early phases of determining the appropriate standards and stakeholder ratio [30]. individuals must face just a small amount of information risk, or no appreciable additional risk, as a result of having their data processed in the databanks. fairness without discrimination for all is the foundation of justice. the key concern is how to deliver the benefits and advancements of precision medicine while maintaining equal access to healthcare. precision medicine requires significant clinical and research work. given the disparities in genetic makeup, habitats, and lifestyles, research data are typically generated from wealthy communities, and results may not apply to other less fortunate people [31]. clinical testing on individuals will be costly, even though greater precision medicine effectiveness may occasionally result in cost savings. in this context, individual rights present one challenging ethical conundrum [32]. can the state mandate that people adopt precision healthy lives in exchange for providing equitable access to precision medicine? precision medicine is an evolving field that raises both professional and patient expectations. instead of being arrogant, the field should carefully manage expectations and avoid making promises that it cannot possibly keep. where does the clinical pharmacist fit? precision medicine has been used in clinical pharmacy practice for many years. as part of standard clinical practice, drug selection and dosage based on patient-specific clinical parameters such as age, weight, renal function, drug interactions, plasma drug concentrations, and nutrition are expected. while epigenetics and pharmacomicrobiomics are still mostly studied in the academic arena, clinical translation of these concepts into clinical practice is anticipated in the future. pharmacogenomics is one of the more recent precision medicine concepts to be used in clinical treatment. the following sections elaborate on the role of pharmacists or clinical pharmacists in the field of precision medicine. chemists must be involved in these advancements as precision medicine research and its clinical applications continue to grow to provide patients with the best, most individualized medication regimens. adverse events in children are frequently unanticipated and patient response to pharmaceutical therapy is very diverse. some patients might experience major side effects from conventional doses of a particular medication and need a lower dose, whilst other patients might need a significantly higher dose of the same medication to get a similar exposure and, hopefully, an analogous therapeutic response [33]. a clinical pharmacist’s understanding of how differences in a single gene, gene networks, and/or the entire genome (pharmacogenomics) may affect drug responsiveness has dramatically improved over the past two decades. the adoption of pharmacogenomic testing in inpatient treatment is also made possible by growing genetic test availability in clinical laboratories (including direct-to-consumer testing) and falling analytical costs. therefore, the promise of precision medicine is gradually making its way into clinical treatment [34]. pharmacogenomics now unquestionably plays a significant role in medication development, regulation, and prescription. pharmacogenomics is proving to be a potential clinical tool for pharmacists who can use it to create individualized treatment plans for children [35]. by choosing the right medication at the right dosage for the right patient, pharmacists can lower their risk of adverse drug events and/or treatment failure. since the original position statement's publication in 2011, the roles that pharmacists play in clinical pharmacogenomics have become more clearly defined, but there are still many opportunities for our profession and practice specialty to promote and establish the role of pediatric pharmacists in pharmacogenomics [36]. pediatric pharmacists routinely recommend medications and dosages as members of the multidisciplinary team based on a variety of clinical considerations, such as a child's age, physiology, concurrent medications, and diagnosis. knowing a patient's pharmacogenomic data, including the ontogeny of various drug-metabolizing enzymes, transporters, and receptors, increases the probability that a prescription will be chosen for the person that is both safe and effective. this information can be used by the pediatric pharmacist to prevent therapeutic failures and proactively lower the likelihood of unwanted adverse medication events [37]. based on their education and expertise, pediatric pharmacists with a working grasp of pharmacogenomics are perhaps the most appropriate members of the medical team to prescribe pharmacogenomic testing and to provide an interpretation of results in the context of a child's pharmacotherapy. additionally, pediatric clinical pharmacists frequently participate in pharmacy and therapeutics committees and collaborate closely with pharmacy it support [38]. in these positions, they are in a good position to suggest jacson role of clinical pharmacists in the precision medicine landscape online first indian j pharm drug studies | 5 gene/drug pairings for institutional use and to develop clinical decision support tools specifically for prescribing doctors, physician assistants, and nurse practitioners. future direction of precision medicine in pharmacotherapeutics precision medicine is a new method for treating and preventing diseases that take into account each person's unique genetic makeup, environmental factors, and way of life. this method will enable medical practitioners and academics to forecast treatment and preventative plans for a condition that a specific person is experiencing more precisely. this strategy will alter clinical pharmacy practice in several significant ways. the link between pharmacogenomics and pharmacodynamics, pharmacogenomics and pharmacokinetics, and pharmacogenomics and pharmacodynamics is the subject of numerous studies that are now being conducted. to further comprehend the uniqueness of therapy response, more recent studies like metabolomics and epigenomics are being done. this new information will be crucial for managing highly effective, risk-free medication in the future. conclusion despite several examples being developed in various fields of pharmacy and medicine, the application of precision medicine in clinical practice is still largely constrained by factors like cost and accessibility of assays. precision, personalized patient care could become a clinical reality thanks to developments in the "omics" sciences and the rising accessibility of health data. references 1. könig ir, fuchs o, hansen g, et al. what is precision medicine? vol. 50, european respiratory journal. 2017; 50(4):1700391. 2. ahn ac, tewari m, poon cs, et al. the limits of reductionism in medicine: could systems biology offer an 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interest: none stated agrawal therapeutic drug monitoring in critical care and transplants online first indian j pharm drug studies | 1 review article therapeutic drug monitoring (tdm): a necessity in critical care and transplant settings rashmi agrawal from, consultant, devmata hospital, bhopal, madhya pradesh, india abstract therapeutic drug monitoring (tdm) personalizes dosage by quantifying plasma concentrations to enhance treatment effectiveness and reduce toxicity. in critically sick patients and transplant recipients, pathophysiological alterations, restricted therapeutic indices, and organ support measures frequently result in unexpected pharmacokinetics (pk). this study emphasizes tdm's significance in critical care and transplantation, concentrating on antimicrobials such as vancomycin, β-lactams, antifungals, and immunosuppressants like tacrolimus, cyclosporine, and mycophenolic acid, substantiated by pubmed-cited literature. key words: tdm, critical illness, transplantation, vancomycin, β-lactams, antifungals, immunosuppressants herapeutic drug monitoring (tdm) is the practice of adjusting a patient's medication dosage to maintain drug concentrations within a specified therapeutic range in the bloodstream [1]. tdm employs a comprehensive analysis of pharmaceutics, pharmacokinetics, and pharmacodynamics to assess the safety and efficacy of a medication across many clinical contexts. for therapeutic drug monitoring (tdm) to be effective, the concentration of a medication must be assessed in several physiological fluids. a depiction of these concentrations in relation to significant clinical considerations is provided [2]. in the last ten years, the theory, practice, and clinical relevance of therapeutic drug monitoring (tdm) have significantly evolved due to the advent of widely accessible and technically proficient modelling, simulation, and dosing software tools [3]. consequently, tdm, once a peripheral area of clinical chemistry, has evolved into a multidisciplinary domain of clinical medicine, enabling laboratory and pharmacometrics experts to provide extremely pertinent clinical information to support pharmacotherapy management. the emerging field is termed “model-informed precision dosing” and is poised to revolutionize medicine through enhancements in performance and usefulness facilitated by machine learning and artificial intelligence technologies. the ultimate objective of the revolution in model-informed precision dosage is to establish individualized, patient-centric therapies [4]. access this article online received – 20th april 2025 initial review – 27th april 2025 accepted – 15th may 2025 quick response code tdm-guided therapy is particularly beneficial for individuals exhibiting significant susceptibility and unique pharmacokinetic characteristics regarding the supplied medicines. examples encompass cancer patients, seriously ill individuals, organ transplant recipients, and people who have undergone significant surgical procedures. moreover, specific groups, like children and those with obesity, should be regarded as subjects for specialized dosage procedures. individuals that possess abnormal biological characteristics, such as pharmacogenomic mutations or liver diseases, need personalized therapy strategies [5]. critical illness alters absorption, distribution, metabolism, and excretion of drugs, making fixed dosing strategies inadequate [6]. post-transplant, maintaining immunosuppressant levels is crucial to prevent rejection while avoiding toxicity. tdm enables dose individualization based on plasma concentrations, integrating pk/pharmacodynamic (pd) principles to optimize therapy [7]. consequently, in both critical care and transplant medicine, the clinical significance of therapeutic drug monitoring (tdm) is highlighted by the administration of medications with a narrow therapeutic index, including aminoglycosides, vancomycin, tacrolimus, and cyclosporine [8]. insufficient exposure in these patients may lead to therapeutic failure, the development of antimicrobial resistance, and graft rejection, while concentrations exceeding the sub-therapeutic threshold are linked to severe toxicities, including nephrotoxicity, hepatotoxicity, and neurotoxicity, __________________________________________________ correspondence to: dr. rashmi agrawal. devmata hospital, bhopal, madhya pradesh, india. email: atharvapub@gmail.com t mailto:atharvapub@gmail.com agrawal therapeutic drug monitoring in critical care and transplants online first indian j pharm drug studies | 2 culminating in end-stage organ failure [9]. the significant interand intra-patient pharmacokinetic heterogeneity in these groups is affected by variables including organ failure, sepsis, polypharmacy, drug-drug and drug-disease interactions, and genetic variants that influence drug metabolism. as a result, personalized concentrationguided treatment is becoming acknowledged as a fundamental aspect of precision medicine in many contexts [10]. by sustaining medication concentrations within specified therapeutic ranges, therapeutic drug monitoring (tdm) boosts clinical efficacy, mitigates adverse drug reactions, shortens hospital stays, and improves overall cost-effectiveness [11]. therefore, incorporating tdm into standard clinical practice is essential for enhancing patient outcomes in critically sick and transplant groups. i. tdm in critical care in the intensive care unit (icu), where patients frequently undergo significant physiological alterations, therapeutic drug monitoring (tdm) plays a crucial role in directing pharmacotherapy. critical illness is linked to hemodynamic instability, hypoalbuminemia, disrupted fluid balance, increased renal clearance, and organ failure, all of which affect pharmacokinetics (pk) and pharmacodynamics (pd) [12]. these alterations render traditional dosage techniques insufficient, necessitating individualized dosing informed by therapeutic drug monitoring as a crucial element of critical care treatment. the icu population is particularly susceptible owing to the severity of illnesses, the frequent administration of lifesustaining treatments, and the prevalence of polypharmacy [13]. extracorporeal techniques, including continuous renal replacement therapy (crrt) and extracorporeal membrane oxygenation (ecmo), significantly modify medication distribution and clearance, resulting in sub-therapeutic exposure or drug toxicity when traditional dosing protocols are utilized [14]. tdm facilitates precise dosage by guaranteeing goal attainment, particularly for medications with narrow therapeutic indices or those necessitating specific pharmacokinetic/pharmacodynamic thresholds for effectiveness [15]. this is especially pertinent for antimicrobials, since under-dosing can lead to therapeutic failure and antimicrobial resistance, whilst overdoing heightens toxicity concerns [16]. a. antimicrobial tdm β-lactams are the most extensively researched antimicrobials in critical care environments. these time-dependent medicines exhibit significant interpatient variability in critically sick patients; therapeutic drug monitoring (tdm) paired with longer or continuous infusions has demonstrated enhancement in pharmacokinetic/pharmacodynamic (pk/pd) goal achievement and perhaps improved clinical outcomes [17]. similarly, vancomycin, aminoglycosides, and colistin necessitate vigilant monitoring owing to their limited therapeutic ranges and possible nephrotoxicity [18]. antifungals like voriconazole and posaconazole benefit from therapeutic drug monitoring due to their variable bioavailability and considerable inter-individual variability [19]. evidence increasingly advocates for the widespread application of antimicrobial therapeutic drug monitoring (tdm) in intensive care unit (icu) treatment, bolstered by recent consensus recommendations from the european society of intensive care medicine (esicm) and the european society of clinical microbiology and infectious diseases (escmid), which offer practical frameworks for implementation [20]. b. non-antimicrobial applications in addition to managing infectious diseases, therapeutic drug monitoring (tdm) plays a vital role in optimizing treatment with anticonvulsants, immunosuppressants, and cardiovascular medications in intensive care unit (icu) patients. agents like phenytoin and valproic acid demonstrate modified protein binding in hypo-albuminemic conditions, requiring the assessment of free drug concentrations to inform dosage [21]. similarly, immunosuppressants like tacrolimus and cyclosporine necessitate monitoring to equilibrate rejection risk with toxicity, especially in post-transplant patients in the icu. digoxin, a cardiac glycoside characterized by a narrow therapeutic index, necessitates meticulous monitoring in critically sick patients with variable renal function. these instances demonstrate the extensive applicability of tdm beyond antimicrobials, underscoring its significance as a fundamental component of personalized medication in critical care [22]. ii. tdm in transplant settings tdm is crucial in managing transplant patients, since it is vital to maintain a delicate equilibrium between sufficient immunosuppression and the prevention of drug-related toxicity. post-transplant patients need prolonged use of immunosuppressive medications, including calcineurin inhibitors (cyclosporine, tacrolimus), mtor inhibitors (sirolimus, everolimus), mycophenolate mofetil, and corticosteroids [23]. drugs such as tacrolimus and cyclosporine exhibit a narrow therapeutic index, considerable inter-individual pharmacokinetic variability, and a substantial risk for drug–drug and drug–food interactions. tdm is essential for personalizing treatment, reducing the likelihood of graft rejection, and preventing side effects such as nephrotoxicity, neurotoxicity, and infections [24]. the pharmacokinetics of immunosuppressants in transplant recipients is affected by various patient-specific and procedural factors, including age, weight, genetic polymorphisms (particularly cyp3a5 variants impacting agrawal therapeutic drug monitoring in critical care and transplants online first indian j pharm drug studies | 3 tacrolimus metabolism), organ function, concomitant medications, and the type of transplanted organ. additionally, the post-operative phase frequently exhibits erratic absorption and metabolism resulting from hemodynamic instability, polypharmacy, and fluctuating gastrointestinal motility. thus, conventional dosage protocols may not consistently forecast drug exposure, rendering personalized tdm-guided modifications crucial for best results [25]. in clinical practice, trough concentrations (c0) are the predominant monitoring metric for calcineurin inhibitors and mtor inhibitors in clinical practice, owing to their simplicity and robust connection with clinical success. nevertheless, several studies indicate that monitoring the area under the concentration-time curve (auc) may offer a more precise assessment of drug exposure, especially for mycophenolate mofetil, as trough levels may not reliably forecast therapeutic effectiveness. advancements in bayesian forecasting and population pharmacokinetic models have facilitated limited sampling tactics for more reliable auc estimation, therefore alleviating patient burden and enhancing accuracy [26]. the application of tdm in transplantation transcends effectiveness and safety, incorporating long-term graft survival. evidence indicates that insufficient immunosuppressant exposure is a primary factor in acute rejection, but chronic overexposure correlates with growing nephrotoxicity and metabolic problems. incorporating tdm into standard post-transplant treatment enables doctors to proactively manage inter-individual variability and customize regimens according to patients' changing clinical conditions. moreover, the integration of pharmacogenomic testing with therapeutic drug monitoring (tdm) presents an opportunity to enhance dosing regimens, advancing the concept of precision medicine in transplantation [27]. overall, tdm in the transplant context is an emerging yet essential instrument. current investigations into innovative biomarkers, non-invasive monitoring techniques, and modelinformed precision dosage aim to improve its efficacy, guaranteeing that transplant recipients have the most effective and safest immunosuppressive protocols for sustained graft function. table 1list of drugs requiring tdm in critical care and transplant settings drug / class / context tdm target / metric when to sample clinical rationale & icu/transplant notes key references vancomycin auc/mic 400–600 (or trough 15– 20 mg/l) trough before 4th dose or steady-state aki risk ↑ with high troughs; auc-based dosing preferred over trough-only methods [4], [18], [29] aminoglycosides (amikacin, gentamicin) peak/mic ≥8–10; trough <1–2 mg/l peak: 30 min postinfusion; trough: before dose nephrotoxicity, ototoxicity risk; altered pk in sepsis, ecmo, crrt [6], [30] β-lactams (pip-tazo, meropenem, etc.) time > mic: 100% for critically ill random / steadystate sampling arc, crrt, ecmo → altered pk; continuous/prolonged infusion often needed [16], [17], [31– 33] linezolid trough 2–8 mg/l pre-dose trough toxicity risk if >10 mg/l; thrombocytopenia, lactic acidosis concerns [34] azoles (voriconazole, posaconazole) voriconazole: 2–5.5 mg/l; posaconazole: >1 mg/l trough after ≥5 days of therapy cyp2c19 polymorphism affects voriconazole levels; toxicity >5.5 mg/l (neurotoxicity) [19], [35], [36] flucytosine peak 30–80 µg/ml; trough <25– 50 µg/ml peak 2h post-dose; trough before next dose myelotoxicity risk if >100 µg/ml; dose adjustment in renal failure [19], [37] tacrolimus (calcineurin inhibitor) trough 5–15 ng/ml (organ/timespecific) 12-h trough (c0) narrow ti; cyp3a5 polymorphism affects metabolism; toxicity: nephro/neurotoxicity, infections [22–27], [38] cyclosporine (calcineurin inhibitor) trough 100–400 ng/ml; some centers use c2 sampling trough (c0) or 2-h post-dose (c2) auc-based dosing may improve outcomes; multiple interactions with drugs/food [39], [43] sirolimus (mtor inhibitor) trough 5–15 ng/ml 24-h trough long half-life; cytopenias, hyperlipidemia; erratic absorption post-transplant [40] everolimus (mtor inhibitor) trough 3–8 ng/ml 24-h trough renal-sparing protocols; post-op pk variability [41] mycophenolate mofetil (mmf) auc0–12 >30–60 mg·h/l bayesian auc or limited sampling trough not reliable for efficacy; aucbased monitoring increasingly used [42] corticosteroids no standard tdm clinical + biomarker endpoints only pk variability post-op; risk of metabolic & infectious complications [7], [23–27] precision medicine & tdm advances bayesian modeling, pharmacogenomics (cyp3a5) limited sampling + model-informed dosing enables individualized regimens, reduced toxicity, improved graft survival [4], [10], [26– 27] agrawal therapeutic drug monitoring in critical care and transplants online first indian j pharm drug studies | 4 challenges and future direction notwithstanding its acknowledged advantages, therapeutic drug monitoring in the intensive care unit encounters several obstacles. the restricted availability of tests for novel pharmaceuticals, inconsistencies in laboratory turnaround times, and the absence of standardized dosage algorithms impede its regular implementation. furthermore, the interpretation of therapeutic drug monitoring necessitates the amalgamation of pharmacokinetic and pharmacodynamic principles with the evolving clinical situation, rather than dependence on fixed reference ranges. improvements in bedside tests, population pharmacokinetic modelling, and bayesian dosing software are expected to increase the accessibility and clinical applicability of therapeutic drug monitoring in real time. subsequent research must prioritize the validation of tdm-guided methods through extensive randomized controlled trials to enhance the evidence base and optimize dose recommendations for various icu populations. conclusion in conclusion, tdm in critical care serves as an essential instrument for precise dosage in patients exhibiting variable pharmacokinetics and a heightened risk of therapeutic failure or toxicity. its use in antimicrobials, anticonvulsants, immunosuppressants and cardiovascular medications underscores its extensive significance within the icu domain. despite ongoing logistical and interpretative obstacles, the incorporation of tdm into clinical workflows—bolstered by technology advancements and global consensus guidelines— provides a pathway to safer, more effective, and personalized medication administration for critically sick patients. references 1. kang js, lee mh. overview of therapeutic drug monitoring. korean j intern med. 2009 mar;24(1):1-10. 2. karvaly gb, vásárhelyi b. therapeutic drug monitoring 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conflicts of interest: none stated i. tdm in critical care bharti et al. hair growth potential of angustifolia leaf gel in rats online first indian j pharm drug studies | 1 original article evaluation of hair growth potential of angustifolia leaves hair gel in rats manu bharti1, anjali bhardwaj2, neha parveen3, ashwani gupta4 from, 1assistant professor, shri venkateswara university venkateswara nagar gajraula, uttar pradesh 244236, 2professor, department of pharmaceutics, shri venkateswara university venkateswara nagar gajraula, uttar pradesh 244236, 3assistant professor, durga college of pharmacy, khaspur sambal u.p., 4associate professor, shri venkateswara university venkateswara nagar gajraula, uttar pradesh 244236. abstract background: hair loss, or alopecia, significantly affects both physical appearance and psychological well-being, particularly in cases such as androgenetic alopecia and alopecia areata. current pharmacological treatments like minoxidil and finasteride have limitations, including side effects and decreased long-term efficacy. objectives: an ethanolic extract of senna angustifolia (eesa), a traditionally used ayurvedic plant, was evaluated for its hair growth-promoting potential using in vivo rat models. methodology: the research began with the successful extraction of phytoconstituents from senna angustifolia leaves using soxhlet extraction. the gel was formulated into two concentrations—2.5% and 5% eesa—and compared with a control and standard 2% minoxidil treatment. hair growth studies were performed on wistar rats by evaluating both qualitative (initiation and completion of hair growth) and quantitative parameters (hair length, follicle count, and histological observations). results: qualitative hair growth analysis indicated that both eesa concentrations accelerated hair growth, with 5% eesa (hg2) showing a significantly faster initiation and completion time (7.01 and 32.4 days, respectively) compared to control (13.8 and 42.6 days) and even slightly outperforming minoxidil (7.07 and 33.0 days). quantitative analysis supported these findings. by day 30, hg2 achieved the greatest hair length (2.8 mm), marginally higher than minoxidil (2.4 mm) and significantly more than the control (1.2 mm). histopathological studies corroborated these findings with increased follicle density and size. conclusion: these results suggest that senna angustifolia extract exhibits promising hair growthstimulatory effects and may serve as a natural alternative or complementary treatment for alopecia with minimal irritation potential. further clinical investigations are warranted to explore its therapeutic application in humans. key words: senna angustifolia, alopecia, ayurveda, ethanolic extract, minoxidil alternative n mammals, hair aids visual and scent communication. humans have less hair, mainly cosmetic. hair loss (alopecia) affects appearance and self-esteem, ranging from patchy (alopecia areata) to complete body hair loss (alopecia universalis). androgens cause alopecia, but treatments like finasteride and minoxidil have side effects and reduced long-term effectiveness [1-5]. human hair is mostly fibrous α-keratin. hair serves thermoregulation, sensing, protection, social cues, and camouflage. humans have ~5 million follicles, 80,000–150,000 on the scalp. hair cycles through anagen (growth), catagen (regression), telogen (rest), and exogen (shedding) stages, driven by cell differentiation, stem cell activation, and epithelial–mesenchymal interactions [6-8]. scalp hair remains in anagen for 2–5 years, catagen for weeks, and telogen for ~3 months. disruptions, like early catagen or shortened anagen, cause hair loss, as seen in telogen effluvium or alopecia areata due to anagen bulb damage [9]. hair loss can have a variety of causes. the most access this article online received – 14th may 2025 initial review – 22nd may 2025 accepted – 14th august 2025 quick response code prevalent conditions, trichotillomania, traumas, extreme stress, childbirth, usage of fertility-stimulating medications, fungal injection, hypothyroidism, sebaceous cysts, some hereditary illnesses, and short anagen syndrome are among them. androgenetic alopecia involves follicle shrinkage, shortened anagen, and prolonged telogen phases, while hirsutism causes excess terminal hair in females [10]. treatment aims to modify anagen duration—shortening it for hirsutism or lengthening it for alopecia. fda-approved drugs are topical minoxidil and oral finasteride. minoxidil prolongs anagen and stimulates follicle growth, though its effects vary by concentration [9, 11-13]. in light of finding herbal alternatives, senna (cassia angustifolia), also called swarna patri in sanskrit, an ayurvedic herb, was employed in the current study. cassia angustifolia belongs to the kingdom plantae and the subkingdom tracheobionata. it is classified under the division magnoliophyta and the class magnoliopsida, within the sub __________________________________________________ correspondence to: dr. anjali bhardwaj. department of pharmaceutics, shri venkateswara university venkateswara nagar gajraula, uttar pradesh 244236. email: anjalisharma071991@gmail.com i mailto:anjalisharma071991@gmail.com bharti et al. hair growth potential of angustifolia leaf gel in rats online first indian j pharm drug studies | 2 class rosidae. the plant falls under the order fabales and is part of the family caesalpiniaceae. its genus is cassia, and the species is angustifolia. it contains anthraquinone, a natural laxative approved by the world health organization (who) and the food and drug administration (fda). native to sudan but named in arabia, senna grows as a perennial after rains [14]. the genus senna has 500 species; 26 contain anthracene derivatives with laxative effects. notable ones include cassia angustifolia (alexandrine/indian senna), cassia fistula, cassia obovata, and others recognized for laxative properties [14]. in the present study, we aimed to assess the quantitative and qualitative effects of senna angustifolia on hair growth in rats. materials and methods 1. identification, collection, authentication, and preparation of leaf extract the leaves of senna angustifolia were gathered in december 2019 at the institute of foreign trade and management (iftm) university botanical garden in moradabad, india. the leaves underwent air drying and washing. the botanical survey of india, scientist in charge, allahabad (central area), india performed the authentication. the department received a voucher specimen (ref: bsi/crc/tech./2019-20/104328). 2000g powdered leaves were soxhlet-extracted with petroleum ether and 95% ethanol for 72 hours. extracts were filtered, concentrated at 40–50°c, and stored at 4°c. 2. preliminary phytochemical screening of the extract the preliminary phytochemical screening of the extract was carried out using standard tests to identify the presence of various bioactive compounds. flavonoids were detected using three methods: the shinoda test (formation of pink, red, green, or blue color upon addition of magnesium and hydrogen chloride), the alkaline reagent test (yellow coloration with sodium hydroxide that turns colorless with acid), and the zinchydrogen chloride test (appearance of a red color). saponins were confirmed by the foam test, where shaking the extract with water resulted in the formation of a stable 1 cm foam layer. alkaloids were tested using dragen dorff’s reagent, producing an orange-red precipitate, and mayer’s reagent, which yielded a white or cream precipitate. for steroids and sterols, the salkowski test (chloroform and sulfuric acid) produced bluish-red or cherry coloration with green fluorescence, while the libermann-burchard test showed a bluish-green color. amino acids were detected by the ninhydrin test, resulting in a blue color upon heating. carbohydrates were confirmed by molisch’s test, which showed a violet ring at the interface with concentrated sulfuric acid. proteins were identified using the biuret test (pink or purple color) and millon’s test, which produced a yellow precipitate on boiling. finally, tannins were detected using ferric chloride (blue-black coloration) and lead acetate, which formed a white precipitate. these tests collectively indicated the presence of multiple phytoconstituents in the extract. 3. thin-layer chromatography (tlc) tlc is a versatile analytical technique for detecting organic/inorganic compounds in micro quantities. plate preparation: silica gel g (acts as absorbent) slurry was applied to plates, dried overnight or for 30 min at 80–90 °c. sample application: 1, 2, and 5 μl of mesm were spotted 2– 2.5 cm from the bottom using a syringe/micropipette. solvent system: non-reactive solvents were used. a 2:1:0.5 mixture of n-hexane:acetone was used for senna angustifolia extract. development chamber: a glass ascending chamber was used. spot detection: plates were placed in an iodine chamber to visualize spots. retardation factor (rf) calculation: rf = distance travelled by the spots from solvent front distance travelled by solvent from the origin front 4. preparation of formulation three herbal gels were made using carbopol base with methylparaben, glycerine, polyethylene glycol, polyvinylpyrrolidone, and triethanolamine. carbopol 934 and extracts were dissolved in water, stirred (800 rpm, 1 hr), glycerin added, then triethanolamine dropwise until neutral. stirring continued until a clear gel formed. senna angustifolia extract amounts varied (table 1). table 1: preparation of formulations formulation hair gel (hg) 1 hair gel (hg) 2 control herbal extract [g] 2. 5% of eesa 5% of eesa -- carbopol 934 [g] 2 2 2 polyvinylpyrrolidone [mg] 5 5 5 methyl paraben sodium [mg] 75 75 75 glycerine [ml] 3 3 3 polyethylene glycol [ml] 6.25 6.25 6.25 triethanolamine [ml] 1.5 1.5 1.5 5. hair growth study preparation for the hair growth study, 150–250 g male or female rats were used from the animal house, iftm university. the experimental protocol was approved by the institutional bharti et al. hair growth potential of angustifolia leaf gel in rats online first indian j pharm drug studies | 3 animal ethics committee (approval no. 2019/837ac/mph/03). rats were kept in standard cages with proper lighting, ventilation, free access to water, and a regular diet. a 7-day acclimatisation period was observed before the study began. 5.1.1 primary dermal irritation study using the organisation for economic co-operation and development (oecd) 404 guidelines, western rats were tested for skin irritation with eesa gel (2.5% and 5%). fur was shaved from a 6 cm² dorsal area 24 hours prior. half a gram of gel was applied, covered with gauze and tape. erythema and edema were scored at 60 minutes, 24, 48, and 72 hours after patch removal. primary dermal irritation index (pdii) was calculated as the average irritation score over 12, 24, 48, and 72 hours: <0.5 = non-irritating; 0.5–2.0 = slightly irritating; 2.1– 5.0 = moderately irritating; 5.0 = severely irritating. 6. treatment for hair growth activity in vivo twenty-four rats were divided into four groups (n=6). hair was removed from a 3 cm² dorsal area using electric shavers and a commercial remover. group 1 (negative control) received plain gel; group 2 (positive control) received 2% minoxidil; groups 3 and 4 received 2.5% and 5% eesa gel, respectively. all treatments were applied topically once daily. the treatment was continued for 30 days, and the hair growth pattern was observed and tabulated. figure 1: treatment for hair growth activity 7. qualitative hair growth study two parameters—hair growth initiation time (i.e., minimum time to initiate hair growth on the denuded skin region) and hair growth completion time (i.e., minimum time taken to cover the denuded skin region with new hair)—were visually observed to conduct a qualitative analysis of hair growth. 8. quantitative hair growth study 8.1 hair length determination rats’ shaved dorsal areas were randomly selected for hair collection on days 10, 20, and 30 of treatment using sterile forceps. after measuring the length of each hair, the findings were expressed as the mean length ± standard error of 25 hairs. 8.2 hair follicle counting at a constant magnification of 100x, digital photomicrographs were captured from representative sections of the slides. following a predetermined area cropping of 1500 μm in width, each image's deep subcutis hair follicles were manually counted. 8.3 histological observation according to the histology observation, each treatment group will have five to seven hair follicles per millimeter of skin. however, there will be a noticeable difference between the treated and control groups in the various cycle phases (anagen/telogen). in all groups, around 40% of hair follicles will be in the anagen phase by the tenth day. 9. statistical analysis one-way anova was used to analyze the data statistically for the test and control groups, and dunnett's test was the next step. data differences were deemed extremely significant when p < 0.05. graphpad instate version 3.06 was the computer program that was utilized. the information is presented as mean ± sem. results the percentage (%) yield of extract from senna angustifolia leaves powder was percentage yield = 3.57 % weighing 735gm. the tlc profile is described in table 2. figure 2: tlc profile of senna angustifolia extract table 2: tlc: senna angustifolia with rf value sr. no. solvent system ratio spot no colour rf value 1. n-hexane and acetone 2:0.5 1 light yellow 0.95 2. n-hexane and acetone 2:0.5 2 yellow 0.88 3. n-hexane and acetone 2:0.5 3 light brown 0.55 4. n-hexane and acetone 2:0.5 4 pale brown 0.45 5. n-hexane and acetone 2:0.5 5 light brown 0.38 bharti et al. hair growth potential of angustifolia leaf gel in rats online first indian j pharm drug studies | 4 moving on to the primary dermal irritation study, using oecd scoring, no erythema or edema was observed in rats after 72 hours of herbal gel application. the pdii was zero, classifying the gel as non-irritating per oecd standards. eesa gel was tested on telogenic rats. anagen onset was indicated by darkening of pink skin. by day 10, hg1 (2.5%) and hg2 (5%) showed more black patches than minoxidil and the control. by day 30, full hair regrowth occurred in hg1 and hg2, while the control showed 60% regrowth. results suggest hg2 promotes early telogen-to-anagen transition. as for the qualitative hair growth study, over 30 days, hair growth initiation and completion were monitored with a magnifying lens. eesa 5% (hg2) treated animals showed significantly faster hair growth start and finish than the control and minoxidil groups. control animals began hair growth at 13.8 days (table 3). table 3: effect of eesa gel formulation on hair growth initiation, completion time, and growth of hair length *p < 0.05 significant when compared to control (anova followed by dunnett’s test values are mean ± sem figure 3: hair growth on 0, 10, 20 and 30th day using hg 2 further, hair follicles, skin thickness, and color were assessed. eesa 2.5% (hg1) showed visible changes from day 20, while eesa 5% (hg2) showed early effects by day 10. hg2 significantly enhanced hair regrowth, follicle density, and subcutis thickness, indicating telogen-to-anagen transition. h&e staining confirmed higher anagenic follicle percentages: hg2 (71%), hg1 (65.1%), minoxidil (69.4%), vs. control (47%), with significant values obtained (p<0.05) for hg2 and the positive control group. increased follicle size and density confirmed hg2’s superior hair growth effect. h&e staining showed increased follicle size and number, indicating telogen-to-anagen transition. the control group had 47% anagenic follicles, while hg2 (69%) and minoxidil (69.4%) showed higher anagen rates and follicle density, confirming enhanced hair growth. discussion senna (cassia angusifolia) is commercially obtained from south india and some from the north part of india. the senna leaves contain rhein, chrysophanol, emodin, aloe emodin, mono treatment hair growth in days percent reduction in hair growth completion time mean length of hair in mm initiation time completion time 10th day 10th day 20th day 30th day negative controlsimple gel (hg3) 13.8 ± 0.42 42.6 ± 1.50 0.0 ± 0.00 0.0 ± 0.00 0.3 ± 0.0 1.2 ± 0.20 positive control standard (2% minoxidil) 7.07 ± 0.42 33.0 ± 0.71 0.6 ± 0.21 0.6 ± 0.21 1.4 ± 0.24 2.4 ± 0.20* 2.5 eesa (hg1) 11.4 ± 0.66 36.0 ± 0.45 0.1 ± 0.0 0.1 ± 0.0 1.4 ± 0.24 1.6 ± 0.24 5% eesa (hg2) 7.01 ± 0.64 32.4 ± 0.51 0.6 ± 0.20 0.6 ± 0.20 1.6 ± 0.24 2.8 ± 0.20* bharti et al. hair growth potential of angustifolia leaf gel in rats online first indian j pharm drug studies | 5 and diglucosides, kaemferol, palmidin, myricyl alcohol and mucilage. the leaves are commercially employed as a hair black dye [15]. the leaves have anti-dandruff, nourishing, conditioning, and strength building properties that can help to grow your hair [16]. gasmi a et al., in 2023, described natural compounds obtained from urtica dioica, humulus lupulus, serenoa repens, vitis vinifera, pygeum africanum, and cucurbita pepo for reducing hair loss [17]. in another study, it was deduced that hair growth-promoting properties of phytochemicals used in various herbal compounds are associated with the tumour growth factor-β (tgf-β) pathway, β-catenin, janus kinasesignal transducer and activator of transcription 3 j (ak/stat3) pathway, extracellular signal-regulated kinase pathway (erk) signaling pathway, and 5α-reductase inhibitory property. some studies proposed that phytochemicals are more effective than conventional hair loss regimens such as minoxidil and finasteride [18]. owing to the presence of the phytochemical properties in senna leaf extract, essa was employed in the current study exploring the hair growth-promoting potential, formulated into a topical gel, and evaluated in vivo using rat models. the study demonstrated that essa, prepared via soxhlet extraction (3.57% yield), contains bioactive compounds like flavonoids, tannins, glycosides, amino acids, and proteins that support hair follicle stimulation. the extract was formulated into gels (2.5% and 5%) and tested against a control and 2% minoxidil. the 5% eesa gel showed superior hair growth activity, initiating and completing growth faster than both control and minoxidil, and producing the longest hair length (2.8 mm vs. 2.4 mm with minoxidil and 1.2 mm in control). histological analysis confirmed its efficacy, with 71% anagenic follicles, increased follicle density, and thicker skin, indicating that senna angustifolia effectively promotes hair regeneration by stimulating the telogen-to-anagen transition. conclusion the ethanolic extract of senna angustifolia (eesa), when formulated as a topical gel, significantly enhanced hair growth in rats, as demonstrated by: faster initiation and completion of hair regrowth, greater hair length over time, and higher density and proportion of anagenic hair follicles, along with absence of dermal irritation or adverse effects. these results suggest that eesa—particularly at 5% concentration—is a promising, safe, and effective natural alternative for hair growth promotion. its efficacy was found to be comparable to minoxidil, a standard fda-approved treatment, thus supporting its potential application in the development of herbal therapies for alopecia and other hair growth disorders. further studies, including mechanistic investigations, clinical trials in humans, and long-term safety evaluations, are recommended to validate these findings and facilitate their translation into commercial use. references 1. olsen, elise a. androgenetic alopecia. disorders of hair growth: diagnosis and treatment. 1994: 257-283. 2. han a, mirmirani p. clinical approach to the patient with alopecia. semin cutan med surg. 2006; 25(1):11-23. doi: 10.1016/j.sder.2006.01.003. 3. bhalerao ss, solanki nh. therapeutic approaches to the management of common baldness. indian drugs. 2002; 39(11):567-73. 4. bagatell cj, bremner wj. androgens in men--uses and abuses. n engl j med. 1996; 334(11):707-14. doi: 10.1056/nejm199603143341107. 5. davies je. the pharmacological basis of therapeutics. occup environ med. 2007; 64(8):e2. doi: 10.1136/oem.2007.033902. 6. franbourg a, hallegot p, baltenneck f, et al. current research on ethnic hair. j am acad dermatol. 2003; 48(6 suppl):s115-9. doi: 10.1067/mjd.2003.277. 7. krause k, foitzik k. biology of the hair follicle: the basics. semin cutan med surg. 2006; 25(1):2-10. doi: 10.1016/j.sder.2006.01.002. 8. reynolds, a.j., jahoda, c.a. hair follicle stem cells? a distinct germinative epidermal cell population is activated in vitro by the presence of hair dermal papilla cells. j cell sci. 1991; 99(2), 373– 385. 9. pause, r., cotsarelis, g. 1999. the biology of hair follicles. n engl j med, 341,491-498. 10. azziz, r. the evaluation and management of hirsutism. obstet gynecol. 2003; 101(1):995– 1007. 11. mori, o., uno, h. the effect of topical minoxidil on hair follicular cycles of rats. j dermatol. 1990; 17(5), 276–281. 12. philpott, m., green, m.r., kealey, t. studies on the biochemistry and morphology of freshly isolated and maintained rat hair follicles. j cell sci. 1989; 93(3), 409–418. 13. kwon os, oh jk, kim m, et al. human hair growth ex vivo is correlated with in vivo hair growth: selective categorization of hair follicles for more reliable hair follicle organ culture. arch dermatol res. 2006; 297(8):367-71. doi: 10.1007/s00403-0050619-z. 14. ram chander, pawan jalwal, and anil midfha. 2017. recent advances on seena as a laxative: a comprahensive review, 6:349-53. 15. gupta a, malviya r, singh tp, et al. indian medicinal plants used in hair care cosmetics: a short review. pharmacognosy journal. 2010; 2(10):361-4. doi: 10.1016/s0975-3575(10)80110-5. 16. senna leaves benefits for hair: a comprehensive guide. natural herbal. 2024. 17. gasmi a, mujawdiya pk, beley n, et al. natural compounds used for treating hair loss. current pharmaceutical design. 2023; 29(16):1231-44. doi: 10.2174/1381612829666230505100147 18. kesika p, sivamaruthi bs, thangaleela s, et al. role and mechanisms of phytochemicals in hair growth and health. pharmaceuticals (basel). 2023; 16(2):206. doi: 10.3390/ph16020206. how to cite this article: bharti m, bhardwaj a, parveen n, gupta a. evaluation of hair growth potential of angustifolia leaves hair gel in rats. indian j pharm drug studies. 2025; online first. funding: none; conflicts of interest: none stated http://dx.doi.org/10.1016/s0975-3575(10)80110-5 https://naturalherbal.info/author/natural-herbal/ http://dx.doi.org/10.2174/1381612829666230505100147 16. senna leaves benefits for hair: a comprehensive guide. natural herbal. 2024. gandrathi et al. press-coated pulsatile ketoprofen tablets online first indian j pharm drug studies | 1 original article design and development of press coated pulsatile release of ketoprofen tablets srujana gandrathi1, padamatini naveen kumar1, chandrasekhara rao baru1, sushma desai2, m. shiroja1, raaga swetha1 from, 1assistant professor, department of pharmaceutics, chilkur balaji college of pharmacy, moinabad hyderabad, telangana, india-500075, 2research scholar, gitam university, rudraram, patancheru, hyderabad, telangana, india502329 abstract ketoprofen pulsatile tablets were developed and evaluated in this work utilizing synthetic polymers grades hydroxy propyl methyl cellulose (hpmc) e5, hpmce15, and natural polymer xanthan gum. the angle of repose and compressibility index were determined to be within the acceptable range by pre-formulation investigations of core tablet granules, demonstrating excellent flow properties for compression. the core tablet underwent evaluation procedures that examined content homogeneity compliance, weight fluctuation within standard deviation (sd) ± 2%, hardness, and friability. evaluations of the coated tablets showed that the weight fluctuation, hardness, and friability all fell within sd ± 2%. in vitro dissolving tests were conducted on formulations f1 through f9, and at the conclusion of the sixth hour, formulation 8 produced a pulsatile drug release. after the allotted period, the medication was released here at once. the stability study, which was conducted for two months for formulation 8 in accordance with international council of harmonisation (ich) requirements, was determined. key words: ketoprofen; press-coated pulsatile release tablet; xanthan gum echnologies are evolving daily in the pharmaceutical industry, leading to the most effective dose form. in that regard, the most widely utilized and effective method of delivery has been oral for more design freedom for dose forms. convenience and simplicity of administration [15]. their pharmaceutics excellence and therapeutic advantages over quick release pharmaceutical medicines have been acknowledged more and more in regulatory approval for commercialization [6-11]. modified-release oral dosage forms have given fresh life to medications that had lost commercial potential because of gastrointestinal problems, toxic effects associated with dose, and the need for frequent administration [12-16]. when it comes to treating conditions like peptic ulcer, asthma, cardiovascular disease, arthritis, diabetes, and hypercholesterolemia, the above regulated and sustained release medication delivery method has limitations because these conditions rely on biological rhythms [17,18]. pulsatile release is the main rationale for its use when continuous drug release, also known as zero-order release, is desired. in this current project, it is hypothesized that pulsatile drug delivery with various polymers ketoprofen drug effectiveness can be improved [19-24]. access this article online received – 04th december 2025 initial review – 08th december 2025 accepted – 10th december 2025 quick response code materials ketoprofen was purchased from infinity pharmaceuticals. lactose monohydrate was purchased from biocon, bangalore, india. microcrystalline cellulose was purchased from sigachi industries. sodium starch glycolate was purchased from maruthi chemicals. pvp k 30 purchased from nabhi chemicals, thane, india. isopropyl alcohol was purchased from rankem. purified talc was purchased from gangotri inorganics. magnesium stearate was purchased from amishi drugs and chemicals. hpmc e5 & hpmc e15 was purchased from jianxin cellulose. xanthan gum purchased from peer chemical industries, hyderabad. methodology pre-formulation studies standard graph preparation: identifying the drug's lambda max: a 100 ml sample of ketoprofen was collected, diluted in 100 ml of 0.1n hcl (1000µg/ml), and then 1 ml of the concentrated sample was made up with 6.8 ph phosphate buffer solution to determine the drug's lambda max. calibration curve: to create the principal stock, 100 mg of precisely weighed medication was added to a 100 ml volumetric flask and topped off with 0.5n hcl (1000 mg/ml). __________________________________________________ correspondence to: sushma desai, gitam university, rudraram, patancheru, hyderabad, telangana, india502329. email: d.sushmapharma@gmail.com t mailto:d.sushmapharma@gmail.com gandrathi et al. press-coated pulsatile ketoprofen tablets online first indian j pharm drug studies | 2 serial dilutions of that solution (1, 2, 3, 4, 5) are prepared and scanned under a uv lamp at λ max 260 nm. this process was then repeated with a 6.8 ph phosphate buffer solution and uv scan at λ max 262 nm and menthol at λ max 257 nm. the fourier transform infra-red spectroscopic study: for compatibility investigations fourier transform infra-red spectroscopic (ftir) is used. for the pellet potassium bromide (kbr) was utilized. the ratio is (1:10). the empty medication was examined without any polymer as carrier, and in addition, drug combinations (1:1) were examined for formulation sample 1 mixture containing pure drug ketoprofen and sample 2 containing lactose + microcellulose phosphate (mccp) + sodium starch glycollate (ssg) + polyvinyl pyrrolidone (pvp) + talc + magnesium stearate + ketoprofen. sample 3 containing drug mixture with hpmc e5 and sample 4 containing drug mixture with hpmc e15 and sample 5 mixture containing pure drug ketoprofen with xanthan gum. moreover, structural elucidation was done. solubility: the medicine ketoprofen has bcs (biopharmaceutics classification system) class ii, which has a weak acid property, poor water solubility presents as (racemic) mixture and good solubility in certain organic solvents, according to a solubility study [25]. using water, acetone, menthol, ethanol, 0.1n hcl, and 6.8 ph phosphate buffer, the sample was extracted and its solubility was examined. drug purity assay: 100 mg of precisely weighed medication was added to a 100 ml volumetric flask and diluted with 6.8 ph phosphate buffer (1000 mg/ml). after extracting 1 milliliter of the sample from that solution, it was diluted in a 100-milliliter volumetric flask and examined under a uv lamp at λ max 262 nm. additionally, the standard sample and this sample were contrasted (identified the difference between the sample and standard/reference sample). preparation of ketoprofen tablet: to create a welldistributed mixture, the powdered ketoprofen, lactose, mccp ph 102, and sodium starch glycolate were run through a 210µm sieve and then thoroughly combined using a pestle and mortar. with constant stirring, a pvp k 30 alcoholic solution should be added to the mixture dropwise. lubricating should be followed by compacting the resulting powdery combinations. a conventional oven was used to dry the granules for six hours at 60ºc. using a traditional single punch press, the dried granules with a size of 25–60 mesh were compacted into tablets after talc and magnesium stearate were added as a lubricant [7]. pre-compression studies angle of repose: after filling a funnel to overflowing, the produced grains were allowed to pass through the opening under the influence of gravity. the height (h) and the radius (r) of the pile was determined from the cone that formed on a graph sheet, which was used to estimate the pile's area [7]. repose angle = tan-1 (h/r) bulk density (bd): twenty grams of precisely weighed grains were added to a 50 ml measuring cylinder. after the first volume (v0) was measured and determined [12]. density of bulk = m/v0 tapped density (td): the grains in the measurement cylinder were tapped 100 times. we measured and determined the cylinder's minimal volume (vt). m/vt is the tapped density. the hausner's ratio. in order to determine hanuser's ratio, bulk density and tapped density were used. hausner's ratio = bd – td [13]. carr's index (ci) or compressibility index (ci): the tapped density and bulk density were used to calculate and assess the compressibility index [16]. ci=(td-bd)/td x 100 ketoprofen pulsatile drug delivery tablet preparation: 400 mg of polymer (hpmc e5, hpmc e15, xanthan gum) and in combination {hpmc e5 and xanthan gum (1:1 and 3:1), hpmc e5 and hpmc e15 (1:1), hpmc e15. xanthan gum (1:1, 2:1, and 3:1)} were removed from the punched tablets, which were used as cores. two phases were employed when using polymer: first, 200 mg of coating polymer was poured into the die, then cores were placed in the middle of the die and slightly pushed to secure the coatings under and around the core. finally, the remaining coatings were filled and compressed [19]. table 1: formulation of ketoprofen tablets: materials f1 f2 f3 f4 f5 f6 f7 f8 f9 ketoprofen 75mg 75mg 75mg 75mg 75mg 75mg 75mg 75mg 75mg lactose 18mg 18mg 18mg 18mg 18mg 18mg 18mg 18mg 18mg mccp ph 102 55.73mg 55.73mg 55.73mg 55.73mg 55.73mg 55.73mg 55.73mg 55.73mg 55.73mg ssg 18mg 18mg 18mg 18mg 18mg 18mg 18mg 18mg 18mg pvp solution q.s q.s q.s q.s q.s q.s q.s q.s q.s talc 1mg 1mg 1mg 1mg 1mg 1mg 1mg 1mg 1mg gandrathi et al. press-coated pulsatile ketoprofen tablets online first indian j pharm drug studies | 3 magnesium stearate 1mg 1mg 1mg 1mg 1mg 1mg 1mg 1mg 1mg hpmc e5 400mg 200mg 200mg 300mg hpmc e15 400mg 200mg 200mg 250mg 300mg xanthan gum 400mg 200 mg 200mg 150mg 100mg 100mg post compression studies: from each formulation, ten tablets were chosen at random, and each tablet's thickness was measured with a vernier caliper. the standard deviation was also computed. test of friability: using tablet friabilator test apparatus, the friability of tablets was assessed. initially ten tablets were first weighed (w initial), they were put into a friabilator. the friabilator was run for four minutes at 25 rpm or until it reached 100 revolutions. weighing the tablets after the friabilation as (w final). friability was then determined. tablets with less than 1% friability are deemed acceptable [21]. in vitro studies: the drug release of ketoprofen from the produced pulsatile tablets was investigated using an eightstation dissolving rate testing device with a revolving paddle at 50 rpm and 25 cm depth in 0.1n hcl for two hours and in phosphate buffer ph 6.8 (900ml) for four hours. a constant temperature of 37.5ºc was maintained during the dissolving process. samples are taken out each at 1hr interval. using a ultra violet (uv) visible spectrophotometer, the drawn samples are measured at 262 nm with 6.8 ph phosphate buffer as reference [21]. stability studies: optimized formulation f8, underwent accelerated stability testing over a two-month period at 40°c ± 2°c and 75% ± 5% relative humidity (rh). dissolution and content uniformity tests were also conducted [21]. results & discussion using various polymers (hpmc e5, hpmc e15, xanthan gum) and a combination of synthetic and natural polymers (hpmc e5 and xanthan gum (1:1 and 3:1), hpmc e5 and hpmc e15 (1:1), hpmc e15 and xanthan gum (1:1, 2:1, and 3:1)), the current study aims to formulate and evaluate ketoprofen pulsatile tablets. determination of lambda max: fig 1: determination of lambda max of ketoprofen drug construction of calibration curve: table 2: construction of calibration curve of ketoprofen s.no concentration absorbance in 0.1n hcl absorbance in 6.8 ph phosphate buffer 1 10 0.098 0.113 2 20 0.191 0.240 3 30 0.293 0.352 4 40 0.398 0.456 5 50 0.487 0.545 fig 2: calibration curve of ketoprofen in 0.1n hcl fig 3: calibration curve of ketoprofen in 6.8 ph phosphate buffer gandrathi et al. press-coated pulsatile ketoprofen tablets online first indian j pharm drug studies | 4 compatibility studies: fig 4: ft-ir of ketoprofen fig 5: ft-ir of ketoprofen+ lactose+ mccp+ssg+pvp k 30+ talc + mg stearate gandrathi et al. press-coated pulsatile ketoprofen tablets online first indian j pharm drug studies | 5 fig 6: ft-ir of ketoprofen +hpmc e5 fig 7: ft-ir of ketoprofen +hpmc e15 gandrathi et al. press-coated pulsatile ketoprofen tablets online first indian j pharm drug studies | 6 fig 8: ft-ir of ketoprofen + xanthan gum the ftir analysis, which was conducted to identify and assess the compatibility of the drug and excipients, revealed no notable alterations in the powder combination of the drug and excipients. for the formulation development, the following excipients were chosen: lactose, mccp, ssg, pvp, talc, magnesium stearate, and polymers: hpmc e5, hpmc e15, and xanthan gum. solubility studies: table 3: solubility of ketoprofen solvent solubility water practically insoluble acetone soluble methanol slightly soluble ethanol soluble 0.1n hcl sparingly soluble ph 6.8 phosphate buffer soluble it was determined that the organoleptic characteristics and solubility of ketoprofen found highly soluble in acetone, ethanol. assay: table 4: study the purity of drug method 1 2 3 avg spectrophotometric method 99.96% 100.1% 99.98% 100.01% limits: ≤ 99 % ≥100.5% pre-compression studies table 5: angle of repose, bulk density, tapped density, hausner’s ratio, compressibility index s.no angle of repose (θ) bulk density (gm/ml) tapped density (gm/ml) hausner’s ratio compressibility index (%) 1 24.680 ± 0.47 0.432 ± 0.004 0.480 ± 0.005 1.112 ± 0.0119 10.067 ± 0.97 all the values are expressed as mean ± sd n=3 gandrathi et al. press-coated pulsatile ketoprofen tablets online first indian j pharm drug studies | 7 research on compatibility, solubility, and organoleptic characteristics was done and the results were satisfactory. angle of repose and compressibility index were determined to be within the range by pre-formulation investigations of core tablet granules, demonstrating excellent flow properties for compression. post compression studies table 6: thickness, diameter, hardness, friability and weight variation of optimized tablet s.no thickness (mm) diameter (mm) hardness (kg/cm2) friability (%) weight variation(mg) 1 3.013 ± 0.009 8.005 ± 0.007 9.9 ± 0.316 0.01 0.185 ± 0.003 all the values are expressed as mean ± sd. n=10 for thickness, diameter and weight variation. hence for hardness n=5, for friability n=1 post compression studies of coated tablet table 7: thickness, diameter, hardness, friability, and weight variation formulation thickness (mm) diameter (mm) hardness (kg/cm2) friability (%) weight variation(mg) f1 3.79 ± 0.03 12.86 ± 0.008 16.9 ± 0.31 0.02 594.6 ± 2.10 f2 3.81± 0.006 12.32± 0.006 17.0 ±0.3 0.01 595.2 ± 1.469 f3 3.87 ± 0.006 12.867 ± 0.04 6.1 ± 0.3 0.04 595.2 ± 2.18 f4 3.748 ± 0.007 12.931 ± 0.005 16.9 ± 0.3 0.03 595.4 ± 1.624 f5 3.815 ± 0.006 12.983 ± 0.006 14.1 ± 0.3 0.04 594.6 ± 1.854 f6 3.893 ± 0.007 12.883 ± 0.06 15.9 ± 0.3 0.04 594.8 ±1.939 f7 3.847 ± 0.006 12.963 ± 0.004 16.9 ± 0.3 0.02 596.2 ± 1.66 f8 3.863 ± 0.004 12.863 ± 0.004 16.9 ± 0.3 0.01 597.4 ± 1.113 f9 3.851 ± 0.005 12.944 ± 0.004 17.0 ± 0.3 0.01 593.3 ± 1.676 a powder mix was assessed for the pre-formulation investigations, which included hausner's ratio (1.112 ±0.0119), compressibility index (10.067 ± 0.97), bulk density (0.432 ±0.004), taped density (0.480 ±0.005), and angle of repose (24.680 ± 0.47). the powder was then punched as tablets. the formulation core tablet's angle of repose and compressibility index (carr's index) were determined to be within the range, demonstrating excellent compression flow properties. the core tablet underwent evaluation trials that included friability of 0.01%, hardness of 9.9 ± 0.316 kg/cm2, weight variation (mg) of -0.185 ± 0.003 falling within 2%, and content uniformity of 103.08 %, which conforms with not less than (nlt) 90.0% to not more than (nmt) 110.0% of the label claim. following assessment tests of the core tablet, the tablet was press-coated utilizing coating materials hpmc e5, hpmc e15, and xanthan gum. thus, evaluation tests for weight fluctuation, hardness, and friability of press-coated tablets were conducted and produced positive findings. in vitro studies: fig: 9: invitro studies graph of f1, f2, f3 formulations fig10: in-vitro studies graph of f4, f5, f6 formulation gandrathi et al. press-coated pulsatile ketoprofen tablets online first indian j pharm drug studies | 8 fig 11: in-vitro studies graph of f7, f8, f9 formulations in vitro dissolving tests were conducted on formulations f1 through f9, whereas formulation 8 provided a 6-hour pulsatile release of the medication. in this case, the medicine was released entirely after the allotted period, indicating that the formulation was deemed satisfactory. for stability investigations, f8 was chosen based on in vitro release tests. stability studies: for stability investigations, f8 was chosen based on in vitro release tests. the formulation 8 (f8), which has a 3:1 ratio of natural polymer (xanthan gum) to synthetic polymer (hpmc e15), exhibits a pulsatile drug release in 6 hours, according to drug release experiments and after two months of accelerated trials, the f8 formulation was discovered to be stable. accelerated studies: (40⁰c ± 2⁰c & 75%±5% rh) table 8: stability studies data for the optimized formulation s.no test parameters optimized formulation 1st month 2nd month 1. appearance almost white colour almost white colour 2. drug content 102.98% 102.91% 3. dissolution at the end of 6th hr released completely at the end of 6th hr released completely the stability study was conducted for two months at 40°c ± 2°c and 75% relative humidity ± 5% acs (accelerated stability studies) in accordance with ich recommendations. during the study time, tablets were assessed for assay and in vitro dissolving research, and no notable alterations were observed. summary ketoprofen pulsatile tablets were developed and evaluated in this work utilizing a variety of polymers, including synthetic and natural polymers. the core tablet underwent evaluation procedures that examined content homogeneity compliance, weight fluctuation within sd±2%, hardness, and friability. evaluations of coated tablets show that the weight fluctuation, hardness, and friability all fall within sd±2%. in vitro dissolving tests were conducted on formulations f1 through f9, and at the conclusion of the sixth hour, formulation 8 produced a pulsatile drug release. after the allotted period, the medication was released here at once. the stability study, which was conducted for two months for formulation 8 in accordance with ich requirements, was determined to be stable. conclusion although controlled and sustained drug delivery systems have been widely used and successful in the medical field, pulsatile systems can be helpful for providing medications in line with the circadian behavior of diseases. there is always a need for new delivery systems to give patients additional therapeutic benefits. one such technique, pulsatile medicine administration, which administers medications at the appropriate time, place, and dose, has the potential to benefit patients with chronic illnesses such arthritis (rheumatoid arthritis) mostly affects women globally. reference 1. rawat s, bisht s, kothiyal p. pulsatile drug delivery “a programmed polymeric device”. indian drugs. 2013; 50(05):522. 2. chaurasia s, arvind k, rahul k, et al. chronopharmaceutics: concept and technologies. journal of chronotherapy and drug delivery. 2011; 2(2):57-69. 3. singh dk, poddar as, nigade su, et al. pulsatile drug delivery system: an overview. international journal of current pharmaceutical review and research. 2011; 2(2):55-80. 4. belgamwar vs, gaikwad mv, patil gb, et al. pulsatile drug delivery system. asian journal of pharmaceutics (ajp). 2008; 2(3). 5. khan z, pillay v, choonara ye, et al. drug delivery technologies for chronotherapeutic applications. pharmaceutical development and technology. 2009; 14(6):602-12. 6. parmar rd, parikh rk, vidyasagar g, et al. pulsatile drug delivery systems: an overview. int j pharm sci nanotechnol. 2009; 2(3):605. 7. senthilnathan b. design and development of pulsatile drug delivery system for anti-diabetic drug (doctoral dissertation, the tamilnadu dr. mgr medical university, chennai). 8. sharma gs, srikanth mv, uhumwangho mu, et al. recent trends in pulsatile drug delivery systems-a review. international journal of drug delivery. 2010; 2(3). 9. rohini rs. formulation and evaluation of pulsatile drug delivery system of pregablin. phramaceutica analytica acta. 2016; 7(10):2-4. 10. rane ab, gattani sg, kadam vd, et al. formulation and evaluation of press coated tablets for pulsatile drug delivery using hydrophilic and hydrophobic polymers. chemical and pharmaceutical bulletin. 2009; 57(11):1213-7. 11. rajput m, sharma r, kumar s, et al. pulsatile drug delivery system: a review. international journal of research in pharmaceutical and biomedical sciences. 2012; 3(1):118-24. 12. shinde pv. evaluation of floating press-coated pulsatile release of aceclofenac tablets. a solution for rheumatoid athritis. asian journal of biomedical and pharmaceutical sciences. 2013; 3(17):58. 13. bandari s, sanka k, jukanti r, et al. formulation and in vitro evaluation of pulsatile colon drug delivery system of piroxicam using 32 factorial design. der pharm lett. 2010; 2(4):177-88. 14. chandani g, ganesh b, preeti k. a comprehensive review of pulsatile drug delivery system. the pharma innovation. 2012; 1(7, part a):99. gandrathi et al. press-coated pulsatile ketoprofen tablets online first indian j pharm drug studies | 9 15. rajput a, pingale p, telange d, et al. a current era in pulsatile drug delivery system: drug journey based on chronobiology. heliyon. 2024; 10(10). 16. arora k, jain ms, sharma m. development and characterization of chronomodulated drug delivery system of salbutamol sulphate. journal of drug delivery & therapeutics. 2017; 7(4). 17. talakoti rk. chronotherapeutic press-coated tablets of tramadol hydrochloride: formulation and in vitro evaluation. asian journal of pharmaceutics (ajp). 2017; 11(02). 18. patel h, pandey s, patel v, et al. pulsatile release of ketoprofen from compression coated tablets using eudragit (r) polymers. int j pharm pharm sci. 2016; 8(2):224-9. 19. madhavi av, reddy dr, venugopal m, et al. formulation and evaluation of pulsatile drug delivery system of zafirlukast. j drug deliv ther. 2020; 10(2):122-8. 20. adil ms, arshad hm, ilyaz m, et al. chronotherapeutics: targeting the disease at its ideal time. the pharma innovation. 2014; 2(12, part a):49. 21. golla c, agaiah g, subhash j, et al. design and evaluation of press coated pulsatile delivery of doxofylline tablets. acta scientific pharmaceutical sciences. 2018; 2(11):58-62. 22. patel p, madan p, lin s. formulation and evaluation of timecontrolled triple-concentric mefenamic acid tablets for rheumatoid arthritis. pharmaceutical development and technology. 2014; 19(3):355-62. 23. patel vp, soniwala mm. pulsatile drug delivery system for treatment of various inflammatory disorders: a review. international journal of drug development and research. 2012; 4(3):67-87. 24. kashyap s, singh a, godbole am, et al. design, development and characterization of release modulated terbutaline sulphate pulsincap device for treatment of nocturnal asthma. research journal of pharmacy and technology. 2018; 11(4):1655-62. 25. soto r, svärd m, verma v, et al. solubility and thermodynamic analysis of ketoprofen in organic solvents. international journal of pharmaceutics. 2020; 588:119686. how to cite this article: gandrathi s, kumar pn, baru cr, desai s, m. shiroja, swetha r. design and development of press coated pulsatile release of ketoprofen tablets. indian j pharm drug studies. 2025; online first. funding: none; conflicts of interest: none stated bafna et al. multiple unit pellet system (mups) online first indian j pharm drug studies | 1 review article a new path for drug delivery by multiple unit pellet system (mups) harshada rajendra bafna1, nitin rajendra shirsath2, vaibhavkumar arun jagtap3 from, 1assistant professor, department of quality assurances, 2assistant professor, department of pharmaceutics, 3professor, department of pharmaceutics, gangamai college of pharmacy, nagaon, dhule, mh, india abstract innovative delivery system that are helpful in enhancing therapeutic objectives and reducing adverse effects are the focus of pharmaceutical research. the advantages of tablets and pellet-filled capsules are combined into a single dose form by mups, one of the more complex and inventive technologies. continuous drug distribution into the bloodstream is made possible by the breakdown of these pills in the stomach and intestine. the current article examines the potential benefits, desirable pellet characteristics, different pelletization methods, mups-influencing factors, drug release mechanisms, and pharmaceutical applications, as well as the challenges associated with compaction and the crucial elements that must be considered for mups production to be successful. key words: mups; innovative delivery system; multiple unit pellet system; benefits; process variables he acronym for multiple-unit pellet system is mups. nonetheless, the term is commonly used to describe mups that have been compressed into tablets from the standpoint of research and the pharmaceutical industry [1]. the name "pellet" has historically been used to refer to a wide variety of well-produced, geometrically defined agglomerates made from various starting materials under infinite processing circumstances [2]. pellets are typically between 0.5 and 1.5 mm in size, though they can be made in a wide range of diameters [3]. pharmaceutical businesses mainly produce pellets for use in oral controlled-release dosage forms that can deliver medications in site-specific, gastroresistant, or sustainedrelease manners. the usage of pellets in dosage form design and development has increased as a result of the growing advancements in pharmaceutical delivery technology [4]. actually, due to its many advantages, a tablet is the solid dosage form that is most commonly used for oral administration. similar therapeutic benefits can be obtained via controlled release capsules, a kind of solid oral formulation that frequently includes a range of coated pellets. around the world, a small number of people and businesses are conducting research on the difficult subject of mups compaction. microparticles, which can be made of natural or synthetic polymers, are tiny, freely-moving particles that range in diameter from 1 to 1000 µm. the advances in genetics and biotechnology have led to the development of many powerful and specialized drugs. because of a number of problems, including the restricted solubility, poor stability, and narrow access this article online received – 09th may 2024 initial review – 01st october 2024 accepted – 31st december 2024 quick response code therapeutic index of many new medications, safer drug delivery is necessary [5–6]. the cores of these pellets, granules, sugar seeds, minitablets, powders, and crystals made of ion exchange resin particles include pharmaceuticals. it is more typical to load multiparticulates into capsule shells rather than compressing them into tablets. because mups have better dispersion, transportation, and surface area, as well as better bioavailability and lower inter-subject variance, they are employed more often than unit dose forms [7-8]. mups improve medication safety because, if a multi-unit dosage form's film covering is destroyed, the drug contained in that small subunit will be released, altering the release behavior of that particular subunit, which makes up a tiny percentage of the entire dose. the entire medicine will be discharged into the stomach if the enteric coating on a single unit or monolithic is destroyed, resulting in irritation or ulceration, dosage dumping, or the loss of the full dose. there may be more consequences if one unit fails than several units. the pharmaceutical industry as a whole started investing money in pellet technology research and, whenever possible, buying state-of-the-art equipment suitable for pellet production after the advantages of pellets over single units became clear. pellets can be made using a variety of production processes, depending on the use and the producer's preferences [9]. the methods used for pelletization and granulation are almost the same. the most often used methods include extrusion, spheronization, solution or suspension stacking, and powder layering. __________________________________________________ correspondence to: harshada rajendra bafna. department of quality assurances, gangamai college of pharmacy, nagaon, dhule, mh, india. email: harshadabafna@gmail.com t mailto:harshadabafna@gmail.com bafna et al. multiple unit pellet system (mups) online first indian j pharm drug studies | 2 2. mups benefits over pellet-filled capsules or conventional modified-release tablets they can be divided into the proper dosage strengths without changing the composition or the procedure. pellets containing the active drug in liquid, capsule, or dissolving tablet form offer significant therapeutic advantages over single unit dose forms. for instance, the extrusion-spheronization process was used to create pantoprazole pellets for the treatment of peptic ulcers [10]. figure 1. benefits of mups 2.1 pharmacokinetic advantages micro pellets in mups travel from the stomach into the small intestine swiftly yet consistently due to their small size, which lowers the possibility of localized discomfort, enhances uniform absorption of medications, and boosts bioavailability. for instance. for both juvenile and geriatric patients, prevacid solutab is a mouth-dissolving mups with a pleasant taste. when using delayed-release formulations, rapid enteric coating is encouraged by the consistent evacuation of micro pellets from the stomach into the small intestine, drug release and degradation, resulting in an early peak time and peak plasma concentration (t-max and c-max). there is less potential for dose dumping, more constant drug release, and inter-subject variance when controlled-release formulations are used [11– 12]. 2.2 pharmacodynamic advantages pellets dissolve medications in the gastrointestinal tract more rapidly and consistently due to their small size and greater surface area, which promotes constant, regulated pharmacological action and uniform drug absorption. compared to a traditional pellet-filled capsule, the mups dosage form has a significantly greater number of pellets, which helps to further reduce intraand inter-subject variability in medication absorption and clinical response. furthermore, there is less chance of partial medication release and dose dumping (in the stomach) [13]. 2.3 patient friendly dosage form a mouth-disintegrating medication with a pleasant flavor, prevacid solutab is appropriate for older people and children who have difficulty in swallowing pills or capsules. the orodispersibl mups can be taken without water to encourage swallowing and salivation, particularly when traveling. lower volume or smaller tablets are more patient-compliant than capsules, and they can be divided into the appropriate dosage strengths without requiring formulation changes. additionally, they can be employed to produce distinct release properties in separate or identical locations of the gastrointestinal system. 2.4 processing advantages because of their roughly spherical shape and ease of conversion into tablets, mups have better flow characteristics than typical granules used for tabletting. furthermore, these formulations require less lubrication during the tablet-making process, which lowers the cost of mups relative to tablets. mups offers all the advantages of tablets over capsules, including improved inert matrix physicochemical and microbiological stability. speed of processing using the existing tableting infrastructure in comparison to capsules. lower processing costs since the product is more resistant to manipulation and processing is completed more quickly. compression causes fewer dust problems than with conventional tablets [14–15]. 2.5 investigation, dissection and assessment pellets offer a high degree of flexibility in designing and developing oral dosage forms such as suspension, sachet, tablet, and capsules [16]. mups offer the chance to examine the change in size, shape, and density of pellets after compaction by retrieving the pellets from highly lubricated compacts or disintegration tubes. 3. mechanism of drug release from mups the following are possible mechanisms for drug release from mups: 3.1 diffusion: on contact with aqueous fluids in the gastrointestinal tract (git), water diffuses into the interior of the particle. drug dissolution occurs and the drug solutions diffuse across the release coat to the exterior [17]. 3.2 erosion: certain coatings have the ability to progressively dissolve over time, releasing the medication that is inside the particle [18]. 3.3 osmosis: when the proper conditions are met, allowing water to enter can cause an osmotic pressure to build up inside the particle [19]. 4. factors influencing design of mups formulation variables: composition, porosity, polymer coating size and amount, nature, and tableting excipient size and amount are the main formulation variables. 4.1 core pellet: pellets are a unique type of granulates that have a smooth surface, low porosity, extremely regular round shape, and a typical size range of 0.2-2 mm. homogeneous and bafna et al. multiple unit pellet system (mups) online first indian j pharm drug studies | 3 inhomogeneous pellets can be distinguished based on the medication distribution within them. 4.1.1 type and composition: enteric coated pellets and a minimum of one tablet excipient make up the multiple unit compositions. 4.1.2 pellet size: the size of the pellets has an impact on both the drug release from the compacted pellets and the compaction properties. 4.1.3 pellet shape: the most crucial feature is the pellets' sphericity, which can be ascertained using a variety of techniques. 4.1.4 pellet porosity: this is another important component that influences the compaction pattern and, in turn, the integrity of the polymer coat during compression. 4.1.5 pellet density: pellet density is very significant, particularly if a prolonged gastric stay is desired. density has little to no significance when it comes to modifying the stomach residence time of medicinal dose forms (both multiple and single units). 4.1.6 elasticity: pellet composition has a direct bearing on elasticity. the center of the pellet should be robust and somewhat flexible. 4.2 coverage: coating of polymers: when it comes to maintaining the integrity of the polymer film during compression, the coating's quantity matters. generally speaking, a thicker coating is more resistant to damage than a thinner one. 4.3 cushioning excipients: tablet formulations include excipients with protecting (cushioning) qualities to preserve the integrity of coated pellets. the mups produced hard tablets with low friability and predictable drug release profiles when 60–70% cushioning grains were added [20]. 5. types of mups there are two types of mups that can exist: mups made of coated pellets. mups made up of pellets of matrix material. while the latter kind of mups is less popular than the former, it does have certain advantages over compaction of polymercoated pellets. 5.1 mups made of coated pellets the ability of sustained-release multiparticulates to release the medication as planned even after compaction is a challenge. polymer-coated pellets were compressed either alone or in conjunction with other excipients to create tablets. 5.2 mups made up of pellets of matrix material pellets that are naturally made of excipients that remain inside the pellet structure's matrix and slow down the release of the medication are referred to as matrix pellets [21]. 6. preparations of mups mups preparation entails the following actions 6.1 preparation of pellets: pelletization is the process of combining excipients and active medicinal substances into spherical beads known as pellets [22-23]. there are numerous methods available for making pellets. some of the traditional coating methods used for pellatization include the following: 6.1.1 layering the layering method involves covering nuclei—which may be inert starting seeds or crystals of the same material—with successive layers of pharmacological entities in the form of liquid, dry powder, or crystals [24]. they include solution/suspension layering and powder layering. example: omeprazole magnesium is suspended in a micronized state and subsequently deposited on 0.250–0.355 mm diameter sugar microspheres. to segregate the omeprazole from the subsequent enteric coating, these pellets are subcoated. pellets are compressed into tablets, film-coated, and mixed with tabletting excipients after a final protective over-coating. 6.1.2 extrusion and spherization extrusion spherization was created as a pelletization technique in the early 1960s. multiparticulates for applications involving controlled drug release were its primary use. for oral solid dosage forms with controlled release and few or no excipients, it is particularly beneficial to make dense granules with a high drug loading. extrusion spheronization is a complicated process that uses wet mass extrusion and spheronization to create evenly sized spherical particles called spheroids, pellets, beads, or matrix pellets, depending on the materials and technique used. the primary advantage over alternative methods of creating drugloaded spheres or pellets is the ability to incorporate large amounts of active components without creating unacceptable large particles (i.e., few excipients are required). despite the wide range of potential applications, improved processing and controlled medication release are most commonly mentioned [25]. the following are the processing steps for pelletization and extrusion: 6.1.2.1 dry mixing all of the ingredients are dry combined to create a consistent powder dispersion using a twin shell blender, plane tray mixer, high speed mixer, and trumbler mixer. this kind of formulation frequently uses lactose monohydrate (lm), microcrystalline cellulose, and glyceryl monostearate (gms). 6.1.2.2 wet massing wet mass extrusion spheronization, sometimes referred to as cold-mass extrusion spheronization, became the method of bafna et al. multiple unit pellet system (mups) online first indian j pharm drug studies | 4 choice for producing dense, spherical pellets with uniform size and shape. wet massing the powder dispersion produces an appropriate plastic mass for extrusion. wet mixing requires the addition of a binding liquid, such as glycerol or water [26]. 6.1.2.3 extrusion the third step in the process is this. the method of extrusion involves pushing a substance through a pre-made hole or aperture until the wet substance forms consistently sized rodshaped particles. since it is believed to be a dependable and repeatable process that may yield high-quality spheroids with a limited size range and plenty of mechanical strength, extrusionspheronization is the most widely utilized technology. additional methods for creating pellets include spray congealing, rotary processing, high/low shear granulation, suspension/solution layering, and powder layering [27]. 6.1.2.4 spheronization the fourth stage of the process, called spheronization, aims to round off the rods produced by extrusion into spherical particles. the process of spheronization, which turns rods into spheres, occurs in several stages. only when the mass is really dry will the rods change to the same degree as dumbbells, and the spheres won't deform. the extrudate rounds into spheres due to frictional forces from particle-particle and particleequipment collisions. cross-linked polyvinylpyrrolidone or crospovidone has been successfully used to aid in spheronization to produce pellets [28]. 6.1.2.5 drying to reach the appropriate moisture content, a drying phase is needed. the pellets can be dried in a fluidized bed dryer, tray drier, or oven at room temperature or at a higher temperature. drying was completed when the exit air reached 50ºc (around 30 minutes). 6.1.2.6 screening in order to attain the intended size distribution, screening may be required; sieves are employed for this purpose. 6.2 selection of excipients because differing tablet disintegration rates result in varying tabletting excipients, there was a little variation in medication release [29]. the group of binders, fillers, dissolves, lubricants, slip agents, and mixtures there of is used to choose excipients. 6.3 compression of mups coated pellet compression is a difficult procedure that requires modifying formulation and process parameters. after compression, it ought should flex and bounce back without endangering the coating. the coated pellets' flat surface is visible in sem photos. the fact that the pellets separated from the cushioning material at 3 kn [30] indicates that they were adequately protected during tableting. there were no obvious cracks or indentations on the pellet's surface as an appropriate tableting excipient to guard against compression-induced damage to coated pellets containing a medication that dissolves easily in water. 7. troubles in developing mups tablets in order to prevent particle attrition, fragmentation, densification, and deformation, the following aspects should be taken into account when designing and manufacturing mups: 1. the coated pellets' resilience in preserving the medication release profile following compression. 2. harmonious mass of pellets and excipients used in tableting are compatible. 3. the tablets' mechanical strength for subsequent processing, including film or functional testing. 4. packing and coating. 8. solutions to overcome challenges in mups 8.1 granulation: good flow and a limited distribution of particle sizes in the tableting mixture inhibit the de-mixing of pellets and extra-granular material. if the coated particles are large in size, size adaptation (controlled by granulation) could be taken into consideration. 8.2 pellet shape: for a good, even distribution, the pellets' shape should be spherical or almost spherical. increased spherical form deviation is not the result of typical release from defects and fractures during compression. 8.3 pellet size: to endure compression pressure, the coated pellets' maximum size is limited to 2 mm. because they separate with tabletting excipients and expose the transmitted force from the upper punch to the lower punch directly, largesized pellets cause the coating to burst. influences the final tablet's content consistency as a result. 8.4 pellet density: compared to pellets with a density of more than 2 g/cm3, those with a density of roughly 1.5 g/cm3 exhibit faster stomach emptying. pellets that have a diameter of less than 2 mm and a density of less than 2 g/cm3 can pass past the pyloric sphincter in both fed and fasted states, similar to how liquids pass through the stomach. 8.5 pellet core and core material: pellets with a low surface to volume ratio are better because they can result in a smaller area of contact between the particles as they are being consolidated. the pellet core should be somewhat flexible in order to accommodate this, allowing it to deform during compression without damaging the coated film. microcrystalline cellulose, both in powdered and granulated form, has been the subject of substantial research by numerous scholars. according to their findings, microcrystalline cellulose offers coated particles in both powder and granulated form better protection and displays plastic deformation when compressed. dicalcium phosphate pellets, for example, should not have a very hard core because this could hinder the flow of the pellets. the surface is subjected bafna et al. multiple unit pellet system (mups) online first indian j pharm drug studies | 5 to compression force in this scenario, which deforms it and modifies the release. 8.6 porosity: due to its important role in compression, pellet porosity is associated with deformation. due to their increased porosity, which makes them denser when compression force is applied and forms as deformed coherent units as a result of noninterfering excipients, medium and high porous pellets showed more deformation than low porous pellets. when fewer porous pellets are compacted, the medication releases much more quickly. this occurs as a result of the pellets seeing reduced deformation and densification. the excipients included in the pellets should not alter their medicine release profile. the deformed pellets and extra granular material must pack as tightly as they can. 8.7 polymer coating and film flexibility: polyarchy and cellulose derivatives are examples of polymers that are commonly used to achieve certain release characteristics. cellulose and its derivatives, including hpmc and hpmcp, form stiff, brittle films that break under compression, in contrast to polyarchy and copolymers of acrylics, which create flexible films that deform readily. plasticizers that help create flexible films include triacetin, polyethylene glycol, and triethyl citrate. a extremely flexible sheet prevents the coating from separating when compressed and ensures elastic properties. polymers like eudragit provide the required degree of elasticity to the film when mixed with triethyl citrate plasticizer. 8.8 selection of solvents: both aqueous and non-aqueous coatings can be applied. aqueous coating has some drawbacks despite its environmental benefits, such as medication deterioration from trapped moisture; temperature also contributes to this degradation when pellets are dried for extended periods of time to eliminate moisture. however, the sol-to-gel thixotropy of the polymer solution in non-aqueous coatings facilitates coating and causes the solvent to evaporate significantly faster than in aqueous solvents. 8.9 mechanical resistance: during compression, the elasticity of the film mechanically stabilizes the pellets. by avoiding particle deformation during compression, high mechanical resistance contributes to the preservation of the film's integrity. larger particle sizes reduce film breakdown by increasing mechanical stability and reducing interparticle interactions. 8.10 coating thickness: the thickness of the coating layer correlates with the pellets' mechanical resistance during compaction. below a certain thickness, even very flexible films tend to break, although films with more thickness retain their elastic properties. the thickness of the coating layer is altered by the compaction-induced deformation of the coated pellets, which affects the drug's release profile. 8.11 extra-granular material and cushioning agents: the stability of films during compression is affected by extragranular material. crystalline materials with sharp edges and abrasive surfaces may damage the coating as the compression force increases. as a result, the characteristics of drug release after compaction into tablets are altered. the sort and quantity of coating agent, the selection of additives such plasticizers, the use of cushioning excipients, and the rate of pressure applied must all be closely monitored because the drug release properties of the subunits help to protect the film. the optimum choice is polyethylene glycol, ideally polyethylene glycol 6000, because cushioning materials are naturally waxy. the coated pellets are shielded from compaction pressures by selectively deforming and/or cracking, or by reorganizing themselves inside the tablet structure. cushioning pellets, which are usually made of excipients, are considered to be softer and more porous than coated medications. the ratio that is believed to be most suitable for reducing coating film damage is 1:3 or 1:4. in medicine, the proportion of drug pellets to cushioning excipients is crucial. 8.12 electrostatic charges: during the tablet compression cycle, the pellet surfaces may become electrostatically charged, which could disrupt their flow. typically, talc is added to address this issue since it functions as a glidant. comparative dissolving experiments should be carried out while developing multiparticulate tablets in order to find any potential variations in the release rates of the tablets and the uncompressed tabletting mixture. to guarantee consistent medication releases, the disparity between the two dissolution profiles must not surpass 10% [31-33]. 9. process variables 9.1 compression force applied: by increasing the force beyond the minimum necessary to create a compact to a specific value which varies depending on the formulation film ruptures and the rate of dissolution are accelerated compression force modifies the dissolving profile according to the intended formulation type and, to a greater extent, causes damage to the polymeric functional coating. when a delayed release formulation is used, a polymer coat rupture causes the medicine to be released into acidic media, which causes the drug to degrade. 9.2 compression speed: to maintain homogeneity in the final mixture during tabletting, the tabletting rate is controlled. the pressure used for tabletting is appropriately set. compression speed is most likely the formulation's ideal value. excessive speed can lead to incorrect die fill. punch heads and compression rollers can come into more touch with one another, which will stop capping and laminating [34]. 9.3 compression velocity: to corresponds more to the dwell duration the amount of time the punch head spends in contact with the compression roller during the compression cycle. mups have a higher propensity to cap during compression. increased dwell time inhibits capping and lamination by promoting the creation of strong bonds between the compressed particles. bafna et al. multiple unit pellet system (mups) online first indian j pharm drug studies | 6 9.4 variables related to equipment: with a few modifications, any tablet compression device can be used to prepare mups. to create pills and tablets, the tablet compression machine uses granulated powder. a die and specific punches work together to form a cavity. the powder inside this hollow is fused when punches are squeezed with a certain amount of power. to compress powder and make tablets, the rotary tablet press machine consists of many rotating stations. tablet compression devices basically work on hydraulic pressure. all tablet machines require this pressure. without being reduced, it passed through the static fluid. it is appropriate to increase pressure since any externally supplied pressure is transmitted in the same direction in all directions through static fluid. to manufacture a tablet, the granulated powder material must be metered into a cavity made by a die and two punches. punches must be pressed firmly together to fuse the material together. there are two varieties of tablet presses: single station and multi station respectively. in terms of feature requirements and production output, these machines function differently. they can also manufacture odd-shaped tablets. to lessen weight variations between batches, they usually have a highpressure system installed. due to several benefits, single-punch tablets are a great choice for small-scale research and manufacturing. single-punch presses are also designed to be as silent as feasible. the weight and quantity of tablets in mups may vary more as a result of the segregation procedure. demixing is usually caused by differences in the sizes, forms, surfaces, and densities of the pellet and additional granular tabletting excipients. when pellets with a limited size distribution are compressed with additives of similar size and shape, mass and content uniformity can be achieved. it is essential to take into account the excipient-to-pellet ratio in addition to the importance of particle and pellet size, shape, and density in order to attain the optimal mups. any tabletting blend must have a minimum of 50% w/w pellet concentration to avoid segregation. for instance, enteric-coated pantoprazole pellets were compressed into orodispersible tablets for usage by elderly and pediatric patients, and pantoprazole (multiunit particulate system) pills were made easier to administer [35]. 10. disintegration and dissolution behaviour of mups mups are anticipated to break down in one of the following ways since they are frequently made with particles that have modified release characteristics: 1. quick disintegration in the mouth, if the mups includes modified-release or taste-masked coated particles that are formulated as a compact in an oro dispersible base (orally dissolving tablets), such as prevacid solutab. 2. quick disintegration in the gastrointestinal tract upon swallowing or oral administration (losec mups). 3. slowly and gradually eroding mups in the git to release particles coated with polymers, like toprol xl, gradually. individual coated multiparticulates that split out as a result of mups disintegration exhibit the desired dissolving behavior, which is frequently determined by the coating type or pellet matrix design [36-38]. conclusion multi-particulate pellet compositions are known as mups (multiple unit pellet systems). these days, mups don't truly reflect an easy choice; rather, they represent a formulation of first choice. the process of compaction of coated pellets into multiunit particles (mups) is actually quite intricate, involving structural deformation or even rupture of the subunits. the increased cohesiveness between the pellets may prevent tablet disintegration and/or significantly alter the drug release profile of the subunits. put differently, pellet compacts must possess a specific crushing strength in order to endure the mechanical shocks that occur throughout the manufacturing, packing, and dispensing processes. this technical project article examines the benefits, necessary preparations, and relevant literature for the manufacturing of mups tablets. references 1. majeed sm, al-shaheen mk, al-zidan rn, et al. multiple unite pellet systems (mups) as drug delivery model. journal of drug delivery and therapeutics. 2020; 10(6):231-5. 2. kállai-szabó n, farkas d, lengyel m, et al. microparticles and multi-unit systems for advanced drug delivery. european journal of pharmaceutical sciences. 2024: 106704. 3. kayrak-talay d, dale s, wassgren c, et al. quality by design for wet granulation in pharmaceutical processing: assessing models for a priori design and scaling. powder technology. 2013; 240:718. 4. shankar ps, ganesh pd, bhanudas sr. pelletization: a most significant technology in the pharmaceuticals. world j pharm res. 2014; 3(6):1972-2003. 5. bathool a, vishakante gd, khan ms, et al. pelletization as a key tool for oral drug delivery: a 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seremeta kp, imperiale jc, et al. novel formulation and drug delivery strategies for the treatment of pediatric povertyrelated diseases. expert opinion on drug delivery. 2012; 9(3):303-23. 18. pai kg, dhirajlal bp. multiple unit particle system (mups): novel technology for preparation of extended release (er) tablets. research journal of pharmacy and technology. 2013; 6(8):856-64. 19. hamman h, hamman j, steenekamp j. multiple-unit pellet systems (mups): production and applications as advanced drug delivery systems. drug delivery letters. 2017; 7(3):201-10. 20. shah sj, shah pb, patel ms, et al. a review on extended-release drug delivery system and multiparticulate system. world j. pharm. res. 2015; 4(8):724-47. 21. mansoori s, dev a, gupta s. an overview of pelletization techniques used in multiparticulate drug delivery system. world j pharm res. 2017; 6(8):720-41. 22. rahman ma, ahuja a, baboota s, et al. recent advances in pelletization technique for oral drug delivery: a review. 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biopharmaceutics. 2010; 75(3):436-42. 29. sangwai m, sardar s, vavia p. nanoemulsified orlistat-embedded multi-unit pellet system (mups) with improved dissolution and pancreatic lipase inhibition. pharmaceutical development and technology. 2014; 19(1):31-41. 30. panda sk, parida kr, roy h, et al. a current technology for modified release drug delivery system: multiple-unit pellet system (mups). j. pharm. sci. and health care. 2013; 3(6):5163. 31. stuiver m, soma k, koundouri p, et al. the governance of multiuse platforms at sea for energy production and aquaculture: challenges for policy makers in european seas. sustainability. 2016; 8(4):333. 32. boyapati i, awasthi r, kulkarni g. formulation, characterization, and in vitro release studies of modified release multiple unit particulate system (mups) of venlafaxine hydrochloride. journal of research in pharmacy (online). 2022; 26(1):75-87. 33. van der merwe cj, steyn jd, hamman jh, et al. effect of functional excipients on the dissolution and membrane permeation of furosemide formulated into multiple-unit pellet system (mups) tablets. pharmaceutical development and technology. 2022; 27(5):572-87. 34. osei-yeboah f, lan y, sun cc. a top coating strategy with highly bonding polymers to enable direct tableting of multiple unit pellet system (mups). powder technology. 2017; 305:591-6. 35. ozarde ys, sarvi s, polshettiwar sa, et al. multiple-unit-pellet system (mups): a novel approach for drug delivery. drug invention today. 2012; 4(12). 36. cal k, mikolaszek b, hess t, et al. the use of calcium phosphatebased starter pellets for the preparation of sprinkle ir mups formulation of rosuvastatin calcium. pharmaceuticals. 2023; 16(2):242. 37. patel s, patel n, misra m, et al. controlled-release domperidone pellets compressed into fast disintegrating tablets forming a multiple-unit pellet system (mups). journal of drug delivery science and technology. 2018; 45:220-9. 38. shinde mb, shinde gv, patel rs, et al. computational predictability of polyethylene glycol encapsulated modified release multiple unit pellets formulation of metoprolol succinate using different multivariate models. materials technology. 2022; 37(12):2090-105. how to cite this article: bafna hr, shirsath nr, jagtap va. a new path for drug delivery by multiple unit pellet system (mups). indian j pharm drug studies. 2024; online first. funding: none; conflicts of interest: none stated pruthviraj et al. synthesis and biopotency evaluation of 1-(4,5-diphenyl-1h-imidazol-2-yl)naphthalen-2-ol online first indian j pharm drug studies | 1 original article synthesis, evaluation of antimicrobial activity, and dft analysis of 1-(4,5diphenyl-1h-imidazol-2-yl)naphthalen-2-ol pruthviraj k1, chethan bs2, lohith tn3, chandrakumar r4, dineshbabu ns4, mohan kumar4, shivaraja g4, sunil k1 from, 1department of chemistry, sri siddhartha institute of technology, ssahe, tumakuru, 2department of basic science (physics), amruta institute of engineering and management sciences, bidadi, bengaluru, 3department of studies in physics, manasagangothri, university of mysore, mysuru, karnataka, 4department of studies and research in organic chemistry, tumkur university, tumakuru. abstract in the present work, we report synthesis of 1-(4,5-diphenyl-1h-imidazol-2-yl)naphthalen-2-ol an asymmetric catalytic block, flurophore and pharmacologically potent triaryl imidazole derivative having naphthalene ring via solvent free eco-friendly method using debusradiszewski condensation reaction of benzil with 2-hydroxy napthaldehyde in presence of molecular iodine as the catalyst and ammonium acetate as the source of nitrogen this method offered the compound in good yield compared to conventional method. the structure of the compound was established based on ftir, multi nuclear nmr (1h &13c) spectral data and mass spectrometry. pharmacological potency was evaluated through in-vitro antimicrobial activity against four different bacterial strains (gram-ve bacteria k aerogenes, e coli, p desmolyticm, gram+ve bacteria s aureus) and two strains of fungi (a flavus and c albicans) exhibiting potent zone of inhibition of 500μg/50μl and 10μg/50μl respectively in comparison with the standard drugs, followed by in silico adme evaluation obeying the lipinski’s rule, molecular docking studies with the binding energy of -8.3 kcal/mo and -8.8 kcal/mol against docked antifungal and antibacterial protein respectively established the good agreement with the in-vitro result. density functional theory (dft), electro static potential diagram (esp) and other computational techniques were utilized to analyze the physicochemical parameters like energy gap, ionization energy, and electron affinity successfully. key words: 1-(4,5-diphenyl-1h-imidazol-2-yl)naphthalen-2-ol, in-vitro antimicrobial activity, in silico adme, molecular docking studies, dft analysis. eterocyclic scaffolds having widespread pharmacyological activities have attracted great attention among imidazole-based heterocyclic scaffolds play a vital role in natural and synthetic organic chemistry, have been well exploited for many medicinal scaffolds exhibiting anti-hiv [1-3], anticancer [4-6], anticonvulsant [7-9], antifungal [10-12] antibacterial [13-15], and anti-tubercular agent [15-17]. this core also has been utilized in other diverse pharmaceutical applications, stands out as a flexible substance with a wide range of uses, including biomedical technology and sophisticated materials. because of its special blend of electron transport, photo stability [18], fluorescence [19], and catalytic qualities [20], it is an important component of scientific research and technological advancement. materials and methods access this article online received – 25th apr 2024 initial review – 17th may 2024 accepted – 02nd jun 2024 quick response code the organic solvents and chemicals were purchased from sd fine and sigma aldrich, standard commercial sources used without further purification.1h and 13cnmr spectra were recorded on ecx500 jeol 400 mhz high resolution multinuclear ft nmr spectrometer with ln2 cooled probe using deuterated solvent (dmso-d6), chemical shifts were expressed in parts per million (ppm) and tetramethylsilane (tms) as an internal standard. the mass spectrum was recorded using waters micromass lct mass detector. experimental synthesis of (4,5-diphenyl-1h-imidazol-2-yl)naphthalen-2-ol [3]: a mixture of benzyl (1) 1mmol, 2-hydroxy-1naphthaldehyde (2) 1mmol, nh4oac (1mmol), were heated in presence molecular iodine i2 (0.1 – 0.05mol) catalyst to70oc. the reaction progress was monitored by thin layer chromatography using n-hexane-ethyl acetate (7:3) solvent system. after the completion of the reaction the mixture was poured aqueous ___________________________________________________ correspondence to: sunil k, department of chemistry, sri siddartha institute of technology, ssahe, tumakuru. email: sunilk999@gmail.com. h mailto:sunilk999@gmail.com pruthviraj et al. synthesis and biopotency evaluation of 1-(4,5-diphenyl-1h-imidazol-2-yl)naphthalen-2-ol online first indian j pharm drug studies | 2 sodium thiosulphate (na2s2o3) as the desired compound separated out with excess of iodine destroyed by the thiosulphate, the crude compound was recrystallized using hot ethanol to obtain a dark yellow solid. scheme 1: synthetic route for iodine assisted synthesis of 1(4,5-diphenyl-1h-imidazol-2-yl)naphthalen-2-ol (3). results chemistry: 1-(4,5-diphenyl-1h-imidazol-2-yl)naphthalen-2ol(3) was synthesized as depicted (scheme-1) in the presence of i2 system. further analytical characterization of the synthesized compound found to be ir (atr): 3332cm-1(br) (imidazole– nh),2920cm-1 (intramolecular hydrogen bonded oh), 1613cm-1 (aromatic, -c=c), 1503 cm-1 (imidazole, –c=n), 1083 cm-1 (co, alcohol stretching) 1h nmr (400mhz, dmso-d6, δ, ppm): 6.071(b, 1h, ar-oh), 7.233-8.901 (m, 16h, arh) 12.010 (s,1h, imidazole nh).13c nmr (100mhz,dmso-d6, δ, ppm ):117.889, 118.866, 119.834, 122.497, 123.829, 128.582, 130.057, 130.153, 132.204, 133.190, 133.401, 134.350, 136.113, 136.640, 154.634, 157.29. calcd m/z from mf (c25h18n2o) = 362.1 found m/z = 363 (m+1). in-vitro anitimicrobial activity: 1-(4,5-diphenyl-1h-imidazol2-yl)naphthalen-2-ol (3) was assessed for their in vitro antibacterial activity against gram-ve bacteria k.aerogenes, e.coli, p.desmolyticm, gram+ve bacteria s.aureus and the antifungal potency of these compounds was also tested against two fungal strains a.flavus and c.albicans using the using the agar well method. the antibacterial activity results of 1-(4,5diphenyl-1h-imidazol-2-yl)naphthalen-2-ol(3)revealed good antibacterial activity against tested bacterial strains group in comparison with the standard ciprofloxacin (cpfx) as shown in table 1. and from the antifungal results, it is evident that compound showed excellent inhibition effects against the tested fungal strains compared to clotrimazole (clt) may be due to the presence of electron-donating –oh group. the results of this antifungal activity were given in (table 2). the measurements were made in triplicate for each compound and their average values are reported. table 1: the antibacterial activity of the 1-(4,5-diphenyl-1h-imidazol-2-yl)naphthalen-2-ol (3) sample treatment zone of inhibition in mm antibacterial activity k. aerogenes e. coli p. desmolyticm s. aureus 3 250μg/50μl 10 ± 0.03** 10 ± 0.03** 12 ± 0.04** 13 ± 0.06** 500μg/50μl 11 ± 0.05** 12 ± 0.00** 14 ± 0.05** 13 ± 0.11** cpfx 5μg/50μl 19 ± 0.5** 18 ± 0.5** 17 ± 0.5** 33 ± 1.0** dmso table 2: the antifungal activity of the 1-(4,5-diphenyl-1himidazol-2-yl)naphthalen-2-ol (3) sample treatment zone of inhibition in mm antifungal activity c. albicans a. flavus 3 5μg/50μl 11 ± 0.33** 14 ± 1.0** 10μg/50μl 25 ± 0.05** 28 ± 1.0** clt 5μg/50μl 18 ± 0.03** 23 ± 0.33** dmso cpfx: ciprofloxacin, clt: clotrrimazole, na: no activity, (±) standard deviation values are the mean of three determinations, the ranges of which are <5% of the mean in all cases. in-silico adme evaluation: the molecular properties and lipinski rule of five for the compounds were determined by swissadme online server [21]. exploration of in-silico adme properties of synthesized compounds in terms of molecular properties and toxicity profile are listed in (table 3). druglikeness is a quantitative parameter that measures a compound’s oral bioavailability. abbot bioavailability score predicts the chance of a compound to have at least 10% oral bioavailability in rat or measurable caco-2 cell line permeability experiment using a model for human intestinal absorption of drugs drug-likeness scores were also calculated by considering (alogp, tpsa, natoms, non, noh/nh, rotb& mw) based on lipinski’s rule for the prediction of bioactivity score. the results of these prediction showed that the compound obeyed lipinski’s rule. this semi-quantitative rule-based score defines the compounds into four probability score classes i.e. 11%, 17%, 55% and 85%. the acceptable probability score is 55% which indicates that it passed the rule of five. further, synthetic accessibility was assessed to quantify the complexity of the molecular structure. the results showed that the score 3.00 revealed that the compounds does not have complex synthetic route [22]. pruthviraj et al. synthesis and biopotency evaluation of 1-(4,5-diphenyl-1h-imidazol-2-yl)naphthalen-2-ol online first indian j pharm drug studies | 3 table 3: in-silico adme properties comp mw alog p nhba nhbd nrb tpsa (å) nviolations 3 362.42 4.01 2 2 3 48.91 0 bioactivity score synthetic accessibility skin sensitization bbb score 0.55 (55%) 3.00 nil 0.044 bbb+ nhba: hydrogen bond acceptor, nhbd: hydrogen bond donor, mw: molecular weight, alogp: logarithm of partition b/w noctanol and water, nrb: no. rotatable bonds, tpsa: topological polar surface area. *mw < 500 dalton, *alog p < 5 *nhba<5 *nhbd< 5, *nrb< 10, * tpsa b/w 40-130 molecular docking studies & computational analysis: the molecule structures were generated based on spectral data multi nuclear nmr (1h & 13c) and mass spectrometry. these structures were drawn in marvin js software and they were cleaned & orientation to 3d. all these molecule structures were prepared in auto dock 4.2 software and exported into pdb file format. the crystal structure of anti-fungal protein target candida albicans nmyristoyltransferase (pdb id: 1iyl), and anti-bacterial protein target – c (30) carotenoid dehydrosqualene synthase from staphylococcus aureus (pdb id: 3acx), were retrieved from protein data bank. these protein structures prepared by removing the water molecules and small molecules in the complex.the molecular docking was performed in auto dock vina 1.1.2 software to evaluate the binding affinity of the synthesized molecules with the protein targets. the kollman charge was added to the protein residues and lamarckian genetic algorithm was incorporated as scoring function. the grid box size for each protein was set significantly to cover the active site residues were standard drug bound. the default values were taken for all other parameters. based on docking the top 10 poses will be generated and ranked based on binding energy.docking results are listed in (table 4) and 2d binding pattern / poses of compound is shown in (figure 1, 2) extracted using schrodinger visualizer, it’s found to be 1-(4,5-diphenyl-1h-imidazol2-yl)naphthalen-2-ol (3) is nearly as potent as other standard drugs with a minute difference in binding score [23-25]. table 4: binding / docking energy comp code pdb id role binding score (kcal/mol) no of interactions interactive residues 3 1iyl anti-fungal protein target -8.3 21 leu 415, glu 109, val 108, tyr 107, gly 212, thr 211, phe 176, phe 117, thr 119, val 449, leu 450, leu 451, leu 357, tyr 335, tyr 225, tyr 354, leu 394, gln 226, hid 227, cys 393, asn 392. clt -9.4 3 3acx anti-bacterial protein target -8.8 19 phe 22, leu 164, gln 165, tyr 248, asn 168, arg 171, asp 172, glu 175, asp 176, arg 265, tyr 129, tyr 183, his 18, tyr 41, cys 44, val 137, arg 45, asp 48 cpfx -9.6 figure 1: 2d docking poses / binding patterns with 3ac pruthviraj et al. synthesis and biopotency evaluation of 1-(4,5-diphenyl-1h-imidazol-2-yl)naphthalen-2-ol online first indian j pharm drug studies | 4 figure 2: 2d docking poses / binding patterns with 1iyl dft analysis: the theoretical calculations using density functional theory (dft) have been utilized to study molecular properties like charge analysis, reduced density gradient (rdg) analysis, along with molecular electrostatic potential surface analysis gives a clear understanding of the structure of the molecule [26-27]. the molecular orbital energies and electrostatic potential of the molecule were calculated in the ground state using dft. the reduced density gradient and global descriptors such as chemical potential, electronegativity, hardness, softness, and electrophilicity index were studied [28-29]. theoretical calculations: the becke’s three parameter hybrid functional (b3) for the exchange part and the lee-young-parr (lyp) correlation function at 6-31g(d,p) is used to perform the density functional theory calculations using gamess-us software [30]. all dft calculations were performed in the gas phase only. the required input for the gamess software was generated using avogadro [31]. the same parameters were used for the optimization structure and to calculate electronic properties. the surface potential and rdg were generated using multiwfn-3.8 [32], and visualized using visual molecular dynamics (vmd) software [33]. frontier molecular orbital (homo-lumo) analysis and chemical reactivity indices: the frontier molecular orbitals (fmo) analysis is very helpful in understanding the nature of orbitals involved in chemical reactions. the fmo energy level of the compounds was computed using the dft method at b3lyp/6-31g(d,p) level of theory in the gas phase. the surface of some important fmo’s along with meps and rdgis shown in (figure 3). the energy gap between the highest occupied molecular orbital (homo) and lowest unoccupied molecular orbital (lumo) was calculated found to be 9.969ev indicating its stability, further promoting high temperature conditions or acidic media for the reactiveness. the chemical reactivity parameters like chemical hardness (η), electronegativity (χ), electronic chemical potential (μ), and electrophilicity index (ω) were also calculated. the chemical hardness is given by η=(elumo-ehomo)/2 is connected with the stability and reactivity of a chemical system [34]. the electronegativity is defined as the ability to attract electrons towards it and is given by the expression χ=-(ehomo+elumo)/2 found to be -0.1967 which is helpful in transport of electrons which is further supported by presence of aromatic rings. the negative of the electronegativity of a molecule is determined by using an equation μ=(ehomo+elumo)/2 is known as chemical potential. parr has introduced the electrophilicity index (ω), is calculated using the electronic chemical potential and chemical hardness from the equation ω=μ2/2η. the calculated values of chemical reactivity parameters are listed in (table 5). table 5: the calculated values of chemical reactivity parameters ehomo (ev) elumo (ev) energy gap (δ) (ev) ionization energy (i) (ev) electron affinity (a) (ev) -5.1814 4.788 9.969 5.1814 -4.788 electronegativity (χ) (ev) chemical potential (μ) (ev) global hardness (η) (ev) global softness (s) (ev-1) -0.1967 0.1967 4.9847 0.200 discussion the synthesized organic derivative via environmentally benign approach found to be potent biological agent exhibiting zoi of 10mm, 10mm, 11mm & 13mm against four different strain of bacteria’s k aerogenes e coli p desmolyticm s aureus respectively and two different fungi strainst c albicans a.flavus clt-1iyl 3-1iyl pruthviraj et al. synthesis and biopotency evaluation of 1-(4,5-diphenyl-1h-imidazol-2-yl)naphthalen-2-ol online first indian j pharm drug studies | 5 found to be susceptible witth the zoi of 11mm and 14mm respectively at 5μg/50μl concentraion in comprison with the statnadrd drug, the evaluation of preliminary adme properties foud to be compound is obeying the lipinski’s rule of five and in-vitro data are further supported with the in silico molecular docking analysis, obtained binding enegy values are supportig the in-vitro data. the dft anaylsis established the molecular paramerters. conclusion in this work, we have successfully prepared 1-(4,5-diphenyl-1himidazol-2-yl) naphthalen-2-olimidazole (3) a versatile asymmetric building blockusing i2 as catalyst. in-vitro antimicrobial activity followed by computer aided drug designing involving admetox and molecular docking studies were performed to establish the pharmaceutical activity and the physiochemical parameters were calculated using dft analysis. in this paper we discuss the synthesis of of 1-(4,5-diphenyl-1himidazol-2-yl)naphthalen-2-ol (3) a versatile building block for assymetric catalyst was synthesized using modified method reported by the only available synthetic report, further the compound was evaluated for its pharmacological potency against bacterial and fungi strains. additionally, computer aided drug discovery approaches like adme, and molecular docking studies were carried out against 2 proteins. results were compared against extensively used antibacterial,and antifungal standard drugs ciprofloxacin (cpfx), clotrimazole (clt) respectively. finally, the synthesized title compound was subjected to dft analysis to establish physicochemical parameters. figure 3: homo-lumo energy gap of compound along with electrostatic potential diagram and rdg references 1. pouria shirvani, afshin fassihi, lotfollah saghaie, et al. synthesis, anti-hiv-1 and antiproliferative evaluation of novel 4nitroimidazole derivatives combined with 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et al. electronegativity: the density functional viewpoint. phys rev sec. 1964; 136:864. how to cite this article: pruthviraj k, chethan bs, lohith tn, dineshbabu ns, chandrakumar r, mohan kumar, shivaraja g, sunil k. synthesis, evaluation of antimicrobial activity, and dft analysis of 1-(4,5-diphenyl-1h-imidazol-2-yl) naphthalen-2-ol. indian j pharm drug studies. 2024; online first. funding: none; conflicts of interest: none stated https://doi.org/10.1016/j.cdc.2020.100452 savvaser et al. anxiolytic effects of phyllanthus niruri leaf extract in mice online first indian j pharm drug studies | 1 original article evaluation of anxiolytic activity of ethanolic extract of phyllanthus niruri leaves in mice spandana savvaser1, rakshitha poojary1, soubiya mahnoor habibullah1, ashoka shenoy m2 from, 1student, 2associate professor, department of pharmacology, srinivas college of pharmacy, mangalore, india abstract anxiety has become one of the most prevalent and widely experienced mental illnesses, affecting 7-30% of the world’s population. it is characterized as a distressing psychological state of mind, which is represented by internal feeling of uneasiness, stress and agony. various theories, including psychodynamic, psychoanalytic, and genetic perspectives, have been proposed to explain the etiology of anxiety disorders. certain drugs used for treatment of anxiety have side effects and limitation such as sedation, central complexity, and habituation and during withdrawal of drug. researchers are actively exploring natural remedies, especially medicinal plants, to address anxiety disorders and minimize potential side effects associated with conventional drugs. hence in the present study we investigate the anxiolytic activity of the plant phyllanthus niruri leaves in mice using various experimental models such as elevated plus maze and light and dark chamber. in the elevated plus maze (epm) model, parameters measured included the number of entries in open and closed arms, along with the time spent in each. in the light and dark chamber, measurements encompassed the number of crossings between light and dark sides recorded for 10 minutes, along with the time spent in each side. the administration of the different doses of the plant extract produced significant difference in the locomotor activity of the mice. key words: anxiety, phyllanthus niruri leaves, epm, light and dark chamber. nxiety is defined as a cognitive emotional response characterized by anatomical activation, typically of the autonomic nervous system.[1] when the manifestation of anxiety significantly disrupts daily life, it is referred to as an anxiety disorder.[2] some symptoms of anxiety are panic attacks, fear, sweating, elevated blood pressure, stress, tension, tremor and vomiting.[3] according to freud theory, anxiety is an unpleasant state of mind. he also mentioned the variability between objective anxiety and neurotic anxiety, primarily by considering whether the origin of the danger stemmed from external circumstances or internal impulses. objective anxiety involves an intricate internal response to the anticipation of injury or harm stemming from an external threat. neurotic anxiety is characterized by feelings of apprehension and heightened physiological alertness.[4] if anxiety is detected early they can be treated as well as the symptoms can be minimized and will provide quality to one’s life.[5] anxiety can emerge as a significant concern for individuals of all ages, including both adults and children. studies have proved that women tend to experience anxiety more frequently than men.[6] to avoid the occurrence of anxiety, drugs like benzodiazepines, serotonin reuptake access this article online received – 09th july 2024 initial review – 28th september 2024 accepted – 03rd october 2024 quick response code inhibitors (ssri’s) serotonin norepinephrine reuptake inhibitors (snri) are the first line drugs that are prescribed for treatment of anxiety. tricyclic antidepressants, monoamine oxidase inhibitor (phenelzine), antihistamines (hydroxyzine), anti-seizure drugs (gabapentin) are mostly prescribed as a second line treatment. injecting this drug alters neurotransmitter levels like serotonin or gaba.[7] prevention of anxiety disorder at early stage is necessarily important.[8] the etiology of anxiety mainly involves psychological factors such as childhood trauma or stressful past events; genetic factors also come into the considerations; physiological factors including alterations in serotonergic and catecholaminergic systems of the body play a role in causing of anxiety.[9] due to the trends seen in those years, several authors called the twentieth century as "the age of anxiety". this description suggests that contemporary life has led to increased anxiety levels. in recent years, individuals appear to be experiencing higher levels of anxiety, with concerns about safety, social acceptance, and job security weighing more heavily on their minds than in previous times.[10] according to the world health organization (who), approximately 450 million people worldwide suffer from anxiety. the current global prevalence rate of anxiety disorders among individuals ________________________________________________ correspondence to: ashoka shenoy m, department of pharmacology, srinivas college of pharmacy, mangalore, india email: shenoyscp@gmail.com a mailto:shenoyscp@gmail.com savvaser et al. anxiolytic effects of phyllanthus niruri leaf extract in mice online first indian j pharm drug studies | 2 aged 18 years and older is 18.1%.[11] approximately oneeighth of the total population is affected by anxiety disorders, which is a heterogeneous type of condition.[12] the anxiety disorders are mainly classified as panic disorder, generalized anxiety disorder, post-traumatic stress disorder, obsessive– compulsive disorder, social phobia, and specific phobias.[13] among anxiety disorders, panic disorder (10.3%), social phobia (2.7%), and generalized anxiety disorder (2.2%) are particularly common.[14] the symptoms and the disorders following anxiety can be due to the impaired regulation of the central nervous system. these physical and emotional symptoms of this dysfunction are due to increased sympathetic stimulation at varying levels. anxiety is the result of an imbalance or abnormal functioning of the neurotransmitters in the body. most commonly, serotonergic and noradrenergic neurotransmitters play a prominent role in the modulatory steps involved. disruption of the gaba system is also one of the actions in the physiology of the body due to anxiety. it is also seen that the corticosteroid level may increase or decrease the activity of various pathways in the brain.[15] plants are said to be rich source of biomolecule which has vast therapeutic uses to cure various illnesses.[16] the synthetic drugs and medications contain multiple side effects; hence the herbal drugs that possess variety of therapeutic values are used in the treatment of anxiety which shows few adverse reactions in the body. for preparation of anxiolytic and antidepressant drugs from plant source requires multidisciplinary approach including ethanopharmacological surveys, careful examination of the folkloric uses of the plant, as well as phytochemical and pharmacological studies.[17] phyllanthus niruri belongs to the family phyllanthaceae.[18] it is a herb that is seen growing in the tropical and subtropical regions of asia, america, and china. phyllanthus niruri is an annual herb that thrives in the wild, following the initial monsoon rains in regions such as jharkhand, bihar, chhattisgarh, and other indian states. there have been reports that this plant also grows habitually in the coastal regions.[19] it is an erect annual herb which grows up to a height of 40-70 cm with ascending herbaceous branch. the leaves are numerous in number; green in colour; sub sessile in structure; they are arranges closely.[20] in the ayurvedic system of medicine in india, chinese traditional medicine, and indonesian medicine, phyllanthus niruri is utilized.[21] it is rich in bioactive compounds, including lignans (such as phyllanthin, hypophyllanthin, and niranthin), flavonoids, glycosides, tannins, alkaloids, ellagitannins, triterpenes, phenylpropanoids, steroids, ricinolic acid, niruriside, and phyltetralin.numerous reports have highlighted the anti-inflammatory, anti-viral, anti-cancer, and anxiolytic potential of various phyllanthus species.[22] among the various bioactive molecules identified from phyllanthus sources, niranthin, a lignan, has shown promise in managing anxiety disorders, primarily through its influence on gaba receptors.[23] chlorogenic acid obtained from p. niruri, 4sinapoyl quinic acid not only is a powerful oxidizing agent but also exhibits anti-inflammatory, anticancer and anti-anxiety properties.[22] materials experimental animals the experiment employed healthy swiss albino mice, weighing between 18 to 25 grams, of either sex, which was obtained from the animal facility at srinivas college of pharmacy in mangalore. these mice were kept in a controlled environment with a temperature of 22±2 degrees celsius, a relative humidity of 60±5%, and a 12-hour light/dark cycle. they were accommodated in clean polypropylene cages filled with sterile paddy husk as bedding material, and they were provided with unrestricted access to a standard pellet diet and water. all the animals were provided with humane care in accordance with the guidelines established in the "guide for the care and use of laboratory animals" which was developed by the ‘national academy of sciences’ and published by the ‘national institute of health’. prior to their involvement in the study, the animals were allowed a minimum of one week for acclimatization. furthermore, all procedures were conducted in compliance with the regulations set forth by the institutional animal ethics committee, as per the directives of the cpcsea (committee for the purpose of control and supervision of experiments on animals), under the ministry of animal welfare division in the government of india, located in new delhi, india. plant material the leaves of phyllanthus niruri belonging to family phyllanthaceae were collected from a local region of kundapura in udupi district on july of 2023. it was authenticated by dr. siddharaju mn, assistant professor and research guide, department of botany, university college, mangalore. chemicals chemical such as diazepam of pure analytical grade will be procured from e merck (india) ltd, mumbai and all other chemicals of analytical grade were procured from local suppliers. methodology preparation of ethanolic extract of phyllanthus niruri leaves the fresh leaves of phyllanthus niruri were collected locally during the season of monsoon. the collected material was further shade dried and powdered coarsely for the process of extraction. the powdered material was extracted by subjecting to cold maceration by using ethanol as solvent for 72 hours. in this procedure, 50g of the powder was soaked in 500ml of savvaser et al. anxiolytic effects of phyllanthus niruri leaf extract in mice online first indian j pharm drug studies | 3 ethanol, with continuous shaking using mechanical shaker. the resultant extract was filtered through whatman no.1 filter paper and the filtrate was subjected to dryness at 50° on water bath.[24] preparation of stock solution of the extract for dosing the ethanolic extract of phyllanthus niruri was weighed and dissolved in required quantity of distilled water.each time fresh preparation of the extract was prepared before administration. the extract was administered post orally at the constant volume of 100mg/kg and 200mg/kg for each animal.[25] experimental designs the swiss albino mice (22-27gms) of either sex were selected. the mice were divided into following groups (n=6) as follows: group i: vehicle control group ii: standard group (diazepam1mg/kg) (i.p) [26] group iii: ethanolic extract of phyllanthus niruri leaves (100mg/kg) (p.o.) [25] group iv: ethanolic extract of phyllanthus niruri leaves (200mg/kg) (p.o.) [25] the treatment of the plant extract was given through oral route. all animals were pretreated for 20 days except diazepam treated animals. on 21st day, animals was treated, 30 min before the evaluation. experimental models 1. elevated plus maze: principlethis model of anxiety has been extensively used for evaluation of novel anxiolytic agents and to investigate psychological and neurochemical basis of anxiety. this test has been proposed for selective identification of anxiolytic and anxiogenic drugs. anxiolytic compounds, by decreasing anxiety, increase the open arm exploration time; anxiogenic compounds have the opposite effect. procedureprior to starting the experiment, the mice was handled daily to reduce stress. two hours after the oral administration of the test drugs and 30 min after the intraperitoneal administration of diazepam, the animal was placed in the center of the maze, facing one of the open arms. thereafter, the results were recorded during the next 5 min. an arm entry being defined when all four paws are in the arm. following parameters measured: 1. number of open and closed arm entries. 2. percentage time spent in open and closed arm. at the end of each trial the apparatus was wiped clean in order to eliminate any olfactory clues, which might modify the behavior of next animal. the procedure was conducted preferably in a sound attenuated room, with observations made from an adjacent room via web camera attached to the computer system.[27] 2. light and dark model: principlecrawely and goodwin (1980) crawley (1981) described a simple behavior model in mice to detect compounds with anxiolytic effects. in a two chambered system, where the animals can freely move between a brightly-lit open field and a dark corner. mice tends to explore a novel environment but toretreat from the aversive properties of a brightly-lit open field and a dark corner, they show more crossings between the two chambers and more locomotor activity after treatment with anxiolytic. the number of crossings between the light and dark sites is recorded. proceduremovements through the partition and the time spent in the dark and light chamber were counted. mice were placed into the cage. the animals were treated 30 min before the experiment with test drugs or vehicle intra-peritoneally and then observed for 10 min, groups of 3 animals are used for each dose. the following behavioral were measured: 1) the number of entries in dark and light chamber. 2) time spent in minutes in dark and light chambers. the procedure was conducted preferably in a sound attenuated room, with observations made from an adjacent room via web camera attached to the computer system.[27] statistical analysis all data were expressed as mean ± sem. the statistical significance between groups were compared using one way anova, followed by dunnett’s multiple comparison test. for all tests a "p" value of 0.05 or less was considered for statistical significance. results phytochemical screening phytochemical analysis is a pivotal aspect of a research, delves into the examination of plant-derived compounds. by the phytochemical screening of the ethanolic extract of phyllanthus niruri leaves (eepnl) it was identified that the extract consists of alkaloids, phenolic acids, tannins, flavonoids, saponins, and glycosides which helps understand the pharmacological activities of the plant and helps paving the way for the development of novel drugs, nutritional supplements. anxiolytic effects in epm (table 1), animals treated with two doses of eepnl (100mg/kg and 200 mg/kg) showed increase in the time spent at open arm of the elevated plus maze model which was significant (33.167±1.537; p<0.05 and 38.0±0.931; p<0.01) when compared with control (22.0±3.512). similarly, animals savvaser et al. anxiolytic effects of phyllanthus niruri leaf extract in mice online first indian j pharm drug studies | 4 treated with diazepam (1mg/kg), as expected, showed a significantincrease in the time spent at open arm of the elevated plus maze model (75.0±3.651;p<0.001) animals treated with two doses (100mg/kg and 200 mg/kg) also showed decrease in the time spent at closed arm of the elevated plus maze model which was significant (109.0±16.73; p<0.01 and 76.0±11.590; p<0.001) when compared with control (173.33±8.33). similarly, animals treated with diazepam (1 mg/kg), as expected, showed a significant increase in the time spent at open arm of the elevated plus maze model (72.00±3.425; p<0.001). animals treated with two doses showed decrease in the number of entries in closed arm of the elevated plus maze model which was significant (6.833±0.60; p<0.01 and 6.167±0.307; p<0.001) when compared with control (11.0±1.211). similarly, animals treated with diazepam (1mg/kg), as expected, showed a significant decrease in number of entries at open arm of the elevated plus maze model (5.5±0.764; p<0.001). animals also showed increase in the number of entries in open arm of the elevated plus maze model which was significant (7.83±0.477; p<0.01 and 8.83±1.249; p<0.001) when compared to control (4.0 ±0.516). similarly, animals treated with diazepam (1mg/kg), as expected, showed a significant decrease in number of entries at open arm of elevated plus maze model(9.5±0.428; p<0.001). fig. 1: comparative profile of number of entries to open and closed arm in epm after oral administration of 100mg/kg and 200mg/kg of eepnl. in ldt (table 2) animals treated with two doses of eepnl (100mg/kg and 200mg kg) showed reduced time spent in dark chamber (4.317±0.303 and 2.1±0.230;p<0.01) and with concomitant increase in time spent in light chamber (1.517±0.166; p<0.01 and 1.86±0.051; p<0.001) when compared with controls (4.633±0.704 and 0.650±0.141). similarly, animals treated with diazepam (1mg/kg) as expected showed reduced the time spent in dark chamber with concomitant increase in time in light chamber (1.2±0.435; p<0.001 and 3.833±0.219; p<0.001) respectively. fig. 2: comparative profile of time spent in open and closed arm in epm after oral administration of 100mg/kg and 200mg/kg of eepnl. fig. 3: comparative profile of number of entries to light and dark chamber in ldc after oral administration of 100mg/kg and 200mg/kg of eepnl. fig. 4: comparative profile of time spent in light and dark chamber in ldc after oral administration of 100mg/kg and 200mg/kg of eepnl. savvaser et al. anxiolytic effects of phyllanthus niruri leaf extract in mice online first indian j pharm drug studies | 5 all animals treated two doses of eepnl (table 10) showed increased number of entries in dark chamber (8.167±.792; p<0.05 and 7.5±0.764; p<0.001) and with increase in number of entries in time in light chamber (10.0±1.15;p<0.01 and 12.33±0.843;p<0.001) when compared with controls (12.0±0.577and 5.5±0.764) respectively. similarly, animals treated with diazepam (1 mg/kg) as expected showed increased number of entries in both dark chamber and light chamber (3.5±0.342; p<0.001 and 15.0±1.1414; p<0.001) respectively. table 1: effect of eepnl on elevated plus maze in mice table 2: effect of eepnl on light dark transition model in mice discussion anxiety encompasses various dimensions, such as cognitive, emotional, physical, and behavioral components. physiological reactions are typically normal and adaptive, but, they can become exaggerated to the extent that they jeopardize an individual's psychosocial well-being.[28] benzodiazepines have been extensively used for the last 40 years to treat several forms of anxiety, but due to their unwanted side effects, alternative treatment strategies with favorable sideeffect profiles, credible benefits and moderate costs are of interest. thus, there is a need to investigate alternatives.[29]in the present study, the anxiolytic potential of phyllanthus niruri was determined using various experimental models. the behavioral models employed for this study were elevated plus maze and light and dark test. the epm is considered to be an etiologically valid animal model of anxiety uses natural stimuli, such as a fear of a new, brightly-lit open space and the fear of balancing on a relatively narrow raised platform, moreover it is known that anxiolytic agent increases the frequency of entries and time spent in open arm of the epm.[29] light-dark exploration test titrates a natural tendency of mice to explore a novel environment, against the aversive properties of a brightly lit compartment. some of the measurable indices of anxiety are number of crossing between chambers, locomotor activity, number of rearing and amount of time spent in the dark area of the apparatus.[30] in the present study it is noted that administration of eepnl (100mg/kg and 200mg/kg) showed dose dependent increase in the time spent in the open arms and the number of entries into open arms when compared with the control. the present study also showed that eepnl (100 mg/kg and 200 mg/kg) increased the time spent in the light area and the no. of entries in to the light suggesting again that eepnl possesses anxiolytic properties. earlier reports on the chemical constituents of plants and their pharmacology suggest that plant contains alkaloids, flavonoids, tannins, phenolic compounds and saponins which sl.no drug treatment dose no. of entries (mean±sem) time spent in seconds (mean±sem) open arm closed arm open arm closed arm 1 control vehicle 4.0 ±0.516 11.0±1.211 22.0±3.512 173.33±8.33 2 standard diazepam 9.5±0.428*** 5.5±0.764*** 75.0±3.651*** 72.00±3.425*** 3 low dose 100mg/kg 7.83±0.477** 6.833±0.601** 33.167±1.537* 109.0±16.73** 4 high dose 200mg/kg 8.83±1.249*** 6.167±0.307*** 38.0±0.931** 76.0±11.590*** values aremean±sem for n=6, expressed as the time (in sec) of 6 animals in each group. data analysis was performed using dunnet’s test. *p<0.05, **p<0.01, ***p<0.001 v/s control sl.no drug treatment dose no. of entries (mean±sem) time spent in min (mean±sem) light dark light dark 1 control vehicle 5.5±0.764 12.0±0.577 0.650±0.141 4.633±0.704 2 standard diazepam 15.0±1.1414*** 3.5±0.342*** 3.833±0.219*** 1.2±0.435*** 3 low dose 100mg/kg 10.0±1.15** 8.167±.792* 1.517±0.166** 4.317±0.303 ns 4 high dose 200mg/kg 12.33±0.843*** 7.5±0.764*** 1.86±0.051*** 2.1±0.230** values aremean±sem for n=6, expressed as the time (in min) of 6 animals in each group. data analysis was performed using dunnet’s test. *p<0.05, **p<0.01, ***p<0.001 v/s control savvaser et al. anxiolytic effects of phyllanthus niruri leaf extract in mice online first indian j pharm drug studies | 6 are active against anxiety and many cns disorders in the body. phyllanthus niruri shows anxiolytic property due to the presence of phenolic compounds and tannins such as chlorogenic acid and niranthin.[22] the phytochemical gallic acid shows anxiolytic activity primarily mediated by the 5ht1 a but not bzd receptors.[31] flavonoids being structural analogues of benzodiazepines bind to gabaa receptor and pharmacologically act as a partial agonist.[29] the effects of eepnl on epm and lightdark test were almost equivalent to that of 1mg/kg diazepam. in the present study, the anxiolytic activity of phyllanthus niruri extract was observed at the dose of 200mg/kg in mice. these observations clearly indicate the phyllanthus niruri exerts anxiolytic activity. it is possible that the mechanism of anxiolytic action of eepnl could be mediated by synergistic action of these phytochemical. the results obtained in the study suggest that the ethanolic extract of phyllanthus niruri possesses anxiolytic property. thus, of eepnl has potential clinic application in the management of anxiety disorder. future investigations are warranted for elucidating the extract mechanism and bioactive compounds. conclusion the present study can be concluded with the fact that ethanolic extract of phyllanthus niruri reveals significant anxiolytic effect in swiss albino mice using animal models of anxiety namely elevated plus maze, light and dark chamber. the data obtained was satisfactory and conclusive so as to achieve our objectives. in the conclusion, present data indicate the administration of eepnl to mice shown anxiolytic activity supporting the folk information regarding anxiolytic activity of eepnl. the extract and preliminary phytochemical studies of eepnl revealed the presence of chemical and phytochemical constituents such as (chlorogenic acid and niranthin), alkaloid, tannins, flavonoid, phenolic compound and saponnins. the exact mechanism underlying anxiolytic activity is not clear but it may be apparently related to active compounds present in the extract. hence further studies would be necessary to evaluate the contribution of active chemical constituents for the observed anxiolytic activity. acknowledgements the authors are grateful to management of srinivas college of pharmacy, mangalore for providing necessary facilities to carry out the experiments and a.shama rao foundation, mangalore for providing 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indian j pharm drug studies. 2024; online first. funding: none; conflicts of interest: none stated bhat & shanbhag biomaterials: revolutionizing cancer immunotherapy and genomics vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 37 review article emerging trends in biomaterials for cancer immunotherapy and genome editing: a comprehensive review ramdas bhat1, preeti shanbhag2 from, 1associate professor, 2pg scholar, department of pharmacology, srinivas college of pharmacy, valachil, post farangipete, mangalore, karnataka, india. abstract this detailed review navigates the dynamic landscape of cancer therapeutics, shifting focus from conventional approaches to the promising frontiers of immunotherapy and genome editing. biomaterials, including nanoparticles and hydrogels, are scrutinized for their role in enhancing therapeutic efficacy across various cancer types. breakthroughs in biomaterial-based interventions are emphasized, with a meticulous examination of critical methodological considerations, such as safety, biocompatibility, and delivery efficiency, laying the groundwork for future research.the narrative emphasizes the need for standardization and regulatory compliance to ensure the safety and reliability of emerging therapies moving towards clinical applications. implications for theory, practice, and future research highlight the potential of biomaterial-based approaches to offer safer and more effective cancer treatments. the integration of personalized medicine is advocated, emphasizing tailored interventions based on individual patient profiles.the review underscores the multidisciplinary collaboration across materials science, immunology, and oncology as essential for a holistic understanding and effective development of biomaterial-based therapies. overall, this review contributes to the ongoing shift towards safer, more efficacious, and personalized cancer therapeutics by exploring innovative avenues in immunotherapy and genome editing, while emphasizing the need for a methodologically rigorous approach in translating these advancements to clinical practice. key words: cancer immunotherapy, precision genome editing, biomaterials, crispr-cas9, combination therapies. ancer remains one of the most formidable challenges in modern medicine, affecting millions of lives worldwide each year [1]. conventional therapies, such as chemotherapy and radiation, have made significant strides in treating certain types of cancer [2]. however, their limitations, such as off-target effects and drug resistance, have underscored the need for novel therapeutic approaches [3]. in recent years, cancer immunotherapy [4] and precision genome editing [5] have emerged as promising fields that hold the potential to revolutionize cancer treatment. the landscape of oncology has undergone a transformative shift with the integration of immune system harnessing and genome editing technologies, notably the revolutionary crispr-cas9 system [6]. three primary facets define this paradigm shift in cancer treatment. immune checkpoint inhibitors, such as nivolumab, access this article online received – 31st jan 2024 initial review – 27th feb 2024 accepted – 15th feb 2024 quick response code pembrolizumab, atezolizumab, durvalumab (imfinzi), ipilimumab (yervoy), and cemiplimab (libtayo), have demonstrated success in treating various cancers, including bladder cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, melanoma, renal cell carcinoma, and advanced cervical cancer, enhancing overall survival rates and response durability [7]. adoptive t cell therapies, exemplified by chimeric antigen receptor (car) t-cell therapy, represent a groundbreaking approach. examples such as daliyno (tilmesogenecel), breyanzi (lisocabtagenedarolumab), and tecartus (breceltinib) utilize a patient's own tumor-infiltrating lymphocytes (tils) or engineered car t-cells to precisely target melanoma, large b-cell lymphoma, and mantle cell lymphoma [8].cancer vaccines, including gvax (rasilmuplasmid), papillomavirus vaccines (gardasil, cervarix), and personalized neoantigen vaccines, stimulate the immune system to identify and eliminate cancer cells, showcasing a personalized treatment approach [9]. __________________________________________________ correspondence to: ramdas bhat, associate professor, dept. of pharmacology, srinivas college of pharmacy, valachil, post farangipete, mangalore, karnataka, india-574143. email: ramdas21@gmail.com, tel.: +91 7795772463. c mailto:ramdas21@gmail.com bhat & shanbhag biomaterials: revolutionizing cancer immunotherapy and genomics vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 38 in the realm of genome editing, the crispr/cas-9 system stands out as a powerful tool, exhibiting promise in correcting mutations and restraining tumor growth in cancer research and treatment. additionally, zinc finger nucleases (zfns), transcription activator-like effector nucleases (talens), base editing, and hdr (homology-directed repair) join the ranks of precision genome editing technologies, expanding the toolkit with their unique capabilities [10-13]. these advancements collectively hold immense potential in advancing cancer therapies, ushering in a new era of personalized and targeted interventions for improved patient outcomes.in cancer, the potential to target and correct genetic mutations implicated in tumor development offers unprecedented opportunities for precision medicine [14]. however, successful cancer immunotherapy and genome editing demand efficient and controlled delivery systems to ensure therapeutic agents reach their targets with minimal side effects [15]. this is where cutting-edge biomaterials play a pivotal role [16]. biomaterials have emerged as essential tools to optimize cancer immunotherapy and genome editing by providing tailored platforms for drug delivery, enhancing immune response, and improving gene editing efficiency [17]. in this review article, we explore the latest advancements in harnessing the power of biomaterials for cancer immunotherapy and precision genome editing [18]. we delve into various types of biomaterials, including nanoparticles [19], hydrogels [20], and viral vectors [21], and their applications in enhancing therapeutic efficacy and minimizing off-target effects. additionally, we discuss the challenges and future directions in the development of biomaterial-based strategies to overcome obstacles faced in cancer treatment. method and materials in conducting this review on "emerging trends in biomaterials for cancer immunotherapy and genome editing," an extensive examination of available literature was carried out, focusing on the integration of immune system enhancement and genome editing technologies in cancer treatment. primary sources included reputable journals, scientific databases, and authoritative publications in oncology, immunotherapy, and genome editing. the search encompassed articles published up to january 2024 to ensure the inclusion of the latest advancements. the review strategically aimed to identify pivotal studies, clinical trials, and research papers elucidating the transformative impact of immune checkpoint inhibitors, adoptive t cell therapies, cancer vaccines, and genome editing tools, with a specific emphasis on the revolutionary crispr-cas9 system in the oncology landscape. meticulous curation of gathered information sought to present a comprehensive overview, emphasizing recent breakthroughs, emerging trends, and notable examples within each category. the narrative was enriched by incorporating specific examples related to lung cancer, breast cancer, and prostate cancer, providing a nuanced perspective on the application of these technologies across various cancer types. beyond academic sources, reports from reputable medical and scientific organizations, scrutiny of clinical trial databases, and exploration of official regulatory publications ensured a well-rounded and evidence-based approach. the synthesis of this information aimed to provide readers with a clear understanding of the current landscape, challenges faced, and future directions in the dynamic intersection of immune system modulation and genome editing, propelling advancements in cancer therapies. the methodology placed a premium on accuracy, relevance, and the inclusion of diverse perspectives, contributing meaningfully to the ongoing scholarly conversation in this transformative field of research. biomaterials for cancer immunotherapy 1. immune modulatory biomaterials: immune modulatory biomaterials are ingeniously designed to manipulate the immune response, creating a favorable microenvironment conducive to the activation of anti-tumor immune cells [17]. their pivotal role in elevating the efficacy of cancer immunotherapy is evident through their ability to deliver immune checkpoint inhibitors and immune-stimulating cytokines with precision. these biomaterials are meticulously engineered to release therapeutic agents in a sustained and controlled manner, ensuring a durable and precisely targeted immune response against the tumor[22, 23]. nanoparticles, operating at the nanoscale, stand as formidable carriers for immune modulatory agents within cancer immunotherapy [24]. laden with immune checkpoint inhibitors such as antipd-1 or anti-ctla-4 antibodies, effectively obstruct inhibitory signals, unleashing the potent activity of tumorspecific t cells [25, 26]. the controlled and gradual release of these antibodies from nanoparticles guarantees sustained immune activation, ultimately leading to profound tumor regression. on the other hand, hydrogels, intricate three-dimensional networks of crosslinked polymers, serve as exceptional vehicles for encapsulating and releasing immune-stimulating cytokines like interleukins (il-2, il-12) or interferon[27,28]. these hydrogels create a localized depot of these vital cytokines precisely at the tumor site, thus fostering the recruitment and activation of immune cells, thereby significantly amplifying the anti-tumor immune response. furthermore, the versatility of hydrogels allows for the engineering of physical support and protection for immune cells, enhancing their survival and functionality within the challenging tumor microenvironment [27]. these advancements in biomaterials hold great promise in revolutionizing the landscape of cancer immunotherapy, offering new avenues to combat cancer with increased precision and effectiveness. bhat & shanbhag biomaterials: revolutionizing cancer immunotherapy and genomics vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 39 2. cancer vaccines: biomaterial-based cancer vaccines have emerged as a compelling approach to augment the body immune response against cancer cells [28]. these innovative vaccines are designed to bolster antigen presentation and stimulate robust immune reactions targeting tumor-specific antigens. one avenue of this strategy involves utilizing biomaterials like liposomes, nanoparticles, and virus-like particles as carriers for tumor-specific antigens [29]. these biomaterial carriers serve a dual purpose by safeguarding the antigens from degradation and enhancing their uptake by antigen-presenting cells (apcs)[30]. this, in turn, facilitates the efficient presentation of these antigens to t cells, igniting a potent and highly specific t cell response directed against cancer cells [31]. additionally, biomaterial-based cancer vaccines can incorporate adjuvants, substances known to enhance immune responses to antigens. adjuvants like toll-like receptor (tlr) agonists and cytokines are commonly integrated into these vaccines [32]. these adjuvants play a critical role in further amplifying the activation of apcs and t cells. by creating a pro-inflammatory microenvironment, they assist in the recruitment and activation of immune cells, ultimately reinforcing the immune assault on cancer [33]. this multifaceted approach harnessing biomaterials and adjuvants holds substantial promise in reshaping the landscape of cancer immunotherapy, offering innovative strategies to combat cancer more effectively and precisely. 3. adoptive cell therapies: adoptive cell therapies (act), a groundbreaking approach in cancer treatment, involve the isolation and manipulation of a patient's own immune cells, primarily t cells, to target and eliminate cancer cells. notably, chimeric antigen receptor (car) t cell therapy has demonstrated remarkable success in specific cancer types [3437]. biomaterials play a pivotal role in the ex vivo engineering of car-t cells, functioning as delivery vehicles for car constructs and other genetic material into t cells [35,36]. this enables the t cells to express specific receptors (cars) designed to recognize and engage cancer antigens effectively. nanoparticles and viral vectors are frequently employed biomaterials, ensuring efficient gene delivery into t cells during this process. moreover, the challenges encountered by car-t cells upon reinfusion into the patient's body within the complex tumor microenvironment are substantial [35-37]. to address this, biomaterials, including hydrogels and scaffolds, can be custom-designed to provide physical support and essential nutrients to car-t cells. this support is crucial for their survival and persistence amidst the harsh conditions of the tumor microenvironment. additionally, these biomaterial-based scaffolds can function as reservoirs for cytokines and other immune-modulating agents, further enhancing the anti-tumor activity of car-t cells [34-37]. this multifaceted approach that combines the power of biomaterials with car-t cell therapy holds immense promise in advancing the field of cancer immunotherapy, offering novel strategies to combat cancer more effectively by equipping engineered immune cells with the tools they need to navigate and conquer the complex tumor landscape. figure 1. biomaterials for cancer immunotherapy. biomaterials for genome editing biomaterials have emerged as essential tools in the field of genome editing, particularly with the advent of crispr-cas9 technology. genome editing aims to precisely modify the dna sequence of living organisms, offering unprecedented potential for treating genetic diseases, understanding gene function, and advancing biotechnology. biomaterials play crucial roles in facilitating efficient and targeted delivery of crispr-cas9 components, protecting these components from degradation, and enhancing gene editing efficiency [12,13]. the various types of biomaterials used in genome editing are: 1. delivery of crispr-cas9 components: efficiently delivering the crispr-cas9 system to target cells or tissues is a central challenge in genome editing, and biomaterials play a pivotal role in overcoming this obstacle [38]. these versatile carriers encapsulate and protect essential crispr-cas9 components, including the cas9 protein or mrna and guide rna (grna) molecules, ensuring their successful delivery to the desired cellular destinations [39]. three commonly used biomaterials in this context are nanoparticles, liposomes, and viral vectors [40]. nanoparticles made from biocompatible materials, such as lipids or polymers, have shown remarkable efficacy as carriers for crispr-cas9 components. they bhat & shanbhag biomaterials: revolutionizing cancer immunotherapy and genomics vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 40 protect the cargo from degradation and allow for efficient uptake by target cells. due to the large molecular weight of the cas9 protein (approximately 4.5 kb in genetic size) and its low stability against serum enzymes and proteins, the entry of the cas9/sgrna or rnp complex into cells is challenging [41,42]. however, nanoparticle delivery systems, such as lipidbased nanoparticles and cationic polymer nanoparticles, have been developed to address these challenges. these delivery systems can be modified to target specific cell types, reducing off-target effects and improving gene editing precision. for instance, a dna nanocell[nc]-based delivery system has been shown to efficiently load the cas12a/crrna rnp [42, 43]. additionally, systemic nanoparticle delivery of crisprcas9 ribonucleoproteins has demonstrated effective tissuespecific genome editing. the development of nanoparticlebased technology for crispr-cas9 delivery holds promising prospects for clinical gene editing, as evidenced by the completion of the first crispr/cas9 clinical trial in 2016 [44]. furthermore, various studies have highlighted the potential of nanoparticle delivery systems for the efficient and targeted delivery of crispr-cas9 components. lipid nanoparticles, polymeric nanoparticles, solid-lipid nanoparticles, nanostructured lipid carriers, and niosomes have all shown great potential in the delivery of crispr compounds to target cells. additionally, polyamidoamineaptamer modified crispr/cas9 and sorafenib-loaded hollow mesoporous silica nanoparticles have exhibited targeted delivery of crispr/cas9 for precise gene editing [45]. 2. liposomes: liposomes, characterized by a lipid bilayer structure, play a pivotal role in genome editing, encapsulating various agents like nucleic acids and proteins. their targeted delivery, responsiveness to environmental cues, and versatility in crispr/cas9 applications make them indispensable. key aspects in crispr delivery include:  pegylation: enhancing efficiency, peg-modified liposomes optimize crispr delivery by improving pharmacokinetics and minimizing immune responses.  endosomal escape: facilitating the release of crisprcas9 cargo into the cellular cytoplasm, liposomes ensure effective genome editing.  stimuli-responsive design: tailored to environmental cues, liposomes provide spatial and temporal control over crispr cargo release.  targeting strategies: surface modifications enable precise delivery to specific cells or tissues, enhancing crispr/cas9 precision.  light-sensitive delivery: innovative light-sensitive liposomes, like those loaded with a photosensitizer, offer precise spatial and temporal control in crispr/cas9 gene editing. despite challenges like low transfection efficiency, the adaptability of liposome formulations allows customization for specific crispr requirements. noteworthy liposome types include:  eg-bearing liposomes: tailored with peg, these liposomes, like the ones carrying crispr components, enhance genome editing efficiency by improving pharmacokinetics and minimizing immune responses.  charged liposomes: positively or negatively charged liposomes, exemplified by those carrying crispr payloads, target specific cells, contributing to precise genetic modifications.  stimuli-responsive liposomes: engineered to respond to environmental cues, liposomes, such as those used in crispr delivery, ensure spatial and temporal control over cargo release, enhancing gene editing precision.  light-sensitive liposomes: innovations like lightsensitive liposomes loaded with crispr components provide high control in gene editing, allowing for flexibility and precision.  ad liposomes and cationic lipids: liposomes derived from ad liposomes, as well as those using cationic lipids like dotap and dlin-mc3-dma, showcase enhanced delivery efficiency in nucleic acid and crispr/cas9mediated gene editing. these liposomes optimize genomeediting efficiency by modulating endocytic pathways. [46,47]. 3. viral vectors: viral vectors, such as adeno-associated viruses (aavs) and lentiviruses, have a well-established history in gene therapy and are now integral to genome editing. these vectors deliver the crispr-cas9 system to target cells with remarkable efficiency and offer the advantage of providing long-term expression of cas9 and grna, making them particularly suitable for genetic diseases requiring sustained correction [42]. the use of viral vectors in gene therapy has seen significant progress, with nearly 70% of clinical trials utilizing viral vectors, highlighting their continued importance in the field. despite their successes, challenges still limit their full potential, and ongoing research aims to address these limitations. viral vectors have been employed for the treatment of various diseases, including metabolic, cardiovascular, muscular, hematologic, ophthalmologic, and infectious diseases, as well as different types of cancer [43,44]. for example, aav-based gene therapy has been used to treat spinal muscular atrophy, a rare genetic disease that causes muscle weakness and wasting. in this case, the aav vector was used to deliver a functional copy of the smn1 gene to motor neurons, resulting in improved motor function and survival in patients. another example is the use of lentiviral vectors in the treatment of hiv/aids. lentiviral vectors have been used to deliver functional copies of the ccr5 gene, which encodes a co-receptor for hiv, to cd4+ t cells. this approach has been shown to protect against hiv infection in animal models and is currently being tested in bhat & shanbhag biomaterials: revolutionizing cancer immunotherapy and genomics vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 41 clinical trials [48]. the development of nanoparticle systems to deliver the crispr-cas9 system to target cells has overcome obstacles such as the large molecular weight of the cas9 protein and its low stability against serum enzymes and proteins. additionally, the use of viral carrier systems has been shown to provide high efficiency in genome editing. while viral vectors have demonstrated significant promise, ongoing research and development are focused on addressing challenges and further improving their applicability. the field of gene therapy continues to see innovative modifications and support from the pharmaceutical and biotech industries, indicating a continued commitment to advancing viral vectorbased therapy. therefore, viral vectors, particularly aavs and lentiviruses, remain crucial tools in the landscape of gene therapy and genome editing, with ongoing efforts to enhance their efficacy and safety for clinical applications [46-48]. 4. gene editing in stem cells: stem cells hold immense potential for regenerative medicine and cell-based therapies stem cells possess the unique ability to self-renew and differentiate into various cell types, making them promising candidates for regenerative medicine and cell-based therapies [49]. precisely modifying their genes via crispr-cas9 unlocks their full therapeutic potential, and biomaterials play a crucial role in achieving this effectively. let's dive deeper into recent developmental examples across different delivery strategies: a. transfection reagents: lipid nanoparticles (lnps): recent advances involve lnps modified with cell-specific targeting ligands. for instance, researchers conjugated folate ligands to lnps for targeted delivery to pluripotent stem cells expressing folate receptors. this led to efficient gene editing with minimal off-target effects [48]. b. polymer-peptide hybrids: novel synthetic polymers with peptide conjugation offer enhanced cellular uptake and endosomal escape. a recent study employed chitosanbased polymers conjugated with cationic peptides for crispr-cas9 delivery to mesenchymal stem cells, achieving high editing efficiency with improved biocompatibility [48,49]. c. electroporation platforms:  microfluidic chips: researchers are now integrating microfluidic chips with temperature control to enhance cell viability during electroporation. a recent study used a temperature-controlled microfluidic chip for crisprcas9 delivery to neural stem cells, demonstrating improved cell survival and editing efficiency compared to conventional methods [50].  conductive hydrogels: development of hydrogels with specific electrical properties allows for localized, tissuespecific crispr-cas9 delivery. scientists developed a conductive hydrogel scaffold for in vivo delivery to cardiac stem cells residing within heart tissue. this approach facilitated targeted gene editing with minimal impact on surrounding tissues [49,50]. d. viral vectors:  engineered aav vectors: adeno-associated viral (aav) vectors are gaining popularity due to their safety and low immunogenicity. recent efforts focus on enhancing their targeting capabilities. researchers engineered aav vectors with stem cell-specific promoters, achieving efficient and selective gene editing in human embryonic stem cells without harming neighboring cell types [51].  hybrid viral vectors: combining different viral vectors leverages their unique strengths. a recent study used a hybrid vector combining aav and lentiviral vectors, achieving sustained and efficient gene editing in hematopoietic stem cells with minimal insertional mutagenesis [52]. e. in-vivo genome editing: in vivo genome editing holds immense promise for treating genetic disorders directly within the patient's body [40]. however, delivering crispr-cas9 components to target tissues or organs in a specific and efficient manner remains a significant challenge. innovative strategies are being developed to overcome these challenges and propel the field forward. f. targeted nanoparticles: these miniature cargo ships can be engineered to carry crispr-cas9 components and adorned with ligands that bind to receptors unique to specific tissues. this targeted delivery approach minimizes off-target effects and enhances overall efficiency. for example, researchers at mit crafted nanoparticles coated with folate ligands, precisely targeting receptors abundant on cancer cells, enabling gene editing specifically within tumors and laying the groundwork for personalized cancer therapies [41,42]. g. controlled-release scaffolds: these biomaterials function as custodians, encapsulating crispr-cas9 components and releasing them gradually over time. this controlled, sustained delivery mechanism ensures localized gene editing within the target area, amplifying the therapeutic impact. for instance, a biodegradable hydrogel scaffold loaded with crispr-cas9 was employed to treat leber's hereditary optic neuropathy, a challenging mitochondrial disease affecting the retina. this localized editing within the eye demonstrated improved vision in animal models, offering promise for conditions previously deemed untreatable [20,35,36]. bhat & shanbhag biomaterials: revolutionizing cancer immunotherapy and genomics vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 42 h. engineered viral vectors: repurposed viruses can be adeptly delivered into cells, with their capsids (outer shells) and promoters (genetic switches) meticulously engineered to target specific tissues. this targeted delivery strategy ensures that the editing machinery reaches its intended destination, minimizing risks and maximizing efficacy. for example, scientists modified an aav vector with a muscle-specific promoter, not only delivering crispr-cas9 but correcting a mutation causing duchenne muscular dystrophy in muscle cells. the result: significant improvements in muscle function observed in animal models [48,49]. i. beyond delivery: researchers are advancing high-fidelity cas9 enzymes and guide rnas with heightened specificity, reducing the risk of unintended edits. strategies involve sustained delivery systems or inducible editing approaches to enhance the persistence of gene editing. careful consideration of ethical implications, especially concerning germline editing and equitable access, is crucial for responsible development [20,28,52]. figure 2. in-vivo gene editing. challenges and future directions in biomaterials for cancer immunotherapy and genome editing. the development of biomaterial-based therapies presents a myriad of complex challenges that necessitate rigorous scientific investigation and innovative solutions [53]. foremost among these challenges is the imperative to ensure safety and biocompatibility. as these biomaterials interact intimately with the patient's immune system and biological processes, a comprehensive assessment of their biocompatibility is essential to mitigate the risk of adverse reactions or immune responses [17,54]. this extends to the imperative of long-term evaluation of the effects of biomaterials, especially when employed in the context of chronic treatments, where potential impacts must be thoroughly understood [17,53,54]. efficient delivery and precise targeting represent pivotal facets of therapeutic success. achieving optimal outcomes hinges on enhancing the delivery efficiency of biomaterials, particularly in the often-hostiletumor microenvironments [55]. innovative strategies must be devised to bolster the targeting specificity and improve tissue penetration, addressing the unique challenges presented by biomaterial-based therapies. the potential immunogenicity of certain biomaterials, notably viral vectors, poses a substantial concern, potentially limiting their effectiveness upon repeated administration [56,57]. therefore, it is imperative to explore avenues for mitigating immunogenicity without compromising therapeutic efficacy [57]. this calls for the development of novel biomaterials engineered to exhibit reduced immunogenic potential or the implementation of immune-evasion strategies to enhance their clinical applicability [58]. off-target effects, a notable challenge in gene editing therapies, where crispr-cas9 may inadvertently edit unintended genomic sites, necessitate continual refinement of the specificity and accuracy of crispr-cas9 systems. this ongoing pursuit aims to minimize off-target effects and bolster the safety profile of genome editing treatments [59]. practical considerations regarding manufacturing scalability and standardization come to the forefront. ensuring bhat & shanbhag biomaterials: revolutionizing cancer immunotherapy and genomics vol 3 | issue 2 | apr – jun 2024 indian j pharm drug studies | 43 reproducibility, quality, and alignment with regulatory standards is imperative to meet the burgeoning demand for clinical trials and future commercialization [60]. standardization of manufacturing processes and strict adherence to regulatory compliance are pivotal for the widespread adoption of biomaterial-based therapies [61]. moreover, the vision of personalized medicine, integral to both cancer immunotherapy and genome editing, requires the seamless integration of biomaterials with patient-specific genomic and immunological data [62]. realizing this ambition hinges on advancements in high-throughput sequencing and bioinformatics, which are poised to play instrumental roles in optimizing biomaterial-based therapies tailored to individual patient profiles [63]. combination therapies that harness the synergy between diverse biomaterials, immunotherapies, and gene editing strategies hold immense promise for enhancing cancer treatment. nevertheless, unravelling the intricacies of optimal combinations and understanding potential interactions between various biomaterials and therapies poses a multifaceted scientific challenge that demands meticulous exploration [64-66]. lastly, as biomaterial-based therapies transition from the realm of research to clinical trials, successfully navigating the complex regulatory landscape and securing approvals from regulatory agencies emerges as a pivotal step [64]. this necessitates collaborative efforts encompassing academic researchers, industry partners, and regulatory authorities to ensure the safe and efficient translation of these pioneering therapies into clinical practice [64,65]. conclusion the combination of biomaterials and breakthrough approaches such as cancer immunotherapy and genome editing are ushering cancer treatment into a new era. biomaterials, which serve as precision transporters for immune-modulating drugs, are meticulously engineered to negotiate the complicated tumor microenvironment. their goal, like that of architects, is to encapsulate and distribute therapeutic substances, thereby boosting the body's immunological response to cancer with precise targeting. this formulation ensures prolonged release for optimal treatment outcomes while also reducing systemic toxicity. despite these advances, obstacles remain, particularly in biocompatibility and delivery efficiency. researchers are actively overcoming these barriers in 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thompson ew, ostrikov k (ken). innovative precision gene-editing tools in personalized cancer medicine. adv. sci. 2020; 7(12). how to cite this article: ramdas bhat1, preeti shanbhag. emerging trends in biomaterials for cancer immunotherapy and genome editing: a comprehensive review. indian j pharm drug studies. 2024; 3(2):37-45. funding: none; conflicts of interest: none stated b.m thejaswini et al. pharmacological review on solanum nigrum vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 95 review article a review on pharmacological potential of solanum nigrum: pharmacological review on solanum nigrum b.m thejaswini1, satish s2, ramkrishna shabaraya a3 from 1pg scholar, 2professor, department of pharmacology, 3principal and head of the department of pharmaceutics, srinivas college of pharmacy valachil, farangipete post, mangalore, karnataka 574143. abstract solanum nigrum, commonly known as black nightshade, is a plant species that belongs to the family solanaceae, used in traditional medicine systems in various parts of the world for its potential pharmacological properties. the present study is an attempt to review the pharmacological potential of solanum nigrum. solanum nigrum contains several bioactive compounds, including phenolic compounds and flavonoids, which exhibit significant antioxidant activity. studies have demonstrated that solanum nigrum possess anti-inflammatory effects. solanum nigrum has shown promising anticancer activity in preclinical studies, its extracts have exhibited cytotoxic effects against various cancer cell lines, including lung, breast, colon, and liver cancers. the phytochemicals present in this plant are believed to induce apoptosis (cell death) and inhibit the growth of cancer cells. several studies have reported the antimicrobial potential of solanum nigrum against various pathogens, including bacteria, fungi, and viruses. solanum nigrum has been investigated for its hepato-protective properties. some research suggests that solanum nigrum may have anti-diabetic properties, reported to possess hypoglycaemic activity. while these studies highlight the potential pharmacological properties of solanum nigrum, it is important to note that further research is still needed to fully understand its mechanisms of action and evaluate its safety and efficacy in humans. key words: black nightshade, pharmacological potential, solanum nigrum, apoptosis. edicinal plants have been used by humans for centuries in folklore medicine [1]. more than 2000 species belonging to the genus solanum (solanaceae family) are found in tropical and subtropical areas of the world. solanum nigrum has been used traditionally to treat various ailments such as pain, inflammation fever and enteric diseases [2]. it is also used against sexually transmitted diseases [3]. throughout history, solanum nigrum has been used in traditional medicine systems in different cultures for its potential medicinal properties. the plant has been employed to treat various ailments, including inflammation, skin disorders, gastrointestinal issues, and respiratory conditions. it has also been used as a diuretic and to alleviate pain [4]. in addition to its historical use in traditional medicine, solanum nigrum has attracted scientific interest due to its access this article online received – 26th may 2023 initial review – 30th may 2023 accepted – 10th june 2023 quick response code phytochemical composition and potebtial pharmacological activities. the plant contains various bioactive compounds, including alkaloids, flavonoids, phenolic compounds, and glycoalkaloids such as solasonine and solamargine. these compounds are believed to contribute to the plant's pharmacological effects, including antioxidant, antiinflammatory, antimicrobial, anticancer, hepato-protective, and anti-diabetic activities [5]. solanum nigrumis a weed of nitrogen rich soils, amentaceous areas and gardens. an infusion of the plant is used as an enema in infants having abdominal upsets. it is a household remedy for anthrax pustules and is applied locally. freshly prepared extract of the plant is effective in the treatmentof cirrhosis of the liver, and also serves as an antidote to opium poisoning [6]. while solanum nigrum shows promise as a medicinal plant, it is important to note that further research is needed to fully understand its mechanisms of action, determine optima dosage, and assess its safety profile. ________________________________________ correspondence to: b.m thejaswini, pg scholar, department of pharmacology, srinivas college of pharmacy valachil, farangipete post, mangalore, karnataka, india. email: bm17thejaswini@gmail.com m mailto:bm17thejaswini@gmail.com b.m thejaswini et al. pharmacological review on solanum nigrum vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 96 vernacular names and taxonomical classification: solanum nigrum belongs to the family solanaceae with common names of black nightshade or garden night shadeis a plant species that belongs to plantae (kingdom}, magnoliophyta (division), magnoliopsida (class), solanales (order), solanum (genus), solanum nigrum (species). vernacular nameincludes black nightshade (english), ganikesopu (kannada), manatakkali (malayalam), makoya, kakamachi (hindi), kaakesoppu (tulu) [7, 8, 9]. the plant solanum nigrum l. (solanaceae), commonly known as ‘herbal mora’, is a common herb that grows wild and abundantly in open fields, whose fruit is used in mexico’s traditional medicine as a nervous tonic [10]. it is a little herbaceous plant that is now spread around the world. tiny white or purple flowers, black tiny berries, and dark green leaves are the distinguishing features of solanum nigrum. habitat and distribution: crop weed solanum nigrum is widespread. it is an annual plant with a brief lifespan reaching a height of 1.25m. these are mostly used as source of vegetables and fruits by harvesting the plants that emerge naturally as weeds in fields that have been cultivated, or in weed plant communities, in moist habitats, along fences and highways, in shaded regions, close to buildings, and on vacant ground. there are a few reports of garden huckleberries being grown for their fruits in north america [4, 9]. only a few countries fully cultivate these species. they are quite prevalent in india, mostly in the states of karnataka, odisha, and tamil nadu (nilgiris). they can be found by riverbanks, damp areas, fallow plains, and arable regions. the solanum nigrum plant is well adopted to the mediterranean region. it is continuously dispersed across india's eastern ghats, as well as in the southern and central areas, which have tropical wet climates [11]. botanical description: black nightshade is a short-lived herbaceous plant or small shrub. plants are about 0.25-1m tall [12]. the entire plant is covered with simple pubescent hairs which are angular and are coarsely pubescent on the stems [13]. it shows tap roots with few branches and numerous small lateral roots pale brown, easily peeled off exposing pale yellow wood. it also has erected glabrous or pubescent green, slightly woody unbranched stem [4]. leaves the leaves are ovate, the bases are cuneate, 4-10 and 3-7cm wide, pubescent, coarsely dentate, the apex is obtuse and shortly pointed [14]. the leaves are dull dark green, and toothless to slightly toothed on the margins [4]. the petiole is about 1-2cm long. the cuneate of the leaf is base wedge shaped to broad and descending to the petiole, with irregular wavy coarse teeth throughout or on each side and smoother soft, and hairy on both sides with five to six veins on both sides (figure 1) [12]. figure 1. solanum nigrum leaves [photo by; own work] phytochemicals in leaves: investigation of the solanum nigrum leaves reported that it contains the substances, such as alkaloid, flavonoids, tannins, saponins, glycosides, proteins, carbohydrates, coumarins & phytosterols. it has been found that solanum nigrum contains substances such as steroid alkaloid, steroidal saponins and glycoprotein [15]. solanum nigrum contains two quercetin glycosides namely, quercetin +.3-o-(2gal-_-rhamnosyl)-_-glucosyl (1_6)-_-galactoside and quercetin 3-o-_-rhamnosyl (l_2)-_galactoside. also, previously known quercetin 3-glucosyl (l_6) galactoside, 3-gentiobioside, 3 galactoside and 3glucoside, were also found [16]. solanum nigrum possesses numerous compounds that are responsible for pharmacological activities. its active components are glycoalkaloids, glycoproteins, and polysaccharides, polyphenolic compounds such as gallic acid, catechin, protocatechuic acid (pca), caffeic acid, epicatechin, rutin, and naringenin [17]. alkaloids: leaves contain alkaloids, including solanine and solanigrine. these alkaloids have been studied for their potential antimicrobial, anticancer, and anti-inflammatory activities [18]. flavonoids: leaves are rich in flavonoids, which are known for their antioxidant and anti-inflammatory properties. flavonoids found in the leaves include quercetin, kaempferol, and rutin [8]. phenolic compounds: the leaves also contain phenolic compounds, which contribute to their antioxidant activity. these compounds help scavenge free radicals and protect against oxidative stress [19]. vitamins: leaves are a good source of vitamins, including vitamin c, vitamin a, and vitamin e. these vitamins contribute to overall health and well-being [20]. b.m thejaswini et al. pharmacological review on solanum nigrum vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 97 minerals: leaves contain various minerals such as calcium, iron, and potassium, which are essential for proper bodily functions [20]. saponins: saponins are another class of phytochemicals found in solanum nigrum leaves. these compounds have been studied for their potential antifungal, antimicrobial and anti-inflammatory activities [21]. the leaves of solanum nigrum have been traditionally used for various medicinal purposes. following is some of the reported medicinal uses of solanum nigrum leaves, it is important to note that while solanum nigrum leaves have a history of traditional use for these medicinal purposes, following are the some of the uses of solanum nigrum leaves. antioxidant activity: solanum nigrum leaves contain antioxidants that help scavenge free radicals and protect against oxidative stress. consumption of the leaves or preparations made from them may contribute to overall antioxidant support in the body [19]. diuretic properties: solanum nigrum leaves have diuretic effects, which means they promote increased urine production. this property has been traditionally used to help flush out toxins from the body and support urinary health [12]. respiratory conditions: the leaves of solanum nigrum have been used to alleviate respiratory ailments like cough, asthma, and bronchitis. they may be consumed as an infusion or decoction to help soothe the respiratory system [7]. gastrointestinal support: solanum nigrum leaves have been used to promote digestive health. they may be consumed to relieve constipation, indigestion, and stomach discomfort. additionally, they are believed to possess mild laxative properties [8]. anti microbial activity: the chloroform: methanol extracts of leaf of solanum nigrum showed maximum inhibitory activity against pseudomonas aeruginosa. the chloroform: methanol extract of stem of solanum nigrum was highly active against bacillus subtilis and the acetone extract against pseudomonas aeruginosa. thus, it revealed that leaves and stem of solanum nigrum possess anti microbial activity [22]. anti-inflammatory properties: the leaves of solanum nigrum possess anti-inflammatory properties and have been used to alleviate inflammation-related conditions. methanolic extract of whole plant of solanum nigrum was investigated using carrageenin-induced rat paw oedema and egg white induced hind paw oedema methods, which demonstrated that plant extract exhibited anti-inflammatory activity [23]. the lipid-soluble extract of solanum nigrum leaves possessed anti-inflammatory activity [24]. hypoglycaemic activity: the crude ethanolic extract of solanum nigrum on blood sugar of albino rat after daily oral administration of dose at the level of 250mg/kg b. wt. for five and seven days respectively. it was noticed that the chronic administration for longer duration leads to significant decrease in blood sugar compared to control. thus, it is concluded that solanum nigrum also has the antidiabetic property. the aqueous and hydro-alcoholic extracts of different parts of solanum nigrum plant, viz leaf, fruit and stem evaluated for hypoglycaemic activity in sprague dawley rats. results indicated that aqueous extracts of leaf and fruit possess significant hypoglycaemic effect in dose dependent manner, followed by hydro alcoholic extracts [25].the stem extract of solanum nigrum has no profound effects [26]. anti-seizure property: of the aqueous extract of the leaves of solanum nigrum was evaluated inchicks, mice and rats. the aqueous leaf extract produced a significantly (p<0.05) dose dependent protection against electricallyinduced seizure in chicks and rats, pentylenetetrazoleinduced seizure in mice and rats and picrotoxin-induced seizure in mice and rats. the anti-seizure property of the extract was potentiated by amphetamine. thus, study suggests that the leaves of this plant may possess anticonvulsant property in chicks, mice and rats [27]. hepatoprotective effects: of solanum nigrum water and methanolic extracts were studied in rats. the water extracts showed a hepatoprotective effect against ccl 4 -induced liver damage, the methanolic extracts of solanum nigrum also had hepatoprotective effects with levels of serum ast, alt, alp and bilirubin decreasing significantly in animals treated with solanum nigrum methanolic extract compared to an untreated group. the solanum nigrum extract significantly lowered the ccl4-induced elevation of hepatic enzyme markers and decreased superoxide and hydroxyl radical generation [28]. mosquito larvicidal activity: the crude and solvent extracts of solanum nigrum l. leaves were evaluated against culex quinquefasciatus. the results indicated that the mortality rates at 0.5% concentration were highest amongst all concentrations of the crude extracts. the results of regression analysis of crude extract of solanum nigrum revealed that the mortality rate is positively correlated with the concentration of the extracts. results of this analysis showed that the ethyl acetate extract of solanum nigrum may be considered as a potent source of a mosquito larvicidal agent [29]. b.m thejaswini et al. pharmacological review on solanum nigrum vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 98 defence against lead acetate induced toxicity: the protective effect of an aqueous leaf extract of solanum nigrum extract was examined against lead acetate swiss albino mice. the results of the present study provide clear evidence of defence provided by solanum nigrum extract against lead acetate induced toxicity in brains of albino mice [30]. flowers the flowers of solanum nigrum have petals greenish to whitish. the inflorescence is extra axillary and composed of 3-6flowers. the total pedicel is about 1–2.5 cm long, and the pedicel is about 5 mm long and pubescent. the calyx is small, shallow cup shaped, about 1.5–2 mm in diameter, the corolla is white. the lobes are ovoid and oblong about 2 mm long. the filaments are short, anthers are yellow, about 1.2 mm long, and about four times the length of the filaments, and the apical hole is inward. the ovary is ovate and about 0.5 mm in diameter, and the style is about 1.5 mm long. the stigma is small (figure 2) [31]. figure 2. solanum nigrum flowers [photo by; own work] phytochemicals in flowers: the flowers of solanum nigrum, contain various phytochemicals that contribute to their potential medicinal properties. while research on the specific phytochemical composition of solanum nigrum flowers is limited, following are some of the phytochemicals that may be present [32, 33] flavonoids: flavonoids are a class of compounds known for their antioxidant and anti-inflammatory properties. glycoalkaloids: solanum nigrum flowers may also contain glycoalkaloids, including solanine and solasonine. these compounds are known to have cytotoxic and antitumor properties. however, it's important to note that these glycoalkaloids can be toxic if consumed in large amounts or if the flowers are not properly processed. phenolic compounds: phenolic compounds, including phenolic acids and phenolic glycosides, are commonly found in plant flowers. saponins: saponins are another class of phytochemicals that may be found in solanum nigrum flowers. they have been studied for their potential antimicrobial and antiinflammatory activities. it's important to note that the phytochemical composition of solanum nigrum flowers may vary depending on factors such as plant variety, growing conditions, and extraction methods. further research is needed to fully characterize the phytochemicals present in solanum nigrum flowers and their potential health benefits. solanum nigrum have also been traditionally used for their medicinal properties. although the flowers are not as widely studied as other parts of the plant, they are believed to possess certain medicinal benefits. following is some of the reported medicinal uses of solanum nigrum flowers: anti-inflammatory properties: like other parts of the plant, the flowers of solanum nigrum are believed to have anti-inflammatory properties. they may be used topically as poultices or extracts to help reduce inflammation and relieve skin conditions such as rashes and irritation [31]. digestive support: solanum nigrum flowers have been used to support digestive health. they are believed to have carminative properties, which can help relieve gas, bloating, and indigestion. they may also be used to stimulate appetite and improve overall digestion [31]. respiratory conditions: the flowers of solanum nigrum have been used in traditional medicine to alleviate respiratory ailments. they may be consumed as an infusion or decoction to help soothe coughs, pulmonary tuberculosis and respiratory congestion [1]. diuretic properties: solanum nigrum flowers are believed to have diuretic effects [13]. which mean they may help increase urine production and promote detoxification. this property has been traditionally used to support urinary health and assist in flushing out toxins from the body. fruit the fruits are berry. these berries are usually broadly ovoid, dull purple and blackish when ripe about 6-10 mm broad, either remains on plants or falls from calyces when ripe [34]. fruit shows thin, papery epicarp, pulpy mesocarp and axle placentation, seeds lie free in pulp of fruit. the berries of solanum nigrum contains 4 steroidal alkaloid glycosides, solamargine, solasonine, α and β-solanigrine. the berries have been found to contain a saturated steroidal genin, which is identified as trigogenin [9]. total 76 steroidal saponins have been isolated and identified in solanum nigrum. unripefruits of solanum nigrum have a high concentration of solasodine (figure 3) [12]. b.m thejaswini et al. pharmacological review on solanum nigrum vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 99 figure 3. solanum nigrum fruits [1] phytochemicals in fruit: the fruits of solanum nigrum contain various phytochemicals that contribute to their potential medicinal properties. following are some of the phytochemicals found in solanum nigrum fruits [31, 35]: solasonine and solamargine: solanum nigrum fruits contain glycoalkaloids such as solasonine and solamargine. these compounds have been studied for their potential anticancer properties. flavonoids: solanum nigrum fruits are rich in flavonoids, which are known for their antioxidant and antiinflammatory properties. vitamin c: solanum nigrum fruits are a good source of vitamin c, also known as ascorbic acid. vitamin c is an essential nutrient that plays a vital role in immune function and acts as an antioxidant in the body. carotenoids: solanum nigrum fruits contain carotenoids, which are responsible for their colour. carotenoids have antioxidant properties and are beneficial for eye health and overall well-being. the fruits of solanum nigrum, have a long history of traditional medicinal use in various cultures. while the fruits are typically consumed in their ripe state, it's important to note that some parts of the plant, including unripe fruits and other plant parts, may contain toxic compounds. following are some of the reported medicinal uses of solanum nigrum fruits. antipyretic activity: solanum nigrum fruits have been traditionally used as a febrifuge, meaning they may help reduce fever. the fruits may be consumed or used in preparations to help lower body temperature during fever episodes [12]. wound healing: the ripe fruits of solanum nigrum have been used topically to promote wound healing. they are believed to possess antimicrobial and anti-inflammatory properties that can help in the healing process [8]. digestive health: solanum nigrum fruits have been used traditionally to support digestive health. they are believed to possess laxative properties and may help relieve constipation, also used to treat gastritis and promote regular bowel movements [13]. it is important to exercise caution when consuming solanum nigrum fruits or using them for medicinal purposes. the following are the scientific research validated fruits effectiveness and safety in animal models antimicrobial activity: the ethanolic fruit extract of solanum nigrum was found to inhibit the gram-positive bacteria. sn extract was found to be most effective against s.aureus showing the maximum zone of inhibition followed by b. sublitis whereas in case of gram negative bacteria, ethanolic extract was found to be most effective against e. coli showing the maximum zone of inhibition followed by p.aeruginosa. interestingly, the ethanolic extract showed high activity against c. albicans [36]. analgesic activity: the solanum nigrum fruit extract was evaluated for its central and peripheral pharmacological actions by using eddy’s hot plate and acetic acid induced writhing respectively, model revealed that the extract possesses analgesic activity [36]. antioxidant properties: the ripe fruits of solanum nigrum are rich in antioxidants. these antioxidants help neutralize free radicals in the body, protecting against oxidative stress and promoting overall health. the anti-oxidant activity of methanolic extract of berries of the plant solanum nigrum was evaluated by tissue biochemical anti-oxidant profile. the methanolic extract of berries of the plant solanum nigrum possessed anti-oxidant activity [37]. anticancer activity: the fruits of solanum nigrum were evaluated for anticancer activity on the hela cell line. the fruits of solanum nigrum methanolic extract were tested for its inhibitory effect on hela cell line. the cytotoxicity of solanum nigrum on hela cell was evaluated by the sulforhodamine b colorimetric assay (srb) and mtt assay. solanum nigrum methanolic extract has significant cytotoxicity effect on hela cell line [38]. antiulcerogenic effect: of the methanolic extract of solanum nigrum berries (sbe) on aspirin induced ulceration in rats with respect to antioxidant status in the gastric mucosa have been investigated. the decreased levels of antioxidant enzymes and increased mucosal injury were altered to near normal status upon pre -treatment with (sbe) when compared to the ulcer induced rats. the results indicated that (sbe) may exert its gastro protective effect by a free radical scavenging action [37]. b.m thejaswini et al. pharmacological review on solanum nigrum vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 100 anti-diarrhoeal activity: of ethanolic dried fruit extract of solanum nigrum against castor oil induced diarrhoea in mice was investigated. the fruit extract showed a significant anti-diarrhoeal activity in mice and in which it decreased the frequency of defecation [14]. cardio protective activity: the cardio protective activity of methanolic extract of berries of the plant solanum nigrum was evaluated by using global in vitro ischemiareperfusion injury carried out using doses of 2.5 and 5.0 mg/kg for 6 days per week for 30 days. the results indicate that the extract exhibited significant (p<0.001) cardio protective activity against global in-vitro ischemiareperfusion injury. the methanolic extract of berries of the plant possessed cardio protective activity [39]. respiratory conditions: the fruits of solanum nigrum have been used to alleviate respiratory ailments, including cough and bronchitis. they may be consumed as a decoction or infusion to help soothe the respiratory system. petroleum ether, ethanol and aqueous extracts of solanum nigrum berries were screened for the treatment of asthma by the various methods. active petroleum ether extract showed presence of anti-asthmatic compound, β-sitosterol. the petroleum ether extract of solanum nigrum berries can inhibits parameters linked to the asthma disease [40]. cytotoxic activity: the ethanolic extract of the dried fruit of solanum nigrum linnwere tested for cytotoxic activity. in the brine shrimp lethality test, the extract showed cytotoxicity significantly with lc50= 63.10µg/ml and lc90= 160µg/ml [41]. seeds the seeds are mostly nearly ovoid, and discoid. about 1.5– 2 mm in diameter, and compressed on both sides. seeds are smooth, minutely pitted and yellow [34]. the seeds have been used in traditional medicine for various purposes (figure 4). figure 4. solanum nigrumseeds [42] phytochemicals in seeds: the seeds of solanum nigrum contain various phytochemicals. however, it's important to note that the seeds also contain toxic compounds such as solanine and solanidine, which can be harmful if the seeds are not properly processed. here are some of the phytochemicals present in solanum nigrum seeds. [18, 31] solasodine: solasodine is a steroidal alkaloid found in solanum nigrum seeds. it possesses anti-inflammatory and antitumor properties and has been studied for its potential therapeutic applications. solanidine: solanidine is another steroidal alkaloid present seeds. it has been studied for its cytotoxic and antitumor activitiesin solanum nigrum glycoalkaloids: solanum nigrum seeds contain glycoalkaloids, including solanine and solasonine. these compounds can have toxic effects if consumed in excessive amounts. however, in controlled and processed forms, they may also have potential medicinal applications. lipids: solanum nigrum seeds contain high lipid content. their protein content and minerals elements (mg being prominent) are considerable and solanum nigrum oil is an important source of linoleic acid. phenolic compounds: solanum nigrum seeds also contain phenolic compounds, which contribute to their antioxidant activity. however, it's important to note that the seeds of solanum nigrum, as well as other parts of the plant, contain toxic compounds such as solanine and solanidine. these compounds can be harmful if consumed in large quantities or if the seeds are not properly processed. as a result, caution should be exercised when using solanum nigrum seeds for medicinal purposes following are some reported traditional uses of solanum nigrum seeds: anthelmintic properties: solanum nigrum seeds have been used traditionally as anthelmintic. however, it's important to note that the seeds should only be used under the guidance due to their potential toxicity [43]. skin conditions: the oil extracted from solanum nigrum seeds has been used topically in some traditional remedies for skin conditions such as eczema, itching, and inflammation. however, more research is needed to validate these uses [8]. it is crucial to consult with a healthcare professional or a qualified herbalist before using solanum nigrum seeds for medicinal purposes, on the appropriate dosage and ensure the safe and responsible use of this plant. the following are the scientific research validated seeds effectiveness and safety in animal models. b.m thejaswini et al. pharmacological review on solanum nigrum vol 2 | issue 3 | jul – sep 2023 indian j pharm drug studies | 101 antiviral effect: methanol and chloroform extracts of solanum nigrum seeds exhibited 37% and more than 50% inhibition of hcv respectively at nontoxic concentration. moreover, antiviral effect of solanum nigrum seeds extract was also analysed against hcv ns3 protease. the results demonstrated that chloroform extract of solanum nigrum decreased the expression or function of hcv ns3 protease in a dosedependent manner results suggest that extract contains potential antiviral agents against hcv and combination of solanum nigrum extract with interferon will be better option to treat chronic hcv thus it is concluded that seeds of solanum nigrum possess anti-viral activity [44]. antifungal activity: thethree solvent extracts from leaf seed and roots of solanum nigrum were assayed for antifungal activity against fungal strains such as penicillium notatum aspergillus niger, fuserium oxisporium and trichoderma viridae. the zone of inhibitions was compared with the standard antibiotics. the organic solvent extracts (ethanol, methanol and ethyl acetate) of seeds were exhibited strong antifungal activity against all the tested fungal strains compared to leaf and root extracts. among all the extracts ethyl acetate seed extract showed high antifungal activity [45]. conclusion the whole plant of solanum nigrum has medicinal properties. the leaves and berries are primarily employed as medicines. solanum nigrum has a wide range of therapeutic potential, including antitumor, analgesic, antiinflammatory, anticancer, antiallergy, antiviral, antioxidant, antibacterial, neuro-protective and hepato-protective activities. consequently, additional research is necessary to separate the active 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pharm drug studies. 2023; 2(3) 95-102. funding: none conflict of interest: none stated bhat et al. self-medication: implications and challenges study vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 24 original article assessing self-medication practices: a cross-sectional study on implications and challenges ramdas bhat1, fazeela mohammed saleem2, ar shabaraya3 from, 1associate professor, department of pharmacology, 2ug student, 3principal and director, srinivas college of pharmacy, valachil, farangipete post, manglore, karnataka, india. abstract aim and background: the survey aimed to explore and understand the perceptions and attitudes of individuals towards selfmedication, and to examine the extent to which they engage in self-medication practices. the survey gathered data on the reasons why individuals choose to self-medicate, the types of medications they use, the sources of information they rely on, and their awareness of the potential risks and benefits of self-medication. methodology: a total of 120 subjects participated in the study, and the majority of respondents belonged to the age group of 18-20 and 21-25. among the respondents, 68.5% of them were female, and 51.4% of respondents belonged to healthcare professions/students. result: when asked about their preference for self-medication, 76.6% of the respondents preferred self-medication, and the majority of respondents (47.7%) opted to self-medicate sometimes. the principal morbidities for which respondents seek self-medication are headache (35.1%), fever, and cold (31.3%). the most commonly used medication for self-medication was analgesics and antipyretics (30.3%) and cough and cold preparations (27%). results of the survey showed that while self-medication is a preferred mode of treatment for many individuals, most of them were unaware of the potential risks associated with self-medication. conclusion: the findings of this study highlight the need for public health interventions aimed at promoting safe and effective medication use. key words: self-medication, prevalence, health outcomes, medication use, survey. elf-medication, the practice of obtaining and using medicines without a prescription or professional medical guidance, has become a prevalent phenomenon with both potential benefits and risks [1]. as noted by the world health organization (who), responsible selfmedication can serve as a valuable tool in preventing and treating certain diseases, offering a convenient and costeffective alternative for managing common illnesses. this nuanced approach acknowledges the positive aspects of selfmedication while recognizing the need for caution in its application [2]. the prevalence of self-medication, notably in developing nations like india, underscores its significance as a primary healthcare strategy, especially in the absence of universal healthcare access [3]. globally, self-medication rates range from 11.2% to 93.7%, with studies in india revealing rates as high as 37% in urban areas and 17% in rural regions [4-9].this widespread reliance on drugs without professional consultation poses significant risks, motivating a focused access this article online received – 19th jan 2024 initial review – 02nd feb 2024 accepted – 15th feb 2024 quick response code study on self-medication practices [10]. the percentage of people using self-medication worldwide is shown in (figure 1) [11]. the appeal of self-medication lies in its inherent convenience, a key factor influencing individuals to address minor health concerns without the necessity of a doctor's visit [12]. in today's fast-paced world, where time is often a precious commodity, the allure of quick and accessible solutions to health issues is undeniable. additionally, the costeffectiveness of self-medication further contributes to its popularity, as over-the-counter medications and home remedies can often provide relief without the financial burden associated with doctor visits and prescription drugs. the timesaving aspect of self-medication is evident in its elimination of the need for scheduling appointments, waiting in clinics, and adhering to the timelines of the traditional healthcare system [13]. particularly for minor ailments and well-known conditions, individuals may prefer the autonomy and immediacy that self-medication offers. however, amidst these apparent advantages, it is crucial to acknowledge and address the potential drawbacks associated with this practice [14]. __________________________________________________ correspondence to: ramdas bhat, associate professor, department of pharmacology, srinivas college of pharmacy, valachil, farangipete post, manglore-574143, karnataka, india. email: ramdas21@gmail.com. s mailto:ramdas21@gmail.com bhat et al. self-medication: implications and challenges study vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 25 studies have consistently indicated that self-medication may lead to delayed diagnosis and treatment of underlying health conditions. the absence of professional notwithstanding these apparent benefits, it is imperative to recognize and manage any possible disadvantages linked to this approach [14]. research has consistently shown that selfmedication may cause underlying medical issues to be diagnosed and treated later than they otherwise would. medical assessment can result in overlooking serious issues that require timely intervention [13]. furthermore, selfmedication poses the risk of interactions between different drugs, emphasizing the importance of seeking guidance from a licensed medical practitioner to mitigate potential risks and ensure the safety and efficacy of the chosen treatment. a notable concern associated with self-medication is the looming threat of antibiotic resistance [15]. a major threat to public health is the emergence of drugresistant bacterial strains, which are a result of the careless use of antibiotics without appropriate medical supervision. it is imperative to address this expanding problem and maintain the efficacy of these drugs to practice responsible antibiotic usage under the supervision of medical specialists [15,16]. this study delves into self-medication trends, scrutinizing factors and attitudes. by uncovering the nuances, it aims to guide health initiatives and policies for a balanced approach to medication use, considering both accessibility and responsible practices for individual and public well-being. figure 1: percentage of people using self-medication worldwide. methodology in conducting this cross-sectional study within the community, we selected srinivas college of pharmacy in valachil, mangalore, as our study site. the research involved a sample size of 120 subjects and spanned 2-5 days. our inclusive criteria encompassed individuals above 18 years of age, comprising both healthcare professionals/students and non-healthcare professionals/students who willingly participated in the study. this diversity in participants aimed to provide a comprehensive understanding of self-medication practices. on the contrary, individuals who did not return the questionnaire within the stipulated time and those unwilling to participate were excluded. data source: for the collection of data in this self-medication study, an online survey was employed, utilizing a meticulously crafted questionnaire created through google forms. the design of the questionnaire drew inspiration from existing literature and research on self-medication, garnering insights from reputable sources such as google scholar and other reliable online databases. this method ensured the incorporation of established knowledge and best practices into the survey instrument, enhancing its validity and relevance. the utilization of google forms facilitated efficient data gathering, allowing participants to provide their responses in a user-friendly digital format. data collection: to ensure a diverse and widespread representation, the survey for this study on self-medication was disseminated across various online platforms, including social media, online forums, and email lists. this approach aimed to engage a broad spectrum of respondents, capturing diverse perspectives on self-medication practices. the questionnaire, designed to extract comprehensive insights, focused on gathering data about the perceptions and attitudes of individuals towards self-medication. key aspects explored included the reasons driving self-medication, the types of medications commonly used, sources of information influencing these choices, and awareness of potential risks and benefits of participants associated with self-medication. a total of 120 subjects actively participated in the study, contributing their responses anonymously to prioritize and uphold privacy and confidentiality. this approach not only encouraged candid feedback but also ensured the ethical handling of sensitive information. data analysis: the collected data underwent a thorough analysis employing statistical tools, with descriptive statistics like percentages and frequencies summarizing key demographic characteristics, including age, gender, and profession of the 120 study participants. detailed scrutiny of preferences for self-medication, types of medications used, and reasons driving self-medication revealed underlying patterns. the findings were then interpreted and presented visually using pie charts and graphs, offering a clear and accessible representation of prevalent self-medication practices. result the survey, facilitated through a structured questionnaire, provided valuable insights into the characteristics and bhat et al. self-medication: implications and challenges study vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 26 preferences of the respondents. the age distribution revealed a predominant representation within the 18-20 and 21-25 age brackets, signifying a predominantly youthful demographic engaged in the study. in terms of gender, the survey exhibited a slight gender imbalance, with 68.5% of respondents identifying as female and 31.5% as male, reflecting varied participation across genders. a notable aspect of the participant demographics was the professional and educational affiliations. approximately 51.4% of respondents were affiliated with healthcare professions or were students in related fields, while the remaining participants belonged to non-healthcare professions or were students from other disciplines. this stratification provided a nuanced understanding of the diverse backgrounds contributing to the survey. the survey delved into the respondent's attitudes towards self-medication, revealing a substantial inclination towards this healthcare approach. a significant 76.6% of participants expressed a preference for self-medication, emphasizing the popularity of this practice within the surveyed population. further exploration into the frequency of self-medication uncovered intriguing patterns. the majority, comprising 47.7% of respondents, indicated a preference for self-medicating "sometimes," while 39.6% chose the option "rarely." notably, a small yet consistent percentage of 3.7% revealed a persistent preference, selecting "always" when it comes to practicing self-medication as shown in (table 1). table 1: distribution of the study subjects according to sociodemographic profile distribution of the study subjects according to sociodemographic profile % age group 18-20 63.1% 21-25 21.6% 26-40 8.1% above 40 7.2% sex female 68.5% male 31.5% stream health care 51.4% non-health care 48.6% preference to selfmedicating yes 76.6% no 23.4% frequency of selfmedicating always 3.7% sometimes 47.7% rarely 39.6% never 9% principal morbidities for which the respondents seek selfmedication are headache (35.1%), fever&cold (31.3%), gi problems (15.3%), and dysmenorrhea (14.7%) as shown in (figure 2). drugs commonly used for self-medication included analgesics and antipyretics (30.3%) and cough suppressants/syrups (27%) followed by antibiotics (14.8%), antacids (12.1%), multivitamins (10.8%), and antihistamines for allergies (5%)a shown in (figure 3). 18% of the total respondents have experienced side effects as a result of self-medication. among these 18% of the respondents, the majority of them have experienced dizziness (28.7%). other side effects experienced were nausea/ vomiting (18.3%), constipation (16.3%), diarrhea (11.6%), itching/rashes (9%), and palpitations (8.6%) as shown in (figure 4). figure 2: principal morbidities for seeking selfmedication. figure 3: drugs used by respondents for self-medication. figure 4: side effects experienced by respondents as a result of self-medication 35.1 31.3 15.3 14.7 3.6 0 5 10 15 20 25 30 35 40 30.3 10.8 14.8 5 27 12.1 0 5 10 15 20 25 30 35 8.6 18.3 28.7 9 11.6 16.3 7.5 0 5 10 15 20 25 30 35 bhat et al. self-medication: implications and challenges study vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 27 about the sources of medicines for self-medication, 61% of the respondents got them from medical stores, 20% of them used left over medicines found at home, 13% of them get the medicines from families and friends and 6% of them shop from online pharmacies as shown in (figure 5). when the attitude of the respondents was analyzed, we got to know that 31% of the respondent’s considered self-medication as a “friend” and 6% of the respondent’s considered selfmedication as a “foe”, while majority of them (63%) considered self-medication botha friend and a foe as shown in (figure 6). when the questionnaire was analyzed, the respondents agreed with various reasons that were against and in favor of self-medication. among the reasons in favor of self-medication, the most favored reasons were no need to visit the doctors for minor illness (48.5%), time saving and quick relief (22.3%), economical (14.3%), crowd avoidance (9%) and 5.9% of them feel that they are confident about his/her knowledge on the medicines as shown in (figure 7). when the reasons against self-medication were assessed, the most important reason were risk for adverse effects (30%), lack of knowledge about the drugs (23.5%), risk of using wrong drugs or using drugs wrongly (18.1%), risk of drug dependence (16.6%) and risk of misdiagnosing (11.7%) as shown in (figure 8). figure 5: source of medicines for self-medication figure 6: respondent's point of view regarding self-medication figure 7: why do respondents think self-medication is a friend? figure 8: why do respondents think self-medication is a foe? discussion several comparative studies have been conducted on the prevalence of self-medication in different countries. a study conducted in puducherry, india, reported a high prevalence of self-medication among illiterate and middle school-educated individuals [17]. another study conducted in bangalore, india, found that the practice of self-medication differed among individuals and was influenced by several factors like age, educational status, gender, family monthly income, level of knowledge, and attitude [18]. a systematic review and metaanalysis of studies conducted worldwide found that the prevalence of self-medication in university students was high [19]. a study conducted in metropolitan areas of thailand reported a self-medication prevalence rate of 88.2% [20]. the pervasive prevalence of self-medication, notably among the youth, underscores the urgent need for targeted health education [21]. gender and professional diversity reveal varied perspectives influencing self-medication practices, with common health concerns apparent, but the diverse array of drugs used raises concerns about informed decision-making and the high prevalence of side effects, particularly dizziness [22-24]. the reliance on medical stores and leftover medicines signals the accessibility of medications, urging regulatory scrutiny. the nuanced perception of self-medication as both a “friend” and a “foe” underscores the importance of balanced medical store 61% left over medicines found in home 20% from relatives and friends 13% online pharmacy 6% friend 31% foe 6% both 63% 48.5 22.3 14.3 9 5.9 0 10 20 30 40 50 60 no need to visit dr. for minor illness quick relief and time saving economical crowd avoidance confident about his/her knowledge of drugs. 23.5 30 18.1 16.6 11.7 0 5 10 15 20 25 30 35 bhat et al. self-medication: implications and challenges study vol 3 | issue 1 | jan – mar 2024 indian j pharm drug studies | 28 healthcare approaches. while convenience and costeffectiveness drive self-medication, contrasting concerns about adverse effects and misdiagnosis highlight the imperative for responsible medication practices. this study emphasizes the need for targeted education, regulatory measures, and future research exploring underlying factors to promote responsible self-care practices, with the potential of digital health interventions playing a pivotal role in improving overall public health outcomes. conclusion prevalence of self-medication is high in the educated youth, despite the majority being aware of its harmful effects. strict policies need to be implemented on the advertising and selling of medications without prescription to prevent this problem. education to help people to decide the appropriateness of selfmedication is required as responsible self-medication can prevent and treat diseases that do not require medical consultation and provide cheaper alternatives. though selfmedication is a convenient alternative, its inappropriate practice may cause detritus effects on health. references 1. bennadi d. self-medication: a current challenge. j basic clini pharm. 2014; 5(1):19. 2. alghanim sa. self-medication practice among patients in a public health care system. east mediterr health j. 2011; 17(5):409-16. 3. akande-sholabi w, ajamu at, adisa r. prevalence, knowledge and perception of self-medication practice among undergraduate healthcare students. journal of pharmaceutical policy and practice. 2021; 14(1):49. 4. ayele aa, mekuria ab, tegegn hg, et al. management of minor ailments in a community pharmacy setting: findings from simulated visits and qualitative study in gondar town, ethiopia. ahmad hassali ma, editor. plos one. 2018; 13(1):e0190583. 5. abdelwahed ae, abd-elkader mm, mahfouz a, et al. prevalence and influencing factors of self-medication during the covid-19 pandemic in the arab region: a multinational crosssectional study. bmc public health. 2023; 23(1). 6. narang p, garg v, sharma a. regulatory, safety and economic considerations of over-the-counter medicines in the indian population. discover health systems. 2023; 2(1). 7. ahmad a, patel i, mohanta gp, et al. evaluation of selfmedication practices in rural area of town sahaswan at northern india. annals of medical and health sciences research. 2014; 4(8):73-8. 8. bhaisare r, rangari g, korukonda v, et al. prevalence of selfmedication in rural area of andhra pradesh. journal of family medicine and primary care. 2020; 9(6):2891. 9. chautrakarn s, khumros w, phutrakool p. self-medication with over-the-counter medicines among the working age population in metropolitan areas of thailand. frontiers in pharmacology. 2021; 12:726643. 10. rathod p, sharma s, ukey u, et al. prevalence, pattern, and reasons for self-medication: a community-based crosssectional study from central india. cureus. 2023; 15(1): e33917. 11. s g, selvaraj k, ramalingam a. prevalence of self-medication practices and its associated factors in urban puducherry, india. perspectives in clinical research. 2014; 5(1):32. 12. zeb s, mushtaq m, ahmad m, et al. self-medication as an important risk factor for antibiotic resistance: a multiinstitutional survey among students. antibiotics. 2022; 11(7):842. 13. muteeb g, rehman mt, shahwan m, et al. origin of antibiotics and antibiotic resistance, and their impacts on drug development: a narrative review. pharmaceuticals. 2023; 16(11):1615-7. 14. aslam b, wang w, arshad mi, et al. antibiotic resistance: a rundown of a global crisis. infection and drug resistance. 2018; 11:1645–58. 15. ayukekbong ja, ntemgwa m, atabe an. the threat of antimicrobial resistance in developing countries: causes and control strategies. antimicrobial resistance & infection control. 2017;6(1):1-8. 16. fekadu g, dugassa d, negera g, et al. self-medication practices and associated factors among health-care professionals in selected hospitals of western ethiopia. patient preference and adherence. 2020; 14:353-61. 17. selvaraj k, kumar sg, ramalingam a. prevalence of selfmedication practices and its associated factors in urban puducherry, india. perspectives in clinical research. 2014; 5(1):32-5. 18. babu ps, balu v, maheswari bu, et al. a comparative study to assess the prevalence, knowledge of impact, and practice of self-medication among adults in urban and rural communities in bangalore. cureus. 2023; 15(5):e39672. 19. behzadifar m, behzadifar m, aryankhesal a, et al. prevalence of self-medication in university students: systematic review and meta-analysis. eastern medite health j. 2020; 26(7):846-57. 20. chautrakarn s, khumros w, phutrakool p. self-medication with over-the-counter medicines among the working age population in metropolitan areas of thailand. front pharmacol. 2021; 12:726643. 21. rather ia, kim bc, bajpai vk, et al. self-medication and antibiotic resistance: crisis, current challenges, and prevention. saudi journal of biological sciences. 2017; 24(4):808-12. 22. nieuwlaat r, wilczynski n, navarro t, et al. interventions for enhancing medication adherence. cochrane database of systematic reviews 2014; 2014(11). 23. senbekov m, saliev t, bukeyeva z, et al. the recent progress and applications of digital technologies in healthcare: a review. fayn j, editor. international journal of telemedicine and applications. 2020; 1–18. 24. amjad a, kordel p, fernandes g. a review on innovation in healthcare sector (telehealth) through artificial intelligence. sustainability. 2023; 15(8):6655. how to cite this article: ramdas bhat, fazeela mohammed saleem, ar shabaraya. assessing selfmedication practices: a cross-sectional study on implications and challenges. indian j pharm drug studies. 2024; 3(1):24-28. funding: none; conflicts of interest: none stated ijpds_4594.pdf samajdar & samajdar in silico activity of a topical formulation using piper longum online first indian j pharm drug studies | 1 original article in silico bioactivity prediction and topical formulation of piper longum root for skin cancer saptarshi samajdar, debojit samajdar from, department of pharmaceutical technology, brainware university, kolkata, india. abstract background: skin cancer is exceedingly common and the incidence is rising rapidly. although the mortality rate for skin cancer is not as large as other cancers but still a steady mortality rate around the world remains. as per various reports, a significant number of skin cancer cases have been reported in india mostly from the northern and eastern regions. objectives: although many treatment strategies for skin cancer have been developed over the years, most of them deal with cell toxicity. so, the main objective of this work is to develop a topical formulation using an alternate herbal source ie, piper longum which can provide solution to skin cancer. methodology: a herbal treatment strategy, using piper longum has been demonstrated by an in silico bioactivity study. further, a set of topical cream formulations using piper longum was prepared and evaluated. results: the study yielded three major compounds, episesamine (-10.4 kcal/mol), fargesin (-9.6 kcal/mol) & pellitorine (-9.1 kcal/mol) from piper longum were highly effective against skin cancer nuclear receptor for vitamin d (1db1). the toxicity predicted by the protox ii tool revealed all ligands with 4-5 level of toxicity and very high ld50 values can be predicted to be safe for human usage. the evaluation of the cream formulation reveals it to be smooth, semisolid nonirritant in nature having high shelf life and spreadability of around 0.3g.cm/s. conclusions: hence from the results we can conclude that piper longum is predicted to have many phytochemicals with multitude of anti-skin cancer effects and its formulation was also found to be stable, but further, in vitro and in vivo research is required for its usage in humans. key words: skin cancer, molecular docking, piper longum, nuclear receptor for vitamin d, protox ii, cream formulation. he skin provides the outer covering of our body covers over 16% of the body's mass and protects our inside organs from different threats. as the skin is the body's most exposed organ, uv radiation, noxious agents, and toxicants can all harm it. these hazardous factors cause molecular and metabolic stress, resulting in genomic changes in skin cells and skin carcinogenesis [1,2]. human skin cell transformation into cancer is a multistep process involving initiation, promotion, and advancement that is thought to be stimulated by oxidative stress in cells, resulting in transformation (into cancer), survival, and metastasis. exposure to ultraviolet radiation (uvr) is a major risk factor for skin cancer. skin cancers are the most commonly diagnosed malignancies in western countries, with a significant increase in the indian subcontinent due to increased exposure to ultraviolet access this article online received 14th apr 2024 initial review 31st may 2024 accepted 05th jun 2024 quick reponses code (uv) radiation [3,4]. skin cancer is defined by an imbalance between inadequate apoptosis and excessive cell proliferation and survival in the epidermis. although uv radiation is the most common cause of skin cancer, viruses, mutagens in food, mutagens in chemicals, and genetic predisposition are also factors [5]. to control skin cancer, multiple synthetic and mechanical therapeutic strategies have been designed, but many of them reel with the problem of cell toxicity [6]. so, natural products from a highly regarded ayurvedic plant piper longum can act as a solution to the problem. p. longum also called indian long pepper or pippali, is a flowering vine in the family piperaceae, cultivated for its fruit, which is usually dried and used as a spice and seasoning which makes it safe for human use. piper longum is first mentioned in ancient indian ayurvedic texts, where its therapeutic and culinary properties are extensively discussed. it ___________________________________________________ correspondence to: saptarshi samajdar, associate professor, department of pharmaceutical, technology, brainware university, 398, ramkrishnapur road, kolkata-700125, india. email: saptarshisamajdar1993@gmail.com t samajdar & samajdar in silico activity of a topical formulation using piper longum online first indian j pharm drug studies | 2 arrived in greece around the sixth or fifth century bce, although hippocrates only mentioned it as a spice and not as a medicine [7,8]. in this study, the in-silico evaluation of the skin cancer receptor of p. longum is observed as well as preparation & evaluation of an herbal topical formulation has been reported. materials and methods materials: authenticated roots of the p. longum plant material were acquired from bixa botanicals and other chemicals were taken from s.d. finechem & spectrochem india. distilled water was used in all the experiments. extraction of p. longum: p longum that had been air-dried and finely powdered was individually added to the maceration pot using petroleum ether, followed by methanol. under lowered pressure and a regulated temperature, the extract was then concentrated until it was completely dry before being stored in a refrigerator [9]. ligands preparation and optimization: as reported by sharma et al., the ligands (9) from p. longum root detected using gcms were reported [10]. three-dimensional structures of the ligands were created in open babel and saved in sdf format for further preparation and molecular docking analysis [11,12]. preparation and optimization of skin cancer receptor: the protein receptor selected for detection of skin cancer potential was the crystallographic structures of the skin cancer nuclear receptor for vitamin d (pdb id: 1db1), downloaded from the protein data bank. the hetatoms as well as selected water molecules were removed from the protein while adding polar hydrogen for charge using biovia discovery studio 2021 visualizer application for its usage in molecular docking [13]. molecular docking analyses and visualization: the molecular docking studies were performed using pyrx application with auto dock vina tool. the protein was firstly saved in .pdb format and loaded to turn it into macromolecule. then the suitable ligands were also loaded their energy was minimized then using the auto dock vina tool the docking study was performed. a grid dimension of 61.06 å x 51.74 å x 46.44 å was chosen for the experiment in order to get the best conforming pattern. the intermolecular interactions between the ligands obtained from p. longum and 1db1 protein were identified and visualized using the discovery studio 2021 client software [14]. toxicity prediction: the ligands were subjected to toxicity prediction using protox ii software in human cells (https://toxnew.charite.de/protox_ii/). the webserver takes a twodimensional chemical structure as input and reports the possible toxicity profile of the chemical for 9 models with confidence scores [15]. formulation of topical herbal cream: white beeswax, liquid paraffin, and p. longum were all put into the first beaker. then, heat on a water bath to ensure even mixing. an oil phase eventually developed after some time. water, borax, and methyl paraben were added to the second beaker. in a water bath, all the ingredients were boiled together to produce the aqueous phase. the oil phase was continually mixed into the water phase to form a semisolid mass. once the mixture has become homogeneous, remove it from the heat and let the cream cool. as the liquid-cooled, menthol crystal was added and thoroughly mixed. two formulations, f1 and f2, were created (table 1) using various beeswax and medication concentrations [16]. table 1: formula of herbal cream ingredients f1 f2 p. longum 2.5 g 5g white beeswax 10 g 7.5g liquid paraffin 22.5 g 22.5g borax 0.35 g 0.35g water 14.5 g 14.5g methyl paraben 0.15 g 0.15g menthol crystal 0.2 g 0.2g evaluation of the cream formulations: formulated herbal creams were assessed further using the physical parameters listed below. physical characteristics color, odor, consistency, and formulation state.visual inspection allowed for the observation of the color for both formulations. the exact pantone shade was confirmed by colorgrab application [17]. the odor of both f1 and f2 formulations was observed by organoleptic evaluations [17]. the state of the cream was visually inspected [17]. the consistency of the formulation was tested by manually rubbing cream on the hand [17]. a digital ph meter (mettler toledo 242) was used to measure the herbal cream's ph. 100 ml of distilled water was used to produce the cream solution, which was then left to sit for two hours. ph was determined in three times for the solution and the average value was calculated [18]. the spreadability of the cream formulation was assessed by sandwiching the sample between two slides and compressing it to a constant thickness with a particular weight for a particular period. the specified time required to separate the two slides was measured as spreadability. the shorter the time taken for separation of two slides results showed better spreadability [18]. to calculate spreadability, use the formula below. spreadability = ml/t, where, m-weight of cream on the slide; llength moved on the slide; ttime taken samajdar & samajdar in silico activity of a topical formulation using piper longum online first indian j pharm drug studies | 3 the washability of cream was applied on the skin surface and observed under the running water and the observation was recorded [19]. the non-irritancy test results for an herbal cream formulation were assessed. the preparation lacked irritation and redness [19]. the viscosity of the cream was determined with the help of brookfield viscometer ldv230 at 6-100 rpm with the spindle no. 63 [19]. the prepared cream was transferred in a suitable wide-mouth container and its phase separation was. set aside for storage there was not any phase separation occurred between the oil phase and water phase [19]. accelerated stability testing of prepared formulations was conducted at 40oc ± 1oc for 60 days. the formulations were kept both at room and elevated temperature and observed on the 0th, 10th, 30th, and 60th day for the different parameters [20]. result in-silico studies pyrx docking was utilized to ascertain the binding affinities and significant interactions between p. longum-derived phytochemical ligands and the skin cancer nuclear receptor for vitamin d (1db1). the binding affinities of the acquired ligands and the standard breast cancer treatment tamoxifen were evaluated. table 2 shows the binding affinity obtained from the protein-bound ligands and the standard drug fluorouracil [21,22]. the binding affinity of the p. longum ranged from -9.4 to -4.2 kcal/mol. the molecular interactions between the most active ligands and the active site of the breast cancer receptor-targeting aromatase inhibitor protein were visualized using the discovery studio 2021 client program (figure 1). these samples showed the expected interactions with the protein& active-region amino acids, indicating strong antagonistic characteristics against the aromatase inhibitor protein, which attacks the nuclear receptor for vitamin d. for the protein coded 1db1, episesamine had the highest binding affinity of -10.4 kcal/mol followed by fargesin (-9.6 kcal/mol) and pellitorine (-9.4 kcal/mol). the ligand with values lowest binding affinity was observed in pipericide (-5.5 kcal/mol). as compared to the standard skin cancer drug fluorouracil (-7.8 kcal/mol), the binding affinities of three natural compounds (episesamine, fargesin, and pellitorine) derived from p. longum were found to be higher indicating their future usage in inhibition of skin cancer (table 2) [23]. the same set of ligands was studied for their toxicity using protox ii software showed that all the ligands had a predicted class 4 to class 5 toxicity with higher ld50 values indicating their safe usage in humans (table 3) [24]. the development and evaluation of a multi-herbal cream were the focus of the current study. the evaluation criteria for the polyherbal pain relief cream, including its viscosity and phase separation, spreadability, washability, non-irritancy test, and physical examination, were listed under the results in (table 4). table 2: docking score of p.longum ligands ligands binding affinity ( g in kcal/mol) 1db1 brachystamide -7.4 caryophyllene -7.2 episesamine -10.4 fargesin -9.6 pellitorine -9.1 pipericide -5.5 piperitine -5.7 piperine -5.9 piperlongumine -6.7 flurouracil -7.8 figure 1: interaction diagram of a. episesamine b. fargesin table 3: toxicity prediction of ligands ligands level of toxicity (1=highly toxic; 6= safe) predicted ld50 (µg/ml) brachystamide 4 760 caryophyllene 5 5300 episesamine 4 1500 fargesin 4 1500 pellitorine 5 4185 pipericide 4 760 piperitine 4 380 piperine 5 3100 piperlongumine 4 1160 evaluation of topical formulation the color of f1 formulation was observed to be brown orange (84831f) and greenish orange (812417f) for f2. the odor of the ayurvedically important herbal topical formulations was observed to have having common characteristic cream and paste smell. thus this ensures the stability of the formulations with no samajdar & samajdar in silico activity of a topical formulation using piper longum online first indian j pharm drug studies | 4 rancid smells. both the f1 and f2 formulations were shown to be in a semisolid state. their appearance was mostly showing plastic flow properties [25-27]. the consistency of the herbal topical formulations was seen to be smooth [25]. the ph of the f1 cream formulation was found to be 7.2 while the ph of f2 formulation was found to be 7.06, both of which are neutral with f2 having a better ph. no change of ph was observed even after four months of testing [28]. it was found that the cream was easily spreadable and had moisturizing properties. the exact values of spreadability were found to be 0.3±0.06 g.cm/s and 0.26±0.04 g.cm/s for f1 and f2 respectively. the spreadability value remains the same even after 4 months of observation [28]. table 4: results of polyherbal cream parameter f1 f2 color brown orange (84674f) pale brown orange (84831f) odor characteristic smell characteristic smell state semisolid semisolid consistency smooth smooth ph 7.20 7.06 spreadability 0.3±0.06 g.cm/s 0.26±0.04 g.cm/s washability sticky non-sticky non-irritancy test non-irritant non-irritant viscosity 2803±0.32cps. 2689±0.18cps phase separation no phase separation no phase separation the washability of f1 formulation was shown to be sticky but f2 got easily removed from the skin surface thereby showing f2 had higher washability, indicating no stain or sticky mass on the skin [29]. in irritancy studies, it was discovered that none of the formulations cause redness, edema, inflammation, or irritation of the skin. there was no sign of redness or other ailments even after 4th month of storage. the viscosity of formulated cream was determined by brookfield viscometer at 20 rpm using spindle no. 63. the viscosity of cream has been reported in the range of 2000 to 4999 cps which indicates that the cream is easily spreadable by a small amount of shear. the formulated creams f1 and f2 show a viscosity of 2803±0.32cps and 2689±0.18cps respectively [30]. the creams were very stable without any kind of phase separation observed. moreover, no phase separation was seen even after four months of storage. there were no observable changes in any of the parameters for the topical cream formulations (f1 and f2) even after 60 days of stability testing [19]. discussion skin cancer is very frequent and is becoming more commonplace. even while skin cancer mortality is not as high as that of other cancers, skin cancer mortality is nonetheless consistently high worldwide. several sources state that a considerable number of skin cancer cases, primarily from the northern and eastern parts of india, have been documented. so, to combat this, a robust treatment strategy is the need of the hour. although there are various synthetic strategies available but most of them faces issue of toxicity and multi drug resistance. so, a new strategy using p. longum root can find better potential. the phytochemicals from macerated extracts were selected for molecular docking studies. from the set of phytochemicals, three phytochemicals fargesin (-9.6 kcal/mol), episesamine (-10.4 kcal/mol), and pellitorine (-9.1 kcal/mol) showed maximum skin cancer activity even higher than the standard flurouracil. the toxicity prediction data showed the extracts to be safe for human use. thus, from these data it can be a promising anticancer treatment strategy [31]. based on these results, a set of topical formulation was designed and the evaluation of topical formulation revealed that the color to be of brown color, having semisolid state. the ph of the formulation f1 was 7.2 while the ph of f2 was 7.06 indicating f2 having a better ph. with f2 having a lower viscosity and no phase separation and nonirritant, non-sticky was selected as a better formulation [32]. thus with further studies like in vitro or in vivo as well as clinical trial, the treatment strategies can be established [33]. conclusion hence from the in-silico studies it could be observed that multiple compounds of p. longum root like fargesin, episesamine, and pellitorine found in p. longum root showed better binding affinity as compared to the standard drug in skin cancer receptors. all the ligands were found to be safe (grade 45) in toxicity prediction studies. overall the compounds showed promising results for their usage in skin cancer nuclear receptors for vitamin d. so, using the promising extract topical creambased formulations were prepared which were in the range of neutral ph and had no irritancy. the viscosity (2689±0.18cps2803±0.32cps) and spreadability values had promises for its wide use on skin. moreover, no changes were observed in 60day accelerated stability studies, indicating their superior shelf life. further in vitro and in vivo studies are required to provide evidence of its usage in humans. references 1. -ortiz a, et al. uv radiation and the skin. int j mol sci. 2013; 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docking studies of phytochemicals from plants salix subserrata and onion as inhibitors of glycoprotein g of rabies virus. biomed biotech res j. 2019; 3(4):269-76. 25. valarmathi s, kumar ms, sharma v, et al. formulation and evaluation of herbal face cream. res j pharm tech. 2020; 13(1):216-8. 26. iriventi p, gupta nv. formulation and evaluation of herbal cream for treating psoriasis. res j pharm tech. 2021; 14(1):167-70. 27. dhase as, khadbadi ss, saboo ss. formulation and evaluation of vanishing herbal cream of crude drugs. am j ethnomed. 2014; 1:313-8. 28. sonalkar my, nitave sa. formulation and evaluation of polyherbal cosmetic cream. world j pharm pharm sci. 2016; 5:772-9. 29. sirsat sv, rathi nm, hiwale as, et al. a review on preparation and evaluation of herbal cold cream. world j pharm res. 2022; 11(5):690-7. 30. satpute kl, kalyankar tm. development and evaluation of herbal cream for the treatment of acne. j pharmacogn phytochem. 2019; 8(3):2618-24. 31. wawszczyk j, wolan r, smolik s, et al. in vitro and in silico study on the effect of carvedilol and sorafenib alone and in combination on the growth and inflammatory response of melanoma cells. saudi pharm j 2023; 31(7):1306-16. 32. okafo se, anie co, alalor ca, et al. evaluation of physicochemical and antimicrobial properties of creams formulated using pterocarpus santalinoides seeds methanol extract j appl pharm sci. 2023; 13(5):126-35. 33. mlozi sh, mmongoyo ja, chacha mn. in vitro evaluation of the herbal cream formulation from methanolic leaf extracts of tephrosia vogelii hook. f for topical application. clin phytosci. 2023; 9(1):3. how to cite this article: saptarshi samajdar, debojit samajdar. in silico bioactivity prediction and topical formulation of piper longum root for skin cancer. indian j pharm drug studies. 2024; online first. funding: none; conflicts of interest: none stated r and k a case on pandas syndrome online first indian j pharm drug studies | 1 case report a case of pediatric autoimmune neuropsychiatric disorder associated with streptococcal infection dr. reshma r1, dr. ajnas k1 from, 1clinical pharmacist, department of prescription audit, indraprastha apollo hospital, delhi, india abstract pediatric autoimmune neuropsychiatric disease associated with streptococcal infection (pandas), is a subtype of acute-onset obsessive-compulsive disorder (ocd) induced by an immunological reaction to group a streptococcal infection. these diseases may be worsen by an autoimmune response brought on by streptococcal infections. one of the recurring diseases linked to pandas is recurrent streptococcal tonsillitis. "strep throat" (acute suppurative tonsillitis) is a tonsil infection typically caused by group a betahemolytic streptococci (gabhs) bacteria, often leading to intense throat pain, fever, and difficulty swallowing for a few days. health care providers frequently use a rapid strep test to check the tonsils of patients experiencing a sore throat for gabhs, as untreated infections can lead to more severe health issues. occasionally, strep throat can result in a body rash (known as scarlet fever) and can also lead to more severe health issues such as arthritis in children, rheumatic fever that can cause long-lasting heart problems, and post-streptococcal glomerulonephritis which may result in kidney failure. here we present a case of 20-year-old male patient with pandas, who underwent tonsillectomy. the morbidity associated with this neuropsychiatric condition can be reduced with prompt management. key words: obsessive compulsive disorder, tonsillectomy, streptococcal infection ediatric autoimmune neuropsychiatric diseases associated with streptococcus (pandas), which explains a collection of illnesses, including scarlet fever and strep throat, that are believed to afflict certain children who have streptococcal infections. although a direct correlation has not yet been shown, there have been some reports of tics, obsessive-compulsive behaviour, and other neurological and mental symptoms developing following pandas. following an infection with streptococcus pyogenes, children experience abrupt and typically substantial changes in personality, behaviour, and mobility. 1 the signs and symptoms occur during a pandas episodes are moodiness and irritability, separation anxiety, adhd (attention deficit hyperactivity disorder)symptoms, sleep disturbances, night-time bed wetting and/or day-time urinary frequency, fine motor changes (writing), joint pain, trouble eating, concentration difficulties and loss of academic abilities. children may have tics, which are uncontrollable, abrupt motions or sounds that they repeatedly make. they could jerk their head or blink a lot. they could repeat sentences, growl, or continuously clear their throat. the child's immune system generates antibodies that specifically attack gabhs bacteria when they are infected. in pandas, access this article online received – 27th july 2024 initial review – 27th september 2024 accepted – 29th november 2024 quick response code it is thought that these antibodies also coincidentally target and cause inflammation in a region of the brain known as the basal ganglia. this region of the brain is responsible for refining deliberate muscle actions and managing behaviour within the realm of thinking and logic. if this area of the brain is affected, its performance decreases and changes, leading to specific involuntary actions and motions. to determine whether the kid has pandas, which can resemble other diseases, a strep bacterial test or a history of the bacterium is required. the doctor should examine additional areas where strep tends to hide, such as the sinuses or child's genitalia and but if a throat swab or antibody test yields no results. even if the child recovered, a blood test might reveal if they had a recent strep infection. if child does not or did not have strep throat, then they do not have pandas. however, their symptoms may still be a result of a malfunctioning immune system response. pandas is a member of a broader category of autoimmune illnesses impacting the brain known as pediatric acute-onset neuropsychiatric syndrome (pans). the symptoms may be alike, but they could be caused by a different factor rather than strep. physician might request blood tests or brain scans to determine if another infection or a different underlying cause is responsible for the symptoms.2,3 __________________________________________________ correspondence to: dr. ajnas k. department of prescription audit, indraprastha apollo hospital, delhi, india. email: ajnas.kth@gmail.com p http://www.nlm.nih.gov/medlineplus/ency/article/001551.htm mailto:ajnas.kth@gmail.com r and k a case on pandas syndrome online first indian j pharm drug studies | 2 despite its rarity, some experts believe it may be responsible for 10% of childhood obsessive-compulsive disorder (ocd) and tic disorders. 4 it affects roughly 1 in 1000 children, and boys are three times more likely than girls to get it. because most children have established streptococcus immunity by adolescence, it is considered a pre-pubertal condition. as a result, the onset is unlikely to occur again, while pandas symptoms may continue to appear on a regular basis.5 the national institute of mental health’s (nimh) criteria for pandas includes: 6 1. presence of tic disorder and/or ocd (obsessive compulsive disorder) 2. prepubertal onset of neuropsychiatric symptoms 3. abrupt onset and a course characterized by dramatic exacerbations 4. the onset or exacerbation is temporally related to group a beta haemolytic streptococci (gabhs) infection 5. neurological abnormalitieshyperactivity, fidgetiness, restlessness or abnormal movements such as choreiform movements may be present during symptom exacerbation. the principle evidence-based therapies for ocd are cognitive behaviour therapy (cbt), which includes exposure and response prevention (erp), and selective serotonin reuptake inhibitors (ssris). 7 antibiotics or tonsillectomy to treat and/or prevent gabhs infection are additional or alternative therapy options when pandas is suspected. 8 therapeutic plasma exchange (tpe), intravenous immunoglobulin (ivig), or anti-cd20 monoclonal antibodies (rituximab) have been used to suppress the immune system in patients with potential autoimmune-based ocd corticosteroids. 9 nonsteroidal anti-inflammatory medicines (nsaids) have also been recommended to help with the psychiatric symptoms of pandas and pans, which is in keeping with the autoimmune etiology explanation. 10 case report a 20-year old male patient was admitted to neurological department, who had history of episodes of sudden staring with deviation of right eye to right side in 2002, when he was 2-year-old and was diagnosed as probable absence seizure though no treatment was started. while initially, these episodes were 1-2 per day, these increased to 5-10 episodes per day over a period of two years, and patient was put on valparin. he had history of aggressive behaviour, which would increase in presence of any infection (had two attacks of hand-foot-mouth disease <5-year age, croup at 6-year age) with eeg report of parieto-occipital spikes and mri brain showing no significant abnormality, and he was diagnosed as having probable adhd (attention-deficit/hyperactivity disorder). from 2006 onwards (6-year age), he started having behaviour disturbances with fluctuating mood, biting clothes, bruxism. he underwent squint surgery in 2008. since 2010 (10-year age), he had significant worsening of scholastic performance, would drink milk excessively, developed slurred speech, memory issues and episodes of bed wetting. over the years, he developed abnormal eye movements, delayed speech and language and was diagnosed as regressive autism and homeopathic treatment was started. in 2014 (14year age), he had increase in aggressive behaviour and was taken to usa for further management. in 2019, cunningham panelanti dopamine receptor d1 increased; anti-tubulin increased; anti-dopamine receptor d2 normal. he had received rituximab, iv steroid, iv-ig, bortezomib. csf was negative for iglon 5 antibody, gfap, gaba-b, nmda glutamate receptor antibody, caspr 2, protein 2, lgi 1, ampa-1 & 2 antibodies. lupus anticoagulant was negative. fdg-pet mri whole body on 21th february 2021hyper metabolism in bilateral striatal nucleiconsistent with limbic encephalitis. he was diagnosed as auto immune encephalitisg6pd deficiencyseborrheic dermatitispandas (pediatric autoimmune neuropsychiatric disorder associated with streptococcal infection). he was on tab.clonidine 0.1 mg, tab. divalproex 250 mg, tab. risperidone 1 mg. he had received 9 doses of rituximab (last29th april 2021), 7 cycles of bortezomib (last19th july 2021) and he was on regular iv ig monthly (last1-3rd september 2021) and weekly iv methyl prednisolone. therapeutic plasma exchange is the primary treatment for severe and life-threatening pandas, and may be used alone or alongside ivig, high-dose iv corticosteroids, and/or rituximab. along with ivig, patients were given preventive antibiotics to avoid worsening of symptoms triggered by infection in the future. they were also provided with typical psychiatric treatment, which involved utilizing antiobsessional drugs and cognitive-behavioural therapy. in order to achieve the best relief from symptoms, a combination of immunomodulatory therapy, antibiotic prophylaxis, and targeted symptom treatments is essential. patient thus came to our neurology department for further evaluation on 7th september 2021. on examination, the patient was conscious and agitated, with bilateral plantar flexor response and no signs of neck rigidity. routine investigation vitamin b12-657, ammonia-42, vitamin d-70.04, tsh-3.15, c-reactive protein (crp)negative, antistreptolysin o (aso) titrepositive (215.07 iu/ml). patient was managed with continuation of tab.resperidal 1 mg, tab. clonidine 0.1 mg, tab. divalproex 250 mg. patient was discharged on 13th september 2021 with advice for opd follow up and review with plan for fdg pet mri and monthly ivig. patient was again admitted for ivig therapy between 29th september to 4th october 2021 and discharged in stable condition and again admitted for monthly ivig cycle (total 120g over 3 days) on 12th november 2021 and patient was managed with ongoing treatment and was discharged on 15th november 2021 with advice for opd follow up and next ivig after 6 weeks (2g/kg total dose, divided over 3 days). r and k a case on pandas syndrome online first indian j pharm drug studies | 3 on 7th december 2021, patient was admitted for tonsillectomy. during the admission, routine hematological and biochemical investigations were normal. serum aso (antistreptolysin o) titre and crp (c-reactive protein) were negative. gram stain, fungal stain, afb stain and stain for cryptosporidium and nocardia were negative from tonsillar pus. serum ebv virus and hv virus rt pcr were negative. aerobic culture from tonsillar pus show growth of alpha hemolytic streptococci, klebsiella, e.coli, rest fungal and afb. anerobic culture shows no growth. serum tpo and anti-thyroid antibody was positive on background of t3-3.2, t4-1.44 and tsh-5.76. patient was managed with appropriate antibiotics, antipsychotics, thyroid replacement and other symptomatic and supportive treatment. on 29th december 2021, patient was again admitted for ivig therapy. immunoglobulin immunorel10 10g (total 10 vials) thrice a day for 3 days were given. serum tsh-1.06, t4-1.48, t3-3.5. advised tab.erythrocin 250 mg twice a day to continue as streptococcal prophylaxis. after receiving the full course of ivig therapy, the patient was discharged in a stable condition. follow up advised after one month. patient condition improved and no notable symptoms showed. discussion during childhood, tics and obsessive-compulsive disorders (ocd) are rather frequent. they affect 1% to 4% of the pediatric population and 2% to 3% of the pediatric population, respectively.11 swedo12 discovered a subset of patients whose tics and/or obsessive-compulsive disorder worsened briefly following group a beta hemolytic streptococcus infections. the acronym pandas has been coined to describe this illness.12 anxiety, emotional lability, focus issues, hyperactivity, and other symptoms are common in these patients.13 our patient had a background of autoimmune encephalitis following criterias of prepubertal onset of neuropsychiatric manifestations, vocal repetitive spells (tics disorder), temporal association between onset or exacerbation of symptoms and prior infection. orvidas et al13 reported two clinical cases of two siblings who suffer from recurrent tonsillitis as well as the pandas syndrome. due to repeated streptococcal pharyngitis, two siblings—one with a tic problem and the other with obsessivecompulsive disorder (ocd)—had tonsillectomy. both patients showed notable improvement in their mental health conditions at the most recent follow-up appointment, which was held 11 months after surgery. their neuropsychiatric problems resolved after their tonsillectomy.13 heubi et al14 describe clinical improvement in two patients with ocd and tics who had recurrent tonsillitis. two siblings who met the criteria for the diagnosis of pandas and had recurrent tonsillitis whose symptoms, which were linked to ts and ocd, were made worse by tonsil infections. according to the pandas diagnostic criteria, a patient must already had a neurological disorder, usually ts or ocd. patients with problematic obsessions and compulsions were said to have ocd, here their tonsils were removed.14 our patient also underwent tonsillectomy to prevent further throat infection. we presented this case to increase awareness of this disorder, which can lead to major declines in academic performance and social adjustment in prepubescent and early adolescent children. after taking antibiotics, some kid’s condition improves fast, but if they have streptococcal infection again, their symptoms can reappear. the majority recover with few long-term problems. for others, it may develop into a persistent issue that need the occasional prescription of antibiotics to manage infections that might result in flare-ups. some children may be unable to function in social or academic settings due to pandas symptoms. if left untreated, pandas symptoms might get worse and cause irreversible cognitive impairment, thus can develop into a chronic autoimmune disease in certain children. conclusion limited reports of pandas in india may stem from inadequate awareness and under-recognition among healthcare providers. in any child presenting with abrupt onset or exacerbation of tics, ocd or late onset of attentiondeficit/hyperactivity disorder (adhd), clinicians need to focus on the history of throat infection and evidence for gabhs infection. pandas is a condition characterized by episodic episodes that can resolve spontaneously; if the child is not in distress or facing disruptions in daily activities, observation may be enough. early consideration of immunomodulatory therapy is recommended, as nsaids or a brief course of oral corticosteroids may be effective for symptom relief in new cases, while individuals with longstanding symptoms may need more intensive and prolonged immunotherapeutic treatments. a proper cross referral between the pediatricians and the psychiatrists can aid in minimizing and eliminating the morbidity and the impairment which are connected with this disease. reference 1. pietrangel a. pandas: a guide for parents [internet]. healthline; march 29, 2019. assessed on january 21, 2022. available from: https://www.healthline.com/health/pandassyndrome 2. pandas: frequently asked questions about pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections. national institute of mental health. accessed 19/01/2022. 3. information about pandas. national institute of mental health. accessed 19/01/2022. 4. michael e pichichero. pandas: pediatric autoimmune neuropsychiatric disorder associated with group a streptococci. uptodate. waltham, ma: uptodate; june, 2016. 5. kulkarni y, warkari r. pandas: a rare case report. international journal of science and research. 2016; 5(2): 148 – 149. r and k a case on pandas syndrome online first indian j pharm drug studies | 4 6. mabrouk aa, eapen v. challenges in the identification and treatment of pandas: a case series, journal of tropical pediatrics, 2009; 55(1):4648, https://doi.org/10.1093/tropej/fmn039 7. pediatric ocd treatment study (pots) team. cognitivebehavior therapy, sertraline, and their combination for children and adolescents with obsessive-compulsive disorder: the pediatric ocd treatment study (pots) randomized controlled trial. jama. 2004; 292(16):1969-76. doi: 10.1001/jama.292.16.1969. 8. pavone p, rapisarda v, serra a, et al. pediatric autoimmune neuropsychiatric disorder associated with group a streptococcal infection: the role of surgical treatment. int j immunopathol pharmacol. 2014; 27(3):371-8. doi:10.1177/039463201402700307. 9. sigra s, hesselmark e, bejerot s. treatment of pandas and pans: a systematic review. neurosci biobehav rev. 2018; 86:51-65. doi: 10.1016/j.neubiorev.2018.01.001. 10. chiarello f, spitoni s, hollander e, et al. an expert opinion on pandas/pans: highlights and controversies. int j psychiatry clin pract. 2017 jun;21(2):91-98. doi: 10.1080/13651501.2017.1285941. 11. catarina prior a, tavares s, figueiroa s, et al. tics in children and adolescents: a retrospective analysis of 78 cases. an pediatr (barc). 2007; 66(2):129-34. doi: 10.1157/13098929. 12. swedo se. sydenham's chorea. a model for childhood autoimmune neuropsychiatric disorders. jama. 1994; 272(22):1788-91. doi: 10.1001/jama.272.22.1788. 13. orvidas lj, slattery mj. pediatric autoimmune neuropsychiatric disorders and streptococcal infections: role of otolaryngologist. laryngoscope. 2001 sep;111(9):1515-9. doi: 10.1097/00005537200109000-00005. 14. heubi c, shott sr. pandas: pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections--an uncommon, but important indication for tonsillectomy. int j pediatr otorhinolaryngol. 2003; 67(8):83740. doi: 10.1016/s0165-5876(03)00158-7. how to cite this article: dr. reshma r, dr. ajnas k. a case of pediatric autoimmune neuropsychiatric disorder associated with streptococcal infection. indian j pharm drug studies. 2024; online first. funding: none; conflicts of interest: none stated https://doi.org/10.1093/tropej/fmn039 patil et al. analytical method development and validation online first indian j pharm drug studies | 1 review article analytical method development and validation rutik nitin patil1, bhupendra l deore2, darshan ganesh gurav1 from, 1m.pharm, 2phd & m pharm, department of quality assurance, a.r.a college of pharmacy, dhule, india. abstract the main objective of the pharmaceutical business is to consistently and economically create goods with the requisite features and quality. the process of finding, creating, and assessing pharmaceutical formulations requires the creation of a methodology. the primary goal of this review article is to examine how the medication development and validation process was carried out, from the beginning of the formulation to the end of the commercial batch. reliability of the results is essential whenever an analytical method is used to generate results for an array of samples related to medicine. in the pharmaceutical sector, good manufacturing practice (gmp) rules and good laboratory practices (glp) are adhered to by means of defined validation policies, forms of validation, and validation criteria. validation is crucial to the efficient operation of the pharmaceutical sector. validation was done everywhere, from the raw material to the final, stable state. the procedure was appropriately designed, and the validation parameters—specificity, accuracy, quantitation, ruggedness, robustness, and system appropriateness testing—are specified in terms of limit of detection (lod), limit of quantitation (loq), robustness, and ruggedness with reference to specific drug examples. both the methodology and evaluation of stability make use of all the validation parameters. key words: analytical validation, method development, validation on hplc, separation technique, various parameters hese drugs may be entirely new or partially structurally modified versions of ones currently on the market. the aim of every analytical measurement is to generate data that is consistent, accurate, and reliable. validated analytical methods are necessary to do this. any legitimate analytical approach must include a means of assessing the precision, dependability, and uniformity of the statistical investigation utilizing its findings of method validation. furthermore, the validation of analytical techniques is a must around numerous laws in addition to criteria pertaining to facilities. the date a medication is added to pharmaceutical protocols and the moment it was initially made available to consumers usually coincide [1]. in such circumstances, the pharmacopoeias could not have specified the requirements and testing methodologies for these drugs. thus, it may be possible to develop fresher analytical methods for these drugs. in the process of finding, developing, and producing medications, analytical methods are crucial. drug combination products, also known as combination products, combine the therapeutic benefits of two or more medications into a single item in order to fulfil the needs of individuals whose work is not formerly treated by them. this requires the invention and development of analytical methods. the analytical chemist in charge of creating and verifying access this article online received – 20th july 2024 initial review – 28th september 2024 accepted – 28th october 2024 quick response code analytical procedures may face significant challenges as a result of these combination products. to verify the identification, purity, potency, and effectiveness of pharmaceutical items, regulatory laboratories employ legitimate test techniques that are produced by these procedures. identification and measurement of contaminants belong to one of the most crucial steps in creating pharmaceutical procedures for quality and safety [1].  basic criteria for new method development for drug analysis a. the medication is unlikely to be listed as official in any pharmacopoeias. b. analytical methods for a medicine may not be permitted in publishing under any specific creation aspect due to interference through formulation excipients. c. due to patent laws, appropriate analytical techniques for the drug could not be accessible. d. there could not be adequate analytical techniques obtainable for assessing the drug through your system.  various reasoning for analytical technique analytical techniques have progressed, and during analytical analyses, hplc procedures produce vast amounts of data. even though hplc is a flexible separation method with many __________________________________________________ correspondence to: rutik nitin patil. department of quality assurance, a.r.a college of pharmacy, dhule, india. email: rutik.patil57@gmail.com t mailto:rutik.patil57@gmail.com patil et al. analytical method development and validation online first indian j pharm drug studies | 2 uses, its many variables, which must be precisely adjusted before each run, can occasionally make the procedure dangerous. consequently, it becomes imperative to comprehend these methods on a deeper level. furthermore, the process of optimizing multiple variables that need to be controlled at the same time in order to achieve the required separations, such as the mobile phase's ph, buffer concentration, rate of flow, column temperature, detector raise length, etc [2]. the chemometric technique is highlighted in all of these studies as a new instrument in the arena of pharmaceutical analysis. the use of laboratory patterns to establish design spaces—a crucial element of the quality with the design approach of analytical methods—was recently covered by rozet et al [3]. figure 1: life cycle of the analytical method [4] objectives: 1. development and validation of analytical methods: from the formulation phase all the way up to the manufacturing of commercial batches, analytical techniques are essential. to make sure that these techniques yield accurate and trustworthy findings, they must be researched and verified. 2. importance of validation: validation guarantees that analytical procedures satisfy specified requirements for system appropriateness, ruggedness, robustness, limit of detection (lod), limit of quantitation (loq), accuracy, specificity, and precision. these criteria are necessary to guarantee the quality of pharmaceuticals. 3. regulatory bodies: validation procedures follow gmp guidelines and are recorded. in order to guarantee the security, effectiveness, and caliber of their goods, pharmaceutical companies must abide by these rules. 4. stages of validation: testing is done on raw materials and final products in every stage of the production method to ensure their stability. this all-encompassing strategy guarantees that the procedure is validated and satisfies necessary requirements. 5. method development: in order to assure that analyses are sensitive, selective, and capable of reliably delivering accurate findings under a variety of the settings window, proper method development entails improving and refining these processes. 6. routine and stability testing: validation parameters are used in stability testing as well as routine analysis, as the latter evaluates the stability of the final product and the pharmaceutical ingredient that is active (api) over time [5,6,7]. method development the development and validation of analytical methods are crucial to the process of making discoveries and producing medications. these techniques are employed to guarantee drug goods' authenticity, potency, purity, and effectiveness. when creating procedures, there are several things to take into account. initially, data on the analyte's physiochemical properties (pka, log p, and solubility) are recorded, and if uv detection is required, the suitable detection mode is chosen, and if uv detection is needed, the appropriate mode of detection is determined. the validation of a stabilityindicating hplc method constitutes the principal focus of analytical development efforts. trying to isolate and quantify the primary active ingredient, any reaction impurities, any synthetic intermediates that are accessible, and any degradants are the objectives of the hplc-method [8, 9]. criteria for the development of new analytical method: the identification of the product is predicated on the evaluation procedure of pharmaceuticals. the addition of a medicine to pharmacopoeias and its commercial release date frequently fall on the same day. this is brought on by new toxicity reports, patient resistance developing over time, potential concerns about the prolonged and increased use of these medicines, and the launch of more potent drugs by rival companies. the novel medication or combination of medications might not be classified in any pharmacopoeia. it's possible that analytical methods for quantifying the drug in body fluids are unavailable. the present tests may require the use of pricey materials and chemicals. it may also need timeconsuming and sometimes unreliable extraction and separation processes [10, 11].  hplc method development 1. recognizing the drug molecule's physicochemical characteristics. when developing a technique, a medicinal molecule's physicochemical characteristics are crucial. the physical characteristics of the drug molecule, such as its ph, polarity, solubility, and pka, must be studied in order to build a method. one of a compound's physical characteristics is polarity. it aids in the determination of the mobile phase's solvent and composition by the analyzer. the polarity of molecules that are provides an explanation for their solubility. solvents that are polar, like water, and nonpolar, like benzene, do not combine. ph is characterized as the negative logarithm patil et al. analytical method development and validation online first indian j pharm drug studies | 3 of the h+ ion concentration to base 10. the ph scale is log10[h3o+]. sharp, symmetrical peaks are consistently observed in hplc when ionizable analytes are selected at the proper ph. quantitative analysis requires sharply symmetrical heights in order to ensure low relative standard deviations between injections, minimal detection limits, and predictable retention durations [12, 13]. 2. the chromatographic conditions chosen: selection of column: in method development, choosing the stationary phase or column is the first and most crucial step. without a reliable, high-performing column, it is difficult to design a robust and repeatable procedure. stable and repeatable columns are crucial to prevent issues with irreproducible sample retention through method development. all samples may normally be separated using a c8 or c18 column, which is highly recommended. these columns are built from carefully purified, lower-acid silica as well as being particularly developed for the separation from basic chemicals [14]. the primary ones are the column diameters, the silica substrate qualities, and the bonded stationary phase features. for a number of physical reasons, silica-based packing is preferred in the majority of current hplc column [15]. 3. improving the mobile stage a) buffer selection: the intended ph determines which buffer to use. when reversed phase on silica-based packaging, the usual ph range is 2 to 8. since buffers regulate ph best at their pka, it is critical that the buffer's pka be somewhat near the intended ph. generally speaking, pick a buffer with a pka value. general consideration for buffer selection: a. phosphate seems to be more soluble in methanol/water than in acetonitrile or thf/water. b. due to their significant hygroscopicity, some salt buffers may result in chromatographic changes, including greater tailing of basic compounds and perhaps different selectivity. c. ammonium salts tend to dissolve more readily in organic/h20 mobile phases [16]. b) effect of ph the pka of the ionizable analyte should be used as a guide to determine the proper mobile-phase ph. by doing this, it is guaranteed that the desired analyte is ionized or neutral. one of the most useful tools in the chromatographer's toolbox is the ability to modify the ph of the mobile phase. this feature enables retention and selectivity to be changed simultaneously, offering a tactical way to maximize separation conditions, especially for important component pairings in the sample. in order to customize chromatographic conditions and provide the required separation results, ph modification is essential [17]. c) effect of organic modifier: in reverse phase hplc, choosing the kind of organic modifier is rather straightforward. methanol and acetonitrile are the most common options (thf is rarely used). since it would not be possible to elute every component between k (retention factor) 1 and ten using a single solvent strength during isocratic conditions, gradient elution is typically used with complicated multicomponent samples [18]. choose the detector the hplc's detector is an essential component. the chemical composition of the analytes, potential interference, required detection limit, detector availability, and/or detector cost all affect the detector choice. the uv-visible detector is a uvvisible dual-wavelength absorbance detector that is flexible and stays hplc. the high sensitivity of this detector is required for routine uv-based applications, such as low-level impurity detection and quantitative analysis. the photodiode array (pda) detector offers advanced optical detection with aqueous analytical hplc, preparative hplc, or lc/ms system solutions. the developments in optics and integrated software allow for the achievement of high spectrum and chromatic sensitivity. the ideal choice for analysing components with little to no uv absorption is the refractive index (ri) detecting device because of its exceptional sensitivity, stability, and reproducibility. the multiwavelength fluorescence detector can measure minute quantities of target chemicals and offers high sensitivity and selectivity fluorescence detection [19, 20]. technique of separation a) isocratic separations analyte-eluent and analyte-stationary-phase interactions remain stable over the course of the run when there is an isocratic, constant eluent composition. this also guarantees equilibrium conditions in the column and the real velocity of compounds flowing through the column. despite the low separation power—the amount of compounds that might be resolved—this increases the predictability of isocratic separations. the peak capacity is modest, and the resulting peak widens as the length of time the component is kept on the column [21]. b) gradient separation a system's separation power is greatly increased by gradient separation, mostly due to the abrupt rise in apparent efficiency (reduction in peak width). peak width decreases in the situation when the chromatographic zone's tail is constantly affected by a more potent eluent composition. peak width is a function of gradient slope, which measures how quickly the eluent composition changes. shifting the gradient since it might not be possible to elute every component between k patil et al. analytical method development and validation online first indian j pharm drug studies | 4 (retention factor) from 1 to 10 using only one solvent's strength during isocratic conditions, gradient elution is used for complicated multicomponent samples. if the ratio is less than 0.25, isocratic would be sufficient; if it is greater than 0.25, gradient will be advantageous [22, 23]. preparing samples for technique development it is necessary for the drug material under analysis to be stable in solution (diluent). the solutions should be prepared in amber flasks during the early stages of method development until it is shown that the active ingredient is stable at room temperature and does not deteriorate under typical laboratory circumstances. it is advised to filter the sample solution using 0.22 / 0.45 µm pore-size filtration to remove any particles. for hplc analysis, filtration is a preventative maintenance technique [24, 25]. optimization of methods determine the shortcomings of approach and use experimental design to strengthen it. evaluate the approach's effects on different samples, equipment setups, and environmental conditions. this iterative process aids in the methodology's improvement and ensures that high-performance liquid chromatography (hplc) analysis is robust, reliable, and applicable under a variety of conditions [26]. validation of methods the procedure of laboratory testing is carried out to show that an analytical method's performance characteristics match the demands of the planned analytical application is known as validation. any new or revised procedure has to be validated to make sure it consistently yields reproducible and trustworthy results, regardless of whether it is performed by several operators in various laboratories using the same utilizing equipment that satisfies requirements, is operational, functional, and accurately calibrated is crucial to the validation process. analytical methods are carefully examined during the validation process, after which they are either approved for use or rejected if they don't match the necessary standards. this ensures the correctness and dependability for statistical results in multiple uses [27, 28]. validation analyst interpretation is used to validate methods because there is no industry standard for assay validation. industry committees, regulatory bodies, and the international conference on harmonization (ich) of technical standards for the registration of medicines in human use have all placed a great deal of emphasis on method validation. methods that have been verified by multiple laboratories are offered by the us environmental protection agency (us epa), the resource conservation and recovery act (rcra), the american association of official analytical chemists (aoac), the us environmental protection agency (usp), and other scientific associations [29]. the food and drug administration (us fda) has put forward criteria on the submission of analytical and sample data for the purpose of technique validation. particular criteria have been established by the united states pharmacopoeia (usp) for technique validation and substance assessment [30]. the four most prevalent types of analytical processes are the focus of the debate on their validity. 1. tests for identification. 2. measurements that quantify the amount of contaminants. 3. limit assessments for impurity control. 4. quantitative analyses of the active ingredient in drug substance or product samples, as well as other specific components within the drug product [31]. various need to be validated. 1. whenever the condition for which the technique has been validated changes, such as an instrument with new features, prior to its introduction into regular usage. 2. whenever modifications are made to the procedure that go beyond its initial parameters [32]. various parameters are given below: a) accuracy accuracy is the level to which the value being measured agrees with the true or recognized value. in practical terms, accuracy is the discrepancy between the actual value and the mean value that was produced. to ascertain accuracy, the process is applied to samples with known analyte concentrations. accuracy is computed as a share of the analyser retrieved by the assay based on test results. it is commonly expressed as the assay-based recovery of the additional analyte levels that are known; this indicates the degree to which the real values are captured by the analytical method [33, 34]. b) precision in analytical procedures, precision is associated with the degree of agreement and dispersion between several measurements taken under particular circumstances from different samples of the same homogenous material. a crucial factor in determining the repeatability of the entire analytical process is precision. precision consists of two elements: intermediate precision and repeatability. repeatability is the difference between two measurements made by one analyst using the same tool these precision assessment components guarantee a thorough comprehension of the analytical method's repeatability and dependability across various operators and settings [35, 36]. %rsd=std dev.*100/mean c) linearity patil et al. analytical method development and validation online first indian j pharm drug studies | 5 a technique for analysis is said to be linear if it can yield test results that are precisely matched to the concentration of any analyte in the sample. one way to demonstrate linearity on the test material is to dilute a standard stock solution, or another method is to weigh each component separately in synthetic blends of the test product. the linearity is confirmed via injecting five to six times at least five standards at doses between 80 and 120 percent of the estimated concentration range. a precise mathematical computation must determine the proportionality of the reaction, or it must be proportional right away to the analyte concentrations. d) limit of detection [loc] analysing an analyte's threshold for detection using a single method is the easiest approach to quantifying it in a sample when it can be identified but not accurately measured. is typically used to express the lod. determination: for noninstrumental approaches, the lowest level at which the analyte may be reliably detected is established, and samples with known concentrations of the analyte are analysed to determine the detection limit. ich standards: the ich outlines a standard procedure that involves comparing the observed signal between samples that have known analyte concentrations and those that are blank. it is determined what the analyte's minimal concentration is at which it may be accurately identified. lod computations pertaining to instrument sensitivity lod(mg/l)=3×noise/signal the linearity samples' lowest concentration a 2:1 or 3:1 signal-to-noise ratio is often considered adequate [37, 38, 39]. e) limit of quantification (loq) minimal level of analyte for the purpose of evaluating low analyte levels in test matrices; this limit functions as a quantitative test parameter. the quantitation limit serves as a threshold for accurate quantitative measurements on analytical procedures and is essential for detecting contaminants and/or impurities in samples [40]. a single analytical procedure's limit of quantitation, also known as its quantitation limit, is the smallest quantity of analyte in an experiment that is capable of being quantitatively measured with appropriate precision and accuracy. the loq is often determined from a s/n ratio (10:1) for analytical processes that display baseline noise, and it is typically validated by injecting standards that both meet an acceptable percent absolute standard deviation and this s/n ratio [41, 42]. f) specificity every development step should see a specificity in the analytical method. the method should be able to conclusively determine if the target analyte is present in the presence of every anticipated component, including sample blank peaks, excipients, degradants, and sample matrix [43]. to ascertain how long each medication would remain in the sample and in a combination, specificity testing was done. when the standard medications were tested separately, their respective retention times were found to be 3.750 min for nitazoxanide and 1.533 min for ofloxacin. when the drugs were tested together, their respective retention times were found to be 3.760 min with nitazoxanide and 1.542 min for ofloxacin. g) range the analytical techniques range is the range of a compound's concentration inside the sample between its highest and minimum levels at which the linearity, precision, and accuracy of the analytical method can be shown to be sufficient. the range is often given in percentages or parts per million, much like the test results generated by the analytical technique. 1. 80–1200% of the test concentration is used in the experiment. 2. consistency of content and test concentration between 70– 130% 3. impurities: 120% of the recommended level is the reporting threshold for impurities. test and impurities: up to 120% of the test-specific reporting criteria. 4. q-20% solubility to 120% [44]. a) b) c) a) linear b) non-linear figure 2. plot for ranges of an analytical technique h) robustness the robustness of an analytical method is defined as the degree of repeatability of test results via the same samples analysed under various standard test conditions, which could include different operators, different laboratories, and possibly different operational along with environmental conditions, while staying within the assay's defined parameters. testing for ruggedness is typically suggested when the process will be used in several labs. ruggedness is evaluated by calculating the degree of outcome repeatability as a function of the test variable. this reproducibility could be used to gauge the analytical method's ruggedness by comparing it to the assay's accuracy under typical conditions [45]. i) system suitability determination tests for system compatibility are an essential component of liquid chromatographic techniques. they are employed to confirm that the chromatographic systems detection sensitivity, resolution, and repeatability are sufficient for the intended analysis. the foundation of the testing is the idea that patil et al. analytical method development and validation online first indian j pharm drug studies | 6 the tools, electronics, processes involved in the analysis, and materials to be examined all work together as a cohesive system that is capable of being assessed as such. measurements have been made of peak resolution, number of theoretical plates can, peak tailing, and capacity to assess the applicability of the used technique [45]. j) forced degradation studies in order to purposefully deteriorate the sample, stress experiments or forced degradation are conducted. by producing possible degradation products, these investigations assess an analytical method's capacity to quantify an active chemical and its breakdown products independently. drug materials are subjected to acid, base, heat, light, and oxidizing agents during method validation, resulting in 10% to 30% degradation of the active ingredient. forced degradation studies are conducted for various reasons, such as developing and validating stability-indicating methodology, identifying drug substances as well as drug product degradation pathways, and differentiating between degradation products in formulations related to drug substances and those related to non-drug substances [45]. k) stability studies the durability of both samples and standards is determined during validation under typical circumstances, under typical storage settings, and occasionally within the instrument to ascertain whether specific storage conditions—such as refrigeration or light protection are required [45]. conclusion the aim of every analytical measurement is to generate data that is consistent, accurate, and reliable. these techniques are employed to guarantee drug authenticity, potency, purity, and effectiveness. these state about various parameters and techniques, which shows that are suitable for the procedure and can be developed for the tests. in this article, we have discussed how validation was carried out with the use of specific validation parameters—including linearity, loq, lod, range, robustness, and system suitability. in the pharmaceutical industry, validation is an essential technique that ensures quality is included in the processes that support drug development and production. it assures all parameters and conducts the phases of trails. references 1. fda guidance for industry. analytical procedures and method validation, 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supplement 2. a. united states pharmacopeial convention. 45. alsante km, ando a, brown r, et al. the role of degradant profiling in active pharmaceutical ingredients and drug products. advanced drug delivery reviews. 2007; 59(1):29-37. how to cite this article: patil rn, deore bl, gurav dg. analytical method development and validation. indian j pharm drug studies. 2024; online first. funding: none; conflicts of interest: none stated objectives: 1. development and validation of analytical methods: from the formulation phase all the way up to the manufacturing of commercial batches, analytical techniques are essential. to make sure that these techniques yield accurate and trustworthy findings,... 2. importance of validation: validation guarantees that analytical procedures satisfy specified requirements for system appropriateness, ruggedness, robustness, limit of detection (lod), limit of quantitation (loq), accuracy, specificity, and precisio... 3. regulatory bodies: validation procedures follow gmp guidelines and are recorded. in order to guarantee the security, effectiveness, and caliber of their goods, pharmaceutical companies must abide by these rules. 4. stages of validation: testing is done on raw materials and final products in every stage of the production method to ensure their stability. this all-encompassing strategy guarantees that the procedure is validated and satisfies necessary requireme... 5. method development: in order to assure that analyses are sensitive, selective, and capable of reliably delivering accurate findings under a variety of the settings window, proper method development entails improving and refining these processes. 6. routine and stability testing: validation parameters are used in stability testing as well as routine analysis, as the latter evaluates the stability of the final product and the pharmaceutical ingredient that is active (api) over time [5,6,7]. method development agrawal and kulkarni ai applications in drug discovery and development online first indian j pharm drug studies | 1 review article application of artificial intelligence in drug discovery, designing, clinical trials and repurposing rashmi agrawal1, meghashyama kulkarni2 from, 1consultant, devmata hospital, bhopal, madhya pradesh, india, 2consulting oral pathologist, bengaluru, karnataka, india abstract drug development, right from discovery, designing, and repurposing, along with various phases to clinical trials, is a complex and timeconsuming process that traditionally relies on the experience of labor forces, along with incessant trial-and-error experimentation. the emergence of various artificial intelligence (ai) tools and techniques is redefining the pharmaceutical industry. the integration of aidriven methodologies into all stages of the drug development pipeline has enhanced the efficiency and effectiveness of the process. with this review, we aim to provide insights into the application of ai at various stages mentioned, with an emphasis on the latest advancements. the role of ai in speeding up the process of drug development while giving distinct and accurate outcomes is highlighted as well. finally, we addressed the current challenges in employing ai along with future perspectives in order to enhance ai-augmented drug development, ultimately offering significant benefits to patients and society. key words: artificial intelligence, drug discovery, clinical trial design, neural networks, drug repurposing rtificial intelligence (ai) involves the least amount of human intervention possible when utilizing a computer to imitate intelligent behaviour [1]. ai has been implemented in the pharmaceutical industry in recent years. one of the important aspects in medicinal chemistry, drug discovery that involves identifying, formulating, and developing new medications, is an arduous, techniquesensitive, and time-consuming process with long-established protocols that require exorbitant resources with several trial and error experimentations needing a good deal of time [2]. this complex process, further requires an average of 12 years and a cost of $2.6 billion to forge ahead a single molecule from inception till food and drug administration (fda) approval. in spite of these strenuous endeavours, the process is marked by unfavourable adverse effects of the drugs, high attrition rates, and tenacious challenges in addressing chronic diseases, including cancers and diabetes mellitus [3, 4]. the emergence of ai marks a revolutionary drift in drug development, with the advent of computational tools designed to supplement human capabilities and intelligence rather than replace them [5]. ai leverages suave algorithms for autonomous decision-making, hence revolutionizing the pharmaceutical industry [6]. ai techniques such as machine learning (ml) and deep learning (dl) offer the potential to accelerate the above-mentioned processes by enabling more coherent, efficient, and precise analysis of large amounts of data [7]. ai has also been able to predict the toxicity of drugs; access this article online received – 23rd july 2025 initial review – 24th july 2025 accepted – 25th july 2025 quick response code besides its role in drug-target interaction predictions, clinical trial design, and drug repurposing, it is aiding in the reformation of drug development approaches [8, 9]. this narrative review aims to unveil the transformative impact of ai on the whole process of drug discovery till the completion of development and sheds light on the role of ai in drug repurposing as well. the paper also emphasizes the importance of ai in accelerating the development of novel therapeutics while juxtaposing it with traditional methodologies. furthermore, we also discuss the ongoing challenges with respect to the usage of ai, along with the future of ai in the pharmaceutical industry and the ways it can significantly restructure the global healthcare system. revolution of ai the dartmouth workshop conducted in 1956 is considered the foundational event for the advent of ai. it is described as intelligence displayed by man-made machines, dedicated to developing theories, technologies, applications, and methods aimed at replicating, enhancing, and extending human intelligence [10]. over the past 70 years, ai has evolved from a theoretical notion into a powerful industrial adaptation, revolutionizing industries such as manufacturing, agriculture, finance, and healthcare [11-13]. ai includes various subtypes, some of which are described as follows: machine learning (ml), natural language processing (nlp), computer vision (cv), and fuzzy logic __________________________________________________ correspondence to: dr. rashmi agrawal. devmata hospital, bhopal, madhya pradesh, india. email: atharvapub@gmail.com a mailto:atharvapub@gmail.com agrawal and kulkarni ai applications in drug discovery and development online first indian j pharm drug studies | 2 (fl). ml works on algorithms trained for decision-making that learn from the already analyzed data. ml is further classified into supervised learning and unsupervised learning. in supervised learning, pre-catalogued/ labelled data is used as input, whereas in the latter, training data is not catalogued/labelled, and the system must perceive and label the said data. a third sub-category exists, known as semisupervised learning, a combination of the two previously explained types [14]. deep learning (dl) is a subgroup of ml based on systems that use artificial neural networks (ann), which imitate the human brain, and act by interpreting and drawing conclusions from the given data. whereas, nlp is a branch of ai that recognizes natural language and builds communication between machines and humans. cv is a subtype of ai that allows computers to discern an image and distinguish the individual elements of that particular image by assigning them a meaning. on the other hand, fl uses nonbinary values to solve issues that normally require tackling with more values, hence deciphers the problems that classical logic cannot solve [15, 16]. figure 1: classification of anns [16, 17] an ml algorithm for any task begins with task definition and ends with model application. different steps with detailed explanations are described in table 1. table 1: steps in machine learning [16, 17] s. no step description 1 pre-processing  ensures that the algorithm easily interprets the datasets  includes data loading, normalization, aggregation, and standardization of the dataset 2 exploratory data analysis (eda)  tests several hypotheses and yields a better understanding of dataset variables and their interrelationships.  check for errors and missing values  helps determine if the statistical techniques that are used for the data analysis are appropriate. 3 model selection  creation of a predictive model  algorithms are often grouped according to the ml techniques used such as supervised or unsupervised etc.  few ml algorithms used in medical research are support vector machine (svm), multinomial logistic regression (mlr), bayesian networks (bn), and decision tree (dt).  few dl algorithms used in medical research are all the sub-types of anns, long short-term memory (lstm), and mixed networks. 4 model processing and evaluation  datasets are divided into test and train sets, and further crossvalidation is done.  if the model shows discrepancies, it can be rebuilt using improvement strategies, which is known as ‘tuning’  model evaluation is done with anns. data, computation, and algorithms, the essential components of ai, serve as the basis of the ai-driven pharmaceutical research [18]. sources of data in the pharmaceutical industry include public and commercial datasets, and research datasets generated through data mining. the evolution of computational power and the advent of various algorithms have provided critical support for ai pharmaceutical companies in carrying out research using the extremely potent tool, ai [19]. challenges faced with traditional drug discovery and development methods traditional drug discovery, as described before, is a complex, time-consuming, laborious, and costly undertaking. to bring a single drug to the market, it typically takes over a decade and an average cost exceeding $2 billion. each and every stage, starting from target identification and validation till preclinical testing and clinical trials, requires immense trial-and-error experimentations with huge irreversible expenses along the way [20]. furthermore, attrition rates of the drug candidates are very high, with almost 90% of them failing owing to insufficient efficacy and/or safety concerns during clinical trials. additionally, financial losses multiply when toxicity is detected after-market release of the drugs [21]. hence, evaluation of toxicity and safety is of paramount importance during drug development, which can be achieved by a better understanding of the protein–ligand interactions and predictability of toxicity. the development of computational methods and ai has emerged as a promising approach to tackle the above-mentioned obstacles while developing a drug. the further sections delve into the applications of ai in all the steps of drug development [21]. role of ai in drug discovery there exist more than 1060 molecules that help in the development of novel drugs [22]. al has the ability to collect https://pubmed.ncbi.nlm.nih.gov/38758610 https://pubmed.ncbi.nlm.nih.gov/38758610 agrawal and kulkarni ai applications in drug discovery and development online first indian j pharm drug studies | 3 positional information about molecules within the space to search for bioactive compounds and select appropriate molecules via virtual screening (vs). a few chemical spaces that have open access are chembank, pubchem, chemdb, and drugbank [17]. quantitative structure-activity relationship (qsar)-based computational models are capable of quickly predicting large numbers of physicochemical parameters of the drug candidates. ai-based qsar models, such as support vector machines (svms), linear discriminant analysis (lda), random forest (rf), and decision trees, are being applied to speed up qsar analysis [23]. deep docking (dd), an open-source protocol for aienabled vs, was developed by gentile and colleagues. it is supposed to be one of the fastest ai-enabled docking platforms being tested on one billion-plus molecular libraries [24]. the input data in dd consists of a particular molecule’s simplified molecular input line entry system (smiles) and the target’s structure. dd executes molecular docking for a small subset of a large library to deduce the ranking of the unprocessed datasets, followed by ligand-based prediction for the rest of the library. in this way, dd scraps off undockable molecular structures without wasting computational resources [25]. further, diverse ml techniques, including naïve bayesian algorithm (nb), k-nearest neighbor algorithm (knns), support vector machine (svms), and anns, can also be used for vs. although anns and svms are commonly considered as the most accurate, nb is excellent in identifying favourable scaffold fragments, while knn is easy to implement and utilizes multi-task learning (mtl) {mtl training, a sub-field of ml, involves training a model to perform multiple tasks at the same time}. amalgamating various ml algorithms is preferred as it can enhance performance [26]. dl algorithms are proven to show increased predictability compared to traditional ml models, especially in distribution, metabolism, excretion, and toxicity (admet) data sets [27]. the recent rise of graph neural networks (gnns) has set in motion a new paradigm in admet model designing. gnns offer an informative and compact representation of datasets [28]. the efficacy of gnn algorithms in predicting physiochemical properties of drugs has been validated by frameworks such as chemi-net and molecule-net, which have superior potential when compared to ml models [29]. role of al in drug designing the most important aspect of drug designing is to predict the structure of the target protein in order to design the drug molecule. ai tools can assist by predicting the 3 dimensional (3d) protein structure, thus helping to predict the effect of a drug molecule on the target prior to its synthesis or production [30]. alphafold is an ai tool based on dnns, used to scrutinize the distance between the adjacent amino acids and the correlating angles of the peptide bonds to predict the 3d target protein structure [17]. the development of alphafold is expected to revolutionize personalized medicine and drug discovery. it represents a significant step forward in the use of ai in structural biology. ai platforms such as self-organizing map (som) are leveraged to link several compounds to numerous targets along with bayesian classifiers and similarity ensemble approach (sea) algorithms, which can be used for integration between the pharmacological profiles of drug molecules and their possible targets [31]. newer methods based on nlp are implemented, which use the amino acid sequences from sequence databases to learn and predict structural and functional patterns. evolutionary scale modeling-based software esmfold was developed by lin et al. in 2022, which utilizes a masked transformer protein language model with a deep understanding of biological properties, trained with over 15 billion parameters [32]. apart from ml algorithms, dl models, such as platform for analytics and distributed machine learning for enterprises (padme) and deepaffinity, have the ability to predict drugtarget interactions by integrating drug and target features. padme helps forecast the strength between drugs and target proteins, leading to accurate predictions of therapeutic efficacy and mechanism of action. deepaffinity combines rnn and cnn with labeled and unlabeled data, providing a subtle understanding of drug-protein interactions [33, 34]. apart from target protein prediction, the prediction of drug– target binding affinity (dtba) is critical for evaluating the efficacy of drug molecules. ai tools such as chemmapper and sea, along with ml and dl techniques such as simboost, deepdta, and padme, have been employed to accurately predict dtba [34]. these ai approaches have provided an upgrade to the traditional methods by deploying computational models for predicting interactions between drugs and protein targets. in recent times, dl methods have been used in de novo drug designing [35]. in 2018, popova et al. developed a reinforcement learning (rl) algorithm for de novo drug synthesis, which involves employing generative and predictive dnns to develop new drug molecules. the generative models are used to produce more unique molecules, whereas the predictive models foretell the properties of the developed compound [36]. the involvement of ai in the de novo drug design is beneficial to the pharmaceutical sector as it provides optimization of the already-learned data while suggesting possible synthesis routes for drug molecules, leading to prompt lead design and development [37, 38]. role of al in clinical trials clinical trials are necessary to prove the safety and efficacy of a drug for a particular disease condition and require anywhere from 6–7 years with substantial financial investments [17]. clinical trial failure rates, especially in oncology and other diseases, can reach as high as 95% which further contributes to financial strain. applying ai in various steps of clinical trials helps to improve patient stratification, intensify recruitment efficiency, and in the long run increase the likelihood of trial success [18]. in silico clinical trials (isct) are anticipated to agrawal and kulkarni ai applications in drug discovery and development online first indian j pharm drug studies | 4 significantly reduce the costs of clinical trials while enhancing overall success rates [39]. isct integrates physiological and pathological data to generate patient-specific predictions that provide decisions regarding diagnosis, dose selection, prognosis, and the identification of suitable patient groups [40]. a newer concept, virtual physiological human (vph), configures virtual patient groups to test the safety and efficacy of new drugs and medical devices. vph acts as an adjuvant to the traditional clinical trials by reducing the number of patients needed for the trials and enhancing the statistical power of the results [41]. additionally, ai contributes to clinical trial success by linking patient genetic data and electronic health records, along with cumulating clinical trial databases to predict drug toxicity, and assisting with patient trial matching and recruitment, plus monitoring patient adherence during trials [18, 42]. one of the major causes of failure of clinical trials is toxicity. in light of this, proctor, a toxicity prediction model, was designed by gayvert, et al. that helps to distinguish between fda-approved drugs and drugs that failed during clinical trials due to toxicity. this enables the design of therapeutic agents with less toxicity [43]. targetox is another toxicity prediction model that produces and unites pharmacological and functional properties in an ml classifier to predict drug toxicity [44]. in order to increase patient adherence in clinical trials, ai-powered facial recognition algorithms are implemented, in which patients need to record a video of themselves swallowing the pills. the ai system then confirms that the correct person has taken the prescribed medication [45]. in a trial of patients with schizophrenia, adherence increased from 50% to 90% within a span of six months, exhibiting the success of ai-driven monitoring approaches [46]. a systematic review in 2024 deduced that ai-derived drugs can have a success rate of 80– 90% in phase i trials, around 40% in phase ii trials, which have shown to have substantially higher success rates than traditionally derived drugs [47]. in the forthcoming years, as more clinical results for ai-discovered drug molecules become available, it will be exhilarating to see how ai technologies will impact the pharmaceutical industry at the drug trial level. table 2: ai tools/techniques employed in drug discovery [1, 17] s. no tool/technique name description 1 machine learning predicts drug-target interactions, helps analyse biological activity, and optimizes lead molecular compounds 2 deep learning helps in de novo drug designing, virtual screening, and predicting drug properties 3 reinforcement learning applied to optimize drug combinations and dosages by taking multiple variables into consideration and maximizing desired outcomes 4 neural graph fingerprints helps in various aspects of drug discovery, such as lead optimization, virtual screening, and drug properties’ prediction 5 deepchem uses a python-based ai system to find a suitable candidate in drug discovery 6 alphafold predicts 3d structures of target proteins 7 potentialnet uses neural networks to predict binding affinity of ligands 8 deeptox software that predicts the toxicity of around 12,000 drugs role of ai in drug repurposing the process of bringing new drugs to the market has significant hurdles in terms of time, labour, and cost. but finding new indications for an already existing drug can considerably reduce these costs by repurposing or repositioning it for other diseases [48]. drug repurposing allows the drug in question to enter phase ii and iii clinical trials instantly, with notably lower development costs, as pharmacokinetic, pharmacodynamic, and toxicity profiles of the drug are already established [48]. reker, et al have developed a method called self-organizing map-based prediction of drug equivalence relationships (spider). the model predicts molecular targets of known drugs, including key-target and off-target proteins [49]. benevolent ai, an ai-enabled drug discovery company, utilizes ai tools to unwrap novel connections within vast, unstructured datasets with respect to drugs, and their clinical trial information, enabling drug repurposing and facilitating the discovery of valuable new indications of the same. benevolent ai, in collaboration with johnson & johnson, has been redeveloping histamine h3 receptor inverse agonist, bavisant, which was originally intended for attention deficit hyperactivity disorder (adhd), and has been repositioned for the treatment of extreme daytime sleepiness in parkinson’s disease [50, 51]. the synergy between ai and drug repurposing is therefore of paramount importance for addressing the unmet medical needs. challenges and obstacles to using ai despite the significant potential of ai in transforming the landscape of the pharmaceutical industry, several challenges are prevalent and need to be addressed. a few of the challenges include resource sustainability, the quality and suitability of data used to train models, and potential for bias, all of which could result in unequal access to medical treatment of certain groups of people, undermining the principles of equality and justice [25]. an example explaining the likelihood of bias is during drug target prediction, when the training dataset contains an excess amount of target data related to a ‘x’ disease while having insufficient data for the disease ‘y’, the model may agrawal and kulkarni ai applications in drug discovery and development online first indian j pharm drug studies | 5 predict targets for the ‘x’ more accurately, while predictions for ‘y’ may be biased, thereby affecting the accuracy, specificity, and efficacy of a new drug development [18]. the use of ai also raises concerns about job losses due to the emergence of automated technologies. additionally, the use of ai in the pharmaceutical industry sparks debate about data privacy and security breaches. as ai systems depend on large amounts of data in order to function, the probability of sensitive personal information being accessed or misused increases. it is imperative that the collection and use of personal data be done in a way that respects the privacy of individuals and is in accordance with the relevant regulations [52]. future perspectives as ai technology continues to evolve, its role in drug discovery and other aspects is expected to expand as well. a major breakthrough was sought recently when the pharmaceutical industry’s first ai-driven drug discovery, developed by insilico medicine, was approved by the fda for clinical trials, which is in phase ii trials at the moment. the drug in question is rentosertib, also known as ism001-055, developed for the treatment of idiopathic pulmonary fibrosis (ipf) using their inhouse generative ai platform called pharma.ai [52]. this development substantially paves the way for further expansion. latest tools, such as quantum computing, could supplement ai’s computational abilities, enabling rapid and more precise predictions [20]. it is very much possible that the future of ai-assisted drug discovery would pivot on developing a virtual human, allowing for accurate predictions of all interactions between drug molecules and exploring all therapeutic capabilities and adverse side effects [2]. association between ai organizations, pharmaceutical companies, and policymakers will be vital for creating an ecosystem in which ai-powered drug discovery and development becomes the customary standard [20]. conclusion artificial intelligence has a crucial role in the various stages of drug development, ranging from drug discovery, design, formulation, clinical trials, repositioning, and up to the final market introduction. various tools assist in all the stages, enhancing efficiency and outcomes, while needing less time and man-power. forging ahead, there is a need to strengthen data management, elucidate superior ai models, ensuring vigorous ethical and legal considerations, with the aim of 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http://dx.doi.org/10.48550/arxiv.1807.09741 https://doi.org/10.1093/bioinformatics/btz111 