barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework mohd taufek et al. (2022) journal of pharmacy, 2(1), 31-35 page 31 validity of claims database compared with the electronic medical record of private health clinics in malaysia: a pilot study nor hidayah mohd taufek1, norny syafinaz ab rahman1, nor ilyani mohamed nazar1, siti halimah bux1, che suraya zin1* abstract introduction: the validity of health insurance claims data in private health sectors has been widely reported in many developed countries to monitor details of private healthcare utilisation. little is known regarding the data validity of private health care services and insurance claims in malaysia. this pilot study aimed to validate the claims data from a private health insurance database, using electronic medical records (emr) at the private clinics as the gold standard. method: patients’ data were retrieved from the pmcare health insurance database from 2016-2019 recorded for international islamic university malaysia employees. patients were sampled from the pmcare database and manually compared with data from emr of selected private panel clinics. data were analysed for descriptive statistics using microsoft excel 2013. results: a total of four panel clinics consented to the study, and data were available for 2016, 2017 and 2019. the number of observations obtained from 118 patients (male = 63, female = 55) was 386, with the most common diagnosis reported in the pmcare database was acute upper respiratory tract infection (63.6%). total accuracy between pmcare and emr data was 91.5%, with an 8.5% difference or inaccuracy. percentage accuracy was varied between different clinics (a= 92.6%, b=84.7%, c=98.6%, d=82.6%). conclusion: data submitted to pmcare claims by private health clinics had high accuracy (>90%) and is acceptable for research and other applications. future studies should investigate the differences in clinic-based practice for documenting the identified types of discrepancies to improve the accuracy of private health insurance databases. article history: received: 20 august 2021 accepted: 31 january 2022 published: 31 january 2022 keywords: validity, claim database, electronic medical record, private health clinics, accuracy. how to cite this article: mohd taufek, n. h., ab rahman, n. s., mohamed nazar, n. i. bux, s. h. & zin c. s. (2022). validity of claims database compared with the electronic medical record of private health clinics in malaysia: a pilot study. journal of pharmacy, 2(1), 31-35. doi: 10.31436/jop.v2i1.107 authors’ affiliation: 1 big data research group, department of pharmacy practice, kulliyyah of pharmacy, international islamic university malaysia, 25200 kuantan, pahang, malaysia *corresponding author: email address: chesuraya@iium.edu.my mohd taufek et al. (2022) journal of pharmacy, 2(1), 31-35 page 32 introduction the healthcare system in malaysia has been primarily divided into private and public sectors. high concentrations of private practices utilised by the community are covered by private health insurance companies (thomas et al., 2011). many private practices have also been affiliated as panel health clinics to government and private organisations which subsidise certain medical insurance coverage as health benefits for their employees with variable premiums charges (chua &cheah, 2012). private health insurance companies record the data on patients’ claims such as diagnosis, prescribed medications and cost submitted by the panel health clinics from their medical records. the format for the claims database could be different between companies, but it generally collects information on prescriptions, procedures and administrative costs of inpatients and outpatients. claims data are useful to monitor details regarding healthcare utilisation, including disease and prescription patterns. however, it requires high accuracy with good validity and reliability components to be tested so that the characteristics and limitations of the dataset are well understood (iwamoto et al., 2015; du et al., 2006). validity studies are crucial owing to increased reliance on health insurance claims, and thus the credibility of such data should be confirmed (koram et al., 2019). there have been limited validation studies reported in the asia pacific region, including australia, south korea, new zealand, thailand, singapore and japan (koram et al., 2019). to our knowledge, there have been no studies available reporting the validity of the claims database in malaysia. little is known whether prescription patterns, medication utilisation, and cost can be accurately obtained from private insurance databases. it is important to test the accuracy of insurance database documentation to ensure the credibility of research utilising databases. validity of database of private insurance companies will facilitate research towards sustainable reimbursement policy, optimal utilisation of medications and patient outcomes. a review has highlighted that the gold standard for database validity research was obtained from medical records, registry data, self-reported questionnaires and other data sources (koram et al., 2019). validated outcomes included variables such as medical conditions (takeda et al., 2016), risk monitoring (tomlin, reith & woods, 2017) and disease‐specific comorbidities (yamana et al., 2017). the present study aimed to investigate the accuracy of the claims data of a private health insurance company, pmcare, compared with electronic medical records (emr) of private panel clinics that are covered by pmcare. methodology this study was approved by the international islamic university malaysia ethical committee (irec-2019212). it was conducted from february to july 2020 at panel private health clinics in two states of pahang and selangor, malaysia. the results were reported in an aggregated manner using de-identified data. informed consent was not required as this study did not involve direct patient interaction. this retrospective pilot study collected data for 20162019 provided by the private health insurance company pmcare for outpatient settings. the pmcare insurance was subsidised by international islamic university malaysia (iium) for its employees. based on the policy, the claim limit was set at ringgit malaysia (rm) 45 per visit per patient. the panel private health clinics (phc) were identified to collect the original data from emr. the inclusion criteria were clinics with a high frequency of attendance by iium employees, used emr and consented to the study. the exclusion criteria were those with low frequency of patients, used manual medical records and did not consent to the study. a total of four panel clinics were included, and data were collected from emr. patient selection the selection of patients was conducted using systematic sampling. three patients per month were selected at the beginning, middle and end of each month from the pmcare database. the inclusion criteria were patients who had three or more medications. the search was conducted using a unique claim code that extracted the correct data regardless of the date recorded for database claim, patient’s visit or delayed submission of claims to pmcare. data collection included information on claim code, date of prescription, name of the patient, patient identification, diagnosis, name of medications, number of medications per prescription, the number of drugs supplied, consultation fee, the total cost of medications and amount paid by the patient. validated outcomes included diagnosis, medication name, number of medications per prescription, the quantity of medication, and the amount claimed to the health insurance. majority of previous studies (83%) have reported similar data collection methods where cases were most frequently validated against diagnoses in medical records (widdifield et al., 2013). accuracy measurement the accuracy was obtained by comparing the pmcare dataset with the original data from emr by dividing the number of observations found to be the same by the total number of data elements of the pmcare database then mohd taufek et al. (2022) journal of pharmacy, 2(1), 31-35 page 33 multiplying that total by 100. the agreement exceeding 90% was considered as a good benchmark when data elements from the two sources were found not to be the same, noting the reasons (for example, unclear data definitions) as reported in previous studies (iwamoto et al., 2015; miller et al., 2009). the discrepancy was determined when the inaccuracies of observations recorded in the emr and pmcare database were identified. the discrepancies were calculated by dividing the number of observations found to be different with the total observations from selected pmcare data then multiplying by 100. results a total of 118 patients (age: mean ±sd, 33.2 ± 12.7) were selected based on the availability of data from emr (table 1). although there were three patients selected per month from 2016-to 2019, many patients had to be excluded due to changes in the recording system used at the clinics, crash of the software and incomplete record of emr. we found that several observations from emr recorded extra medications (415 observations) compared to the pmcare database, which was 386 observations. electronic medical records contained original data entered from patients’ prescriptions. the number of observations recorded in the emr was higher than the pmcare database because the emr captured the total number of medications prescribed to each patient. in contrast, only a few medications were recorded for insurance claims to pmcare for the coverage limit of rm45. therefore, after removing extra observations from emr for accuracy measurement, the total number of observations for both emr and pmcare database was 386. as presented in table 2, the number of patients included in the present pilot study was sufficient as reported by a similar study that measured accuracy and validity of a hospital database (cook et al., 2002). the advantage of using emr was that additional information was available and comprehensively recorded for each visit. we found that consultation fees were varied between clinics and between clients depending on the number of medications prescribed and adjustment to the insurance eligibility. data from emr provided the amount of out-of-pocket money paid by the patients if the total charges for consultation and medications exceeded the insurance coverage of rm 45, including the detailed price for each medication with a specific quantity. on the contrary, the pmcare database did not have this information. discrepancies were found due to differences in recording the name and quantity of medications, diagnosis, the number of medications and the missing information in either emr or pmcare database for the variables investigated. the most common diagnosis reported in the pmcare database was acute upper respiratory tract infection (urti) (63.6%), followed by acute gastroenteritis and colitis (13.5%). table 1: demographics of patients from four private medical clinics. characteristics n = 118 frequency (%) gender: a. male b. female 63 55 53.4 46.6 age (years): mean (sd) median (range) 33.2 (12.7) 35 (4 – 61) diagnosis: a. acute upper respiratory infection, unspecified b. infectious gastroenteritis and colitis, unspecified c. unspecified abdominal pain d. anemia, unspecified e. others (urticaria, low back pain, acute tonsillitis, viral infection unspecified, dermatitis and eczema, conjunctivitis, gout, hemorrhoids, hordeolum (externum) (internum) of eyelid, gastritis, myalgia, mycosis) 75 16 3 3 21 63.6 13.5 2.54 2.54 17.8 discussion the accuracy of the pmcare database has never been validated to determine its credibility to be used for database research in malaysia. the present study reported that the pmcare claims database had overall 91.5% accuracy and 8.5% discrepancies compared with emr data as a gold standard for four different panel clinics for the segregated patient samples collected in 2016, 2017 and 2019. this outcome indicated an ideal accuracy value that was set as above 90% to be considered valid (miller et al., 2009). our finding was lower than that reported by a study in japan, that demonstrated 99% accuracy in estimating opioid consumption among cancer patients (iwamoto et al., 2015). another study validated claims algorithm and clinical operative reports with 97% accuracy (miller, saigal & warren, 2009). however, a different study has indicated a lower range of good accuracy with 80% and above for a pilot study reporting database validity (cook et al., 2002). the analysis for an individual clinic showed that accuracy could achieve above 98% with a systematic and appropriate recording of claims data according to emr (table 2). mohd taufek et al. (2022) journal of pharmacy, 2(1), 31-35 page 34 table 2: discrepancies of data from emr of respective clinics in comparison to pmcare database variables clinic a clinic b clinic c clinic d total no. of patients 30 15 50 23 118 year of observations 2016 2016 20162017 2019 no. of observations from emr 94 59 147 86 386 no. of observations from pmcare claims database 94 59 147 86 386 no. of observations with discrepancies 7 9 2 15 33 types of discrepancies name and quantity of medicat ion. name and quanti ty of medic ation, diagn osis. name of medicat ion. name and quantity of medicat ion, diagnos is. percentage of discrepancies (%) 7.4 15.3 1.4 17.4 8.5 percentage of accuracy (%) 92.6 84.7 98.6 82.6 91.5 investigation of data discrepancy showed that types of discrepancy tend to vary between clinics. this could be due to the nature of pmcare claims data, where data were created and submitted later from the date of the patient’s visit, whereas the emr data showed the actual medications dispensed daily. although the differences in characteristics exist, both data were prone to human error in entering the details into the online system, which resulted in discrepancies (iwamoto et al., 2015). it is also important to note that claims databases could only cater for specific data covered by the insurance company for certain medications and diseases. a review of previous studies has also highlighted the critical measures of diagnostic accuracy (sensitivity, specificity, and positive and negative predictive values) and disease prevalence in reporting validity of administrative database algorithms (widdifield et al., 2013). our study identified the discrepancies by including the missing names of medication or different medications recorded when compared between emr and pmcare database. the diagnosis error was included when the diagnosis submitted to the pmcare database was different or unspecified compared to emr. the limitation of this study was that we did not have specific details regarding the options given for each data category in the claim database system, such as general option (e.g. unspecified abdominal pain) or specific option for accurate diagnosis (e.g. dyspepsia, gastritis). we did not consider that specific, or general diagnosis of similar nature or location was different unless the difference was obvious such as abdominal pain (emr) versus urti (pmcare). from the perspective of the claims database and emr, we propose that details listed in the claims database should be the same as those recorded in emr. it was expected that the number of observations in emr would be greater than those in the claim database since the insurance company covers only certain medications. the consistency in recording the data will improve the accuracy and validity of the data from both sources. the major limitation of our pilot study was that the sample size was relatively small compared to previous studies (iwamoto et al., 2015; takeda et al., 2016) but was sufficient to report validity for a pilot study (cook et al., 2002). our study was multi-centred, with a standardised data format from the pmcare database, which allowed the comparison of varying practices between settings. future studies may identify interventions for corrective actions to ensure high accuracy of the claims database. the results suggest that pmcare database is valid and accurate as a valuable resource to be used for research purposes but with careful analysis and interpretation considering the data characteristics. the identified discrepancies were common for certain variables in database validity studies as previously reported in other studies (cook et al., 2002; gabriel et al., 2005). however, improved documentation practice is required at the specific settings to improve the accuracy of the claims database. conclusion data from the health insurance claims database was valid and applicable for research purposes with careful analysis and interpretation. the overall high accuracy of the health insurance claims database in this study indicated its validity. however, variation in accuracy level was found between different clinics attributable to varying practices in recording the data. future studies are required to identify effective interventions to improve the accuracy of claims databases with gold standards. acknowledgement the authors would like to thank the pmcare database health insurance company and private panel health clinics of the international islamic university malaysia for their assistance in completing this research. csz was supported by a research grant from iium health wellness centre. mohd taufek et al. (2022) journal of pharmacy, 2(1), 31-35 page 35 data availability statement request to access the datasets should be directed to international islamic university malaysia research ethical committee. the de-identified data could be shared with interested researchers after obtaining approval from the above ethical committee. the restriction on public data deposition is due to the privacy and confidentiality of patients’ health data. conflict of interest the authors have no conflict of interest to declare. the funder had no role in study design, data collection and analysis, decision to publish or prepare the manuscript. references chua, h.t., & cheah j.c. h. (2012). financing universal coverage in malaysia: a case study. bmc public health, 12(suppl 1), s7. https://doi.org/10.1186/1471-2458-12-s1-s7 cook, s.f., visscher, w.a., hobbs, c.l., & williams r.l. 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(2017). validity of diagnoses, procedures, and laboratory data in japanese administrative data. journal of epidemiology, 10, 476–482. https://doi.org/10.1016/j.je.2016.09.009 barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework siddiqui (2022) journal of pharmacy, 2(1), 8-15 page 8 prescribing practices of antibiotics for acute diarrhea in children aged less than five years old in pakistan durr-e-shahwar siddiqui1* abstract introduction: childhood diarrhea accounts for 16% of child deaths in pakistan. irrational prescribing of antibiotics, prescribing of antibiotics for viral infections, self-medication using antibiotics, prescription sharing, and refilling are very common practices in pakistan. the aim of this study was to evaluate the prescribing practices of antibiotics for acute diarrhea in children less than five years of age at a secondary healthcare hospital of pakistan and to assess the compliance of prescribers with authentic clinical guidelines of treatment for childhood acute diarrhea. method: a cross-sectional study was conducted for a period of one year, from august 2020 to august 2021, at a secondary care hospital of karachi, pakistan. it was based on the collection of outpatient clinic prescriptions of children aged less than five years and suffering from acute diarrhea. the regimens or suggested therapies by prescribers for acute diarrhea were assessed as per the national institute of care and health excellence and world health organization guidelines. a brief questionnaire was also distributed among prescribers, pharmacists, and caretakers of children to extract their opinions regarding antibiotic prescribing in acute diarrhea. results & discussion: antibiotics were inappropriately prescribed for acute diarrhea among children in pakistan because p-value was less than 0.05 (p<0.05) as calculated by descriptive statistical tools using z-test. more than 90% prescriptions of acute diarrhea in children less than five years of age failed to comply with the authentic treatment guidelines. due to the limited knowledge of prescribers regarding treatment guidelines and compliance of parents with antibiotic prescribing for diseases in children, irrational prescribing of antibiotics for acute diarrhea in children less than five years of age is frequent in pakistan. healthcare professionals must be adequately trained to ensure the proper management of acute diarrhea by following authentic clinical guidelines. conclusion: antibiotics are irrationally prescribed for acute diarrhea in children less than five years of age in pakistan and prescriptions do not comply with authentic clinical guidelines. article history: received: 31 october 2021 accepted: 24 january 2022 published: 31 january 2022 keywords: acute diarrhea, clinical guidelines, prescribing practices, antibiotics. how to cite this article: siddiqui, d. (2022). prescribing practices of antibiotics for acute diarrhea in children aged less than five years old in pakistan. journal of pharmacy, 2(1), 8-15. doi: 10.31436/jop.v2i1.116 authors’ affiliation: 1 supervisor pharmacist, al-khidmat pharmacy services, karachi, pakistan *corresponding author: email address: silver_shine27@hotmail.com siddiqui (2022) journal of pharmacy, 2(1), 8-15 page 9 introduction diarrhea is the frequent passage of three or more loose or watery stools per day which is more than normal for an individual. according to the world health organization (who), diarrhea is the second leading cause of death in children less than five years of age. its causes may include infection by parasite, virus or bacteria, malnutrition, contaminated water, and unhygienic conditions (world health organization, 2017). when increased bowel movement terminates within three weeks, it is called acute diarrhea, but if it persists for more than three weeks it is called chronic diarrhea (marsha & anthony, 2012). symptoms of chronic diarrhea include uncontrollable bowel movements, nausea, abdominal pain, cramps, vomiting, shivering and fever. these symptoms can lead to severe dehydration and mal absorption (national institute of diabetes and digestive and kidney diseases, 2017). antibiotic-associated diarrhea is mild diarrhea that can occur in children and adults and does not cause decrease in weight or dehydration and terminates within one to two days of stopping the antibiotic (fliesher & o’ryan, 2017). childhood diarrhea under two years of age accounts for 72% deaths globally (walker et al., 2013). it also leads to impairment in the growth of children as well as cognitive development (checkley et al., 2008) (lorntz et al., 2006). diarrheal diseases in children below five years of age in low or middle-income countries need immediate treatment to reduce mortality rate (ugboko et al., 2020). in pakistan, childhood diarrhea accounts for 16% of child deaths and it was suggested that a cost-effective, standardized, and accessible mode of treatment along with public education and awareness regarding childhood diarrhea must be established (quadri et al., 2013). diarrhea in children can be prevented up to major extent by vaccination against escherichia coli, shigella spp. and rotavirus, promoting health hygiene, consuming noncontaminated water and good sanitary conditions (mokomane et al., 2018). acute diarrhea should not necessarily be treated by antibiotics in children because rehydration therapy is proven to be the mainstay treatment and usually the symptoms subside without requiring any specific therapy. in case of severe diarrhea, traveler’s diarrhea, antibiotic-associated diarrhea, or chronic diarrhea, one may opt to antibiotics or antimicrobial therapy. oral doses of co-trimoxazole or metronidazole or parenteral doses of ceftriaxone or ciprofloxacin can be a choice for empiric therapy in severe cases of diarrhea (bruzesse et al., 2018). according to the national institute for care and health excellence (nice) guidelines of 2009, acute diarrhea must be treated primarily by oral rehydration salt as 50ml/kg over 4 hours, diarrhea usually subsides within 2 weeks and if this duration is crossed then healthcare professional must be consulted (the national institute for care and health excellence, 2009). oral rehydration salt (ors) is the primary element of care in children with acute gastroenteritis and acute diarrhea because it has been reported that the efficacy of oral rehydration is comparable to the iv rehydration in severe dehydration while severe dehydration may require iv rehydration therapy (guarino et al., 2014). according to who, patients must obtain medicines as per their clinical necessity in appropriate doses and frequency for adequate duration of therapy with the lowest possible expense to promote rational use of medications. prescribing of medicines without authentic clinical guidelines, polypharmacy, preference of injections when oral dosage form of same drug could be used, irrational use of antibiotics, self-medication and non-compliance with prescribed regimen are some of the examples coming under irrational use of medications. this worldwide concern can be resolved mainly by ensuring that the medications are prescribed according to clinical guidelines, public awareness about medications, enforcement of policies and regulations by multidisciplinary national body for rational use of medicines, essential drugs list, pharmacy and therapeutic committee in healthcare areas, problem-based training of pharmacotherapy, supervision, and audits (world health organization, 2002). a diarrhea control plan was launched by world health organization (who) and united nations international children’s emergency fund (uniicef) in 2009 and global action plan for pneumonia and diarrhea (gappd) in 2013 realizing the increased prevalence of these diseases in children (world health organization & unicef, 2013). national essential medicines list (neml) of pakistan includes low osmolarity ors, 250mg and 500mg oral amoxicillin trihydrate and 20mg zinc sulphate as recommended by unicef and who for pneumonia and diarrhea (drug regulatory authority of pakistan, 2016). government of pakistan has provided this list at primary as well as secondary healthcare settings in the country. a study conducted in pakistan revealed that healthcare education to communities, health interventions for children and intensive supervision or healthcare audits can improve mortality rate in children less than 5 years of age suffering from diarrhea and pneumonia. it also suggested promoting rational use of medicines, activities of healthcare workers in community, training of healthcare workers, surveillance, and improvement in socio-economic conditions of people to reduce the risks and deaths caused by diarrhea and pneumonia (hansen et al., 2020). irrational prescribing of antibiotics, prescribing of antibiotics for viral infections, self-medication using antibiotics, prescription sharing, and refilling are very common practices in pakistan (hameed et al., 2016). inappropriate prescribing of antibiotics in low and middleincome countries is highly prevalent (8% to 100%) at primary healthcare settings which needs strategies for proper prescribing practices and implementation of national prescribing guidelines or recommendations of treatment by siddiqui (2022) journal of pharmacy, 2(1), 8-15 page 10 who (sulis et al., 2020). healthcare professionals must be trained periodically upon the proper management of diarrhea along with prescribing or indication of antibiotics for children under five years of age (udoh & meremikwu, 2017). the aim of this study was to evaluate the prescribing practices of antibiotics for acute diarrhea in children less than five years of age at a secondary healthcare hospital of pakistan and to assess the compliance of prescribers with authentic clinical guidelines of treatment for childhood acute diarrhea. methodology this study was conducted for a period of one year at a secondary care hospital of karachi, pakistan. it was a cross-sectional study based on the collection of outpatient clinic prescriptions at pharmacy from august 2020 to august 2021. the prescriptions were collected based on the inclusion and exclusion criteria such as pediatric prescriptions of acute diarrhea for both gender of children aged less than five years were included in the study while all such prescriptions of children greater than five years of age were excluded. total of 500 prescriptions were collected along-with the verbal consent and demography of children from their caretakers or attendants. the age, weight, duration of diarrhea, concomitant diseases, therapies given by caretakers at home for diarrhea and medications prescribed by prescribers were also noted. the compliance of prescribers with authentic clinical guidelines while prescribing treatment of acute diarrhea in children less than five years of age was evaluated. the appropriateness and anti-microbial spectrum of prescribed antibiotics as per age of children, episodes of diarrhea and duration of diarrhea were noted. regimens or suggested therapies by prescribers for acute diarrhea were also assessed as per nice guidelines. moreover, the opinions of prescribers, pharmacists, and caretakers of the children for prescribing antibiotics in children less than five years of age were also recorded through questionnaire. the questionnaire contained some open-ended questions such as satisfaction with the prescribed regimen for acute diarrhea, episodes of diarrhea, nature of diarrhea, duration of diarrhea, knowledge about guidelines for childhood diarrhea and antibiotics, brief demography of case and concomitant diseases. the questionnaire contained scoring from 1 to 5 for each question. descriptive statistics was used to describe the variables of the study. p values were calculated through z-test using spss software and pvalues less than 0.05 were considered as statistically significant. results table 1 shows the antibiotics prescribed for children less than 5 years of age for acute diarrhea. the prescribed antibiotics did not comply with nice guidelines when assessed on the basis of nature, duration and episodes of acute diarrhea. although the prescribing practices of injectable preparations in acute diarrhea was less than that of oral dosage forms but 480 prescriptions out of total 500 prescriptions contained un-necessarily prescribed antibiotics that can lead to antibiotic resistance in pediatric population. the combination of diloxanide furoate and metronidazole as syrup dosage form was the most frequently prescribed antibiotic for childhood acute diarrhea because prescribers considered that the anti-microbial coverage of this combination would be more effective than other single generic formulae of antibiotics. table 1: prescribed medications for acute diarrhea in children less than 5 years of age medications number of prescriptions (n=500) percentage of prescriptions oral diloxanide furoate + metronidazole combination 187 37.4% oral ciprofloxacin 75 15% cefixime suspension 125 25% oral metronidazole 65 13% metronidazole infusion 5 1% ceftriaxone infusion 3 0.6% nitazoxanide suspension 15 3% ciprofloxacin infusion 5 1% in table 2, it is evident that the probability of inappropriate prescribing of antibiotics in childhood diarrhea was higher because p-value was less than 0.05 as calculated by descriptive statistical tools. p-value was highly significant as determined by z-test using spss software. the main concomitant symptoms were dehydration; vomiting and abdominal pain but these symptoms did not need antibiotic prescribing. most of the children suffering from acute diarrhea had normal body weight and the duration of diarrheal episodes was than 3 days. the prescriptions of general practitioners for childhood acute diarrhea deviated more from nice guidelines than that of pediatricians. only 4% prescribers had compliance with clinical guidelines for acute diarrhea in children less than 5 years of age while 96% prescribers irrationally prescribed antibiotics as shown in figure 1. a brief questionnaire distributed among prescribers, pharmacists and caretakers of children revealed their opinions for the rationale of prescribing antibiotics in children less than 5 years of age for acute diarrhea. siddiqui (2022) journal of pharmacy, 2(1), 8-15 page 11 the rationale put forward by most of the prescribers for antibiotic prescribing in such cases was to prevent further risks and chances of chronic diarrhea while some of them documented that antibiotic prescribing was for the satisfaction of caretakers as 90% of the parents complied with antibiotic prescribing in acute diarrhea and considered it effective for treatment. only 3% prescribers accepted it being due to limited knowledge of authentic and updated clinical guidelines. some of the pharmacists considered these practices as blindly following the past practices of other prescribers while most of them had opinions that it is due to the information provided by medical representatives to the prescribers about the benefits of the prescribed medicine (figure 2). figure 1: prescribers’ compliance with guidance for childhood acute diarrhea discussion the frequent passage of three or more loose or watery stools per day is referred to as diarrhea and when this increased bowel movement ends within three weeks, it is called acute diarrhea. as per the authentic treatment guidelines for acute diarrhea in children under five years of age, oral rehydration therapy is the mainstay of treatment. irrespective of etiology, treatment with diosmectite and probiotic powders or solutions must continue along with rehydration therapy. clinical conditions of child and host-related aspects must be considered while prescribing treatment for acute diarrhea. antibiotic therapy must not be started when clear indications for the need of antibiotic therapy are absent because symptoms of acute diarrhea usually subside with oral rehydration therapy and probiotics. antibiotics can be considered for treatment if diarrhea becomes chronic, child is at risk or is severely ill but microbiological investigations are necessary. world health organization does not recommend the use of antibiotics in children for acute diarrhea except for those with severe clinically identified cases. antibiotics can be considered for acute diarrhea with bloody stool, severe traveler’s diarrhea, diarrhea with some other super-imposed infection for which antibiotic is essential, diarrhea with high grade fever and for immunocompromised children. oral zinc in the form of zinc sulphate suspension can also be prescribed as zinc is severely lost from the body during diarrheal condition. childhood diarrhea accounts for 16% of child deaths in pakistan. the prescribing practices of injectable preparations in acute diarrhea is although less than that of oral dosage forms in secondary healthcare setting of pakistan but irrational prescribing of antibiotics for diarrhea in pediatric population is common. this study revealed that the combination of diloxanide furoate and metronidazole was the most frequently prescribed antibiotic for childhood acute diarrhea. the two major reasons told by most of the prescribers for prescribing antibiotics were to prevent the transformation of acute diarrhea to chronic diarrhea and the compliance of parents with antibiotics for resolution of diarrheal condition. pharmacists considered that the prescribing of antibiotics in acute diarrhea to children less than five years of age was due to the limited knowledge of prescribers for clinical guidelines, following the past practices of other prescribers or due to the information provided by the medical representatives from different pharmaceutical industries for their products. more than 90% prescriptions of acute diarrhea in children less than five years of age failed to comply with the authentic treatment guidelines. healthcare professionals must be adequately trained to ensure the proper management of acute diarrhea by following authentic clinical guidelines, such as guidelines by who or nice. healthcare education and awareness programs, health interventions and healthcare audits are necessary to promote rational use of medications in childhood acute diarrhea. vaccinations of children against escherichia coli, shigella spp. and rotavirus must be regarded mandatory by the government. presence of neml must be assured at all the healthcare settings and all the medications mentioned in it for diarrhea must be available at every healthcare area of pakistan. the therapy for acute diarrhea in children less than five years of age must be according to the severity of clinical condition and oral rehydration therapy must be ensured. parents or caretakers of children must be convinced by healthcare professionals to discourage the irrational use or prescribing of antibiotics for acute diarrhea in children less than five years of age in pakistan. prescribers' compliance with guidelines for acute diarrhea compliant prescriptions non-compliant prescriptions siddiqui (2022) journal of pharmacy, 2(1), 8-15 page 12 table 2: demography, concomitant symptoms, and prescribers’ qualification characteristics number of prescriptions inappropriate prescriptions percentage of inappropriate prescriptions p value diarrhea 500 p<0.05 age (months) 1-12 130 125 96% 13-24 142 140 98% 25-36 107 106 99% 37-46 52 49 94% 47-60 69 60 86% fever 60 dehydration 456 vomiting 391 abdominal pain 442 weight under-weight 113 normal weight 387 overweight 0 duration of diarrhea less than 3 days 289 3-7 days 200 8-14 days 11 prescirbers’ qualifications p<0.05 general practitioners 215 208 96% pediatricians 285 272 95% siddiqui (2022) journal of pharmacy, 2(1), 8-15 page 13 figure 2: reasons for prescribing antibiotics for acute diarrhea in children aged less than 5 years 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% limited knowledge of clinical guidelines past practices of prescribers parents’ compliance with antibiotics information provided by medical representatives prevention of severe chronic diarrhea reasons for prescribing antibiotic in acute diarrhea reasons for prescribing antibiotic in acute diarrhea siddiqui (2022) journal of pharmacy, 2(1), 8-15 page 14 conclusion antibiotics are irrationally prescribed for acute diarrhea in children less than five years of age at secondary healthcare hospital of pakistan and prescriptions do not comply with the authentic clinical guidelines due to the limited knowledge of prescribers regarding treatment guidelines and compliance of parents with antibiotic prescribing for diseases in children. conflict of interest the author has no conflicts of interest to report. references bruzesse e, giannattasio a, guarino a. (feb 2018). antibiotic treatment of acute gastroenteritis in children. f1000 res, 7 (1): 193. doi: 10.12688/f1000research.12328.1. ecollection 2018. checkley w, buckley g, gilman rh, assis am, guerrant rl, morris ss, molbak k, valentiner-branth p, lanata cf, black re. (2008). multi-country analysis of the effects of diarrhoea on childhood stunting. international journal of epidemiology, 37: 816–830. drug regulatory authority of pakistan; ministry of national health services, regulations and coordination. (2016). national essential medicines list, government of pakistan. fliesher gr and o’ryan mg. (jun 2017). patient education: acute diarrhea in children. available on: https://www.uptodate.com/contents/acutediarrhea-in-children-beyond-the-basics guarino a, ashkenazi s, gendrel d, vecchio al, shamir r, szajewska h. (jul 2014). european society for pediatric gastroenterology, hepatology, and nutrition/european society for pediatric infectious diseases evidence-based guidelines for the management of acute gastroenteritis in children in europe: update 2014. journal of pediatric gastroenterology, 59(1): 132-52. doi: 10.1097/mpg.0000000000000375. hameed a, naveed s, qamar f, alam t, abbas ss and sharif n. (2016). irrational use of antibiotics in different age groups of karachi: a wakeup call for antibiotic resistance and future infections. journal of bioequivalence & bioavailability, 8:5. doi: 10.4172/jbb.1000302 hansen cl, mccormick bjj, azam si, ahmed k, baker jm, hussain e, jahan a, jamison af, knobler sl, samji n, shah wh, spiro dj, thomas ed, viboud c & rasmussen za. 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(feb 2017). symptoms & causes of chronic diarrhea in children.niddk, national institutes of health. available on: https://www.niddk.nih.gov/healthinformation/digestive-diseases/chronic-diarrheachildren/symptoms-causes quadri f, nasrin d, khan a, bokhari t, tikmani ss, nisar mi, bhatti z, kotloff k, levine mm, zaidi akm. (jul 2013). health care use patterns for diarrhea in children in low-income peri urban communities of karachi, pakistan. american journal of tropical medicine and hygiene, 89(1 suppl): 49–55. doi: 10.4269/ajtmh.12-0757. sulis g, adam p, nafade v, gore g, daniels b, daftary a, das j, gandra s and pai m. (2020). antibiotic prescription practices in primary care in lowand middle-income countries: a systematic review and meta-analysis. plos medicine, 17(6). doi: https://doi.org/10.1371/journal.pmed.1003139 the national institute for care and health excellence. (apr 2009). diarrhoea and vomiting caused by gastroenteritis in under 5s: diagnosis and management. nice clinical guidance (cg84). available on: https://www.nice.org.uk/guidance/cg84 udoh ee and meremikwu mm. (2017). antibiotic prescriptions in the case management of acute watery diarrhea in under-fives. international journal of contemporary pediatrics, 4(3). doi: http://dx.doi.org/10.18203/23493291.ijcp20171685 siddiqui (2022) journal of pharmacy, 2(1), 8-15 page 15 ugboko hu, nwinyi oc, oranusi su, oyewale jo. (apr 2020). childhood diarrhoeal diseases in developing countries. heliyon, 6(4). doi: 10.1016/j.heliyon.2020.e03690. walker c.l.f., rudan i., liu l., nair h., theodoratou e., bhutta z.a. (apr 2013). global burden of childhood pneumonia and diarrhoea. lancet, 381(9875): 1405–1416. world health organization & united nations international children’s emergency fund. (2013). ending preventable child deaths from pneumonia and diarrhoea by 2025. the integrated global action plan for pneumonia and diarrhoea (gappd). available on: https://apps.who.int/iris/bitstream/handle/10665/ 79207/who_fwc_mca_13_01_eng.pdf;jsessi onid=c42f0cb545c0b7a46ac597b9ef5879 f6?sequence=1 world health organization. (may 2017). diarrhoeal disease. available on: http://www.who.int/newsroom/fact-sheets/detail/diarrhoeal-disease world health organization. (2002). promoting rational use of medicine. available on: https://www.who.int/activities/promotingrational-use-of-medicines barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework fahrurazi et al. (2022) journal of pharmacy, 2(1), 1-7 page 1 factors affecting waiting time in outpatient pharmacy at hospital raja perempuan zainab ii (hprzii) fairul ezwan fahrurazi1*, nur husna ibrahim1, nurul musfirah mafauzy1, wan nor ain wan ismail1 and syauqin syazwani mohamed rusli1 abstract introduction: world health organization (who) has identified that patient waiting time as one of the most important measurements of a responsive health system for healthcare services. outpatient pharmacy is associated with patient waiting time as the indicator for satisfaction of the services. this study aimed to determine the factors affecting waiting time in hospital raja perempuan zainab ii, kelantan. method: a cross-sectional study was conducted by collecting prescription received in outpatient pharmacy from 1st october 2020 till 31st december 2020. all prescriptions prescribed manually were excluded. multiple linear regression was performed to determine the factors affecting waiting time and the data were analysed using spss version 25. results: a total of 248 prescriptions were collected in outpatient pharmacy. the mean waiting time in outpatient pharmacy was 23.0 minutes (sd = 11.0). waiting time was found to be associated with number of medications in the prescription, number of staff working on that day, prescriptions which required intervention and filling personnel. conclusion: the waiting time at the outpatient pharmacy of hrpz ii indicated the acceptable range of quality services which met the patient’s satisfaction. future studies are needed to confirm the satisfaction level of patients and further improve quality of the service. article history: received: 30 july 2021 accepted: 27 january 2022 published: 31 january 2022 keywords: waiting time, association, outpatient pharmacy. how to cite this article: fahrurazi, f. e., ibrahim, n. h., mafauzy, n. m., wan ismail, w. n. a. & mohamed rusli, s. s. (2022). factors affecting waiting time in outpatient pharmacy at hospital raja perempuan zainab ii (hprzii). journal of pharmacy, 2(1), 1-7. doi: 10.31436/jop.v2i1.105 authors’ affiliation: 1 department of pharmacy, raja perempuan zainab ii hospital, ministry of health malaysia, 15586 kota bharu, kelantan, malaysia. *corresponding author: email address: fairulezwanfahrurazi@gmail.com fahrurazi et al. (2022) journal of pharmacy, 2(1), 1-7 page 2 introduction outpatient pharmacy refers to the pharmacy department in charge of dealing with patients that do not occupy the beds in hospitals or other inpatient settings (hammouda & hammouda, 2012). the outpatient pharmacy is often associated with the waiting time as the degree to which the patients are satisfied with the care received and strongly related to the quality of the waiting experience (nosek jr & wilson, 2001). the aim and vision to improve healthcare quality has always been a great concern in healthcare services. quality itself can be defined as conformance to specific requirements and standards given (alodan et al., 2020). therefore, pharmacy unit needs to improve the services quality as regulated according to the ministry of health. several studies used different techniques to overcome healthcare services problems and improve quality. a study utilized six sigma processes and able to reduce waiting time by 50% in an outpatient pharmacy in a local hospital specialized in cancer treatment in pakistan (arafeh, barghash, sallam, & alsamhouri, 2014). suss et al. (2017) implemented a patient flow project to improve efficiency spends in pharmacy queues to reduce waiting time. every problem was given solutions and a framework was provided to evaluate pharmacy performance based on simulations (suss, bhuiyan, demirli, & batist, 2017). world health organization (who) has identified that patient waiting time as one of the most important measurements of a responsive health system for healthcare services (sun et al., 2017). waiting time in outpatient pharmacy has been defined as the length of time from when the patient received the queue number at the counter to the time the patient being called to the counter (afolabi & erhun, 2003). excessive waiting time shows the lack of efficiency of pharmacy services. such delay leads to dissatisfaction, loss of patronage and poor patients’ compliance (kusumowardhani & ilyas, 2019; lin et al., 1999). therefore, waiting time should be addressed as part of good management practice. the waiting time in the outpatient pharmacy is usually attributable to the tedious process of packaging, labelling and prescription intervention (ndukwe, tayo, & sariem, 2011). in addition, internal operational factors also contribute to the increase in time spent, for example, prescription requires extemporaneous preparation, low percentage of staff at work and long list of medication in one prescription (ndukwe et al., 2011). the factors affecting the waiting time are somehow multifactorial and to date, studies determining factors affecting waiting time have been inadequate especially in local setting in malaysia. therefore, we aim to determine the mean waiting time as well as factor affecting waiting time in outpatient pharmacy in hospital raja perempuan zainab ii (hrpz ii), kelantan, malaysia. methodology design and study population a cross sectional study was conducted for 3 months from 1 october 2020 until 31 december 2020. the inclusion criterion was all electronic prescriptions received at the outpatient pharmacy during the study period whereby any manual prescription was excluded. data collection the waiting time in hrpz ii’s outpatient pharmacy was defined as the time taken from when the patient received the queue number at the counter to the time the patient was called at the counter using queue management system (qms). the qms was utilised to determine the length of time taken for each prescription being called at the counter. there are four main dispensing counters available. all prescriptions were given numbers based on the number of medications and the type of diseases in the prescription. prescriptions with 3 medications or less with acute diseases were given queue number 2, 3 medications or less with chronic disease were given queue number 3, more than 3 medications were given queue number 4 and queue number 5 was given to prescriptions which requires extemporaneous preparation. for each queue number, systematic random sampling was applied to pick the prescriptions for this study. the sampling interval was determined by dividing the average daily number of prescriptions with the number of samples required for each queue number. then, random starting point was determined using random table and the sampling interval was repeated to choose subsequent prescription. a form which consists of all the necessary data and possible factors affecting waiting time was collected and attached with the copied prescription. sample size calculation was calculated for each objective. single mean formula was used for determination of mean waiting time. the values of two-tailed α = 0.05, σ = 20 and d = 2.5 were entered in the equation which yielded a minimum of 246 prescriptions (ndukwe et al., 2011) .for the second objective, 200 was set as the minimum sample size based on the rule of thumb as suggested by green for any regression analysis (green, 1991). thus, by comparing the samples required for both objectives, the minimum prescriptions needed was 246. statistical analyses data analyses were carried out by using spss version 25.0 (ibm corp, 2017). the descriptive statistics were presented in categorical and numerical data. the categorical data were summarised in frequencies and percentages. meanwhile, numerical data were presented with mean and standard fahrurazi et al. (2022) journal of pharmacy, 2(1), 1-7 page 3 deviation (sd). multiple linear regression was utilised to determine the factors associated with waiting time. statistical significance was set at 95% confidence level. ethical approval this research has obtained ethical approval from the medical research and ethics committee (mrec), ministry of health malaysia (nmrr-20-2610-57076) while permission to conduct the study at the site was granted by the director of hrpz ii. results sociodemographic characteristics a total of 248 prescriptions were collected during the study duration. the mean age of the patients was 41.9 years (sd = 21.7) and more than half of them were female. half of the prescriptions were obtained from queue number 2 and majority of them were collected on wednesday. the average number of staff was around 23 people. the mean waiting time for outpatient pharmacy was 23.0 minutes (sd = 11.0). most of the prescriptions were chronic diseases, with ≤ 5 medications, contained standard medications, from outpatient clinics, only one clinic and arrived during peak hours. in addition, the majority of them were new prescriptions, did not require spub, contained no extemporaneous preparations, not required intervention, medication filled by others and counterchecked by pharmacists (table 1). table 1: sociodemographic characteristics of patients/prescriptions (n=248) characteristics n % age (year) 41.9* 21.6# gender male 110 44.4 female 138 55.6 queue number 2000 – 2999 124 50.0 3000 – 3999 38 15.3 4000 – 4999 65 26.2 5000 – 5999 21 8.5 day of visit sunday 27 10.9 monday 42 16.9 tuesday 59 23.8 wednesday 79 31.9 thursday 41 16.5 number of medications 3.3* 2.4# types of medication standard medications 218 87.9 others (e.g. special medications) 30 12.1 number of staff 23.7* 1.2# types of patients discharged 18 7.3 outpatient 230 92.7 sources of prescription one clinic 243 98.0 multiple clinic 5 2.0 patient’s arrival time peak hour (10am1pm) 132 53.2 non-peak hour (8am10am, 1pm5pm) 116 46.8 types of prescription new 195 78.6 refill 53 21.4 spub prescription yes 18 7.3 no 230 92.7 extemporaneous prescription yes 22 8.9 no 226 91.1 intervention prescription yes 12 4.8 no 236 95.2 filled by pharmacist 102 41.1 others 146 58.9 counterchecked by pharmacist 191 77.0 others 57 23.0 *mean #standard deviation (sd) factor associated with waiting time in univariable analysis, there were no statistically significant differences in types of patients, sources of prescriptions, types of prescriptions, spub prescriptions and extemporaneous prescriptions towards waiting time in outpatient pharmacy (table 2). the multivariable analysis was carried out for all significant variables to determine which factors associated with waiting time. total number of medications, total number of staff, prescription requiring intervention and filling personnel were found to be significant factors which associated with waiting time. an increase of one medication in the prescription, will increase the waiting time by 1.4 minutes (95% ci = 0.90, 1.94). the waiting time will be reduced by 1.8 minutes (95% ci = 2.87, -0.65) if the number of staff is increased by one person. prescriptions with intervention increased the waiting time by 10.0 minutes (95% ci = 4.09, 15.94). the waiting time is reduced by 3.1 minutes (95% ci = -5.68, -0.52) if the medication in the prescription is filled by pharmacist in comparison to other personnel such as prp, pharmacy assistant and student. fahrurazi et al. (2022) journal of pharmacy, 2(1), 1-7 page 4 table 2: factors associated with waiting time in outpatient pharmacy (n=248) variables slra mlrb bc 95% ci p-value adj. bd 95% ci p-value types of disease acute chronic 2.87 0.14, 5.60 0.040 number of medications 1.49 0.96, 2.03 < 0.001 1.42 0.90, 1.94 < 0.001 types of medications special medication standard medication -3.26 -7.45, 0.93 0.126 number of staff -1.09 -2.27, -0.09 0.070 -1.76 -2.87, -0.65 0.002 types of patients discharged outpatient 1.77 -3.53, 7.06 0.512 sources of prescriptions one clinic multiple clinic 0.92 -8.85, 10.69 0.853 arrival time non peak hour peak hour 8.60 6.07, 11.13 < 0.001 types of prescriptions new refill -1.94 -5.28, 1.40 0.253 spub prescriptions yes no -1.29 -6.57, 4.00 0.633 extemporaneous prescriptions yes no -2.03 -6.8, 2.79 0.408 intervention prescriptions no yes 10.56 4.31, 16.82 0.001 10.01 4.09, 15.94 0.001 filling by others pharmacist -2.47 -6.26, -0.49 0.081 -3.10 -5.68, -0.52 0.019 countercheck by others pharmacist -4.74 -20.07, .10.60 0.544 asimple linear regression bmultiple linear regression (r2 = 0.182; the model reasonably fits well; model assumptions are met; there are no interactions between independent variables and no multicollinearity problem) ccrude regression coefficients cadjusted regression coefficients fahrurazi et al. (2022) journal of pharmacy, 2(1), 1-7 page 5 discussion this study reported the mean (sd) waiting time of 23.0 minutes (sd = 11.0) which is lower than waiting time reported in other studies in malaysia. yi et al. (2021) reported that the waiting time in klinik kesihatan bandar mentakab and klinik kesihatan temerloh were 29.7 minutes and 28.4 minutes respectively (yi et al., 2021). a study by huang (1994) concluded that patients were satisfied if the waiting time is not more than 37 minutes (huang, 1994) whereas a study by afolabi & erhun (2003) mentioned that the average waiting time should be between 10-30 minutes for patient’s satisfaction (afolabi & erhun, 2003). our findings were within the recommended range of waiting time, but we did not investigate the level of satisfaction in our population. different population may have different perceptions on the length of time they need to wait to get their medication. there were four factors which had significant effect on the waiting time in our study which were 1) number of medications in the prescription, 2) number of staff, 3) prescriptions required intervention and 4) filling process. the number of medications in the prescription influenced the waiting time by increasing the time by 1.4 minutes per item in the prescription. according to a study conducted by loh et al. (2019), a reduction in average waiting time was shown when a fast-track dispensing window was introduced to dispense prescription with three medications or less (loh et al., 2017). another study by yang et al. (2019), also suggested that prescriptions with less item reduced the chances of medication error and thus reduces the waiting time (yang, liao, lin, & wu, 2019). this is because number of errors tend to increase with more numbers of drug orders in each prescription. as a result, waiting time increased due to the medication errors, with some of the processes need to be repeated before medications being dispensed. another study also proved that a fast-track counter containing items less than 3 medications showed a reduction in the waiting time from 20 to 23 minutes to an average of just 4 minutes (sadi, harb, el-dahiyat, & anwar, 2019). in our study, it was found that the number of staff working reduced the waiting time by 1.8 minutes when the staff was increased by one person. understaffing creates instability in the pharmacy operations leading to long queues and long prescription preparation times (arafeh et al., 2014). this then causes longer waiting time. another study has shown that a higher number of pharmacy technicians was associated with higher percentage of prescription served less than 30 minutes (loh et al., 2017). this indicated that pharmacy technician plays an important role in medication dispensing activity, assisting in recording, packaging and labelling of medications (loh et al., 2017). another study suggested that lack of staffing is a known major contributor to lengthy waiting time in hospital outpatients and public health clinics (ahmad, khairatul, & farnaza, 2017). therefore, a recommendation was made to increase the number of registration staff to two members to cope with the tasks (ahmad et al., 2017). a previous study revealed that waiting time was highly dependent on the number of pharmacists. it was suggested that employing 2 additional pharmacists, could lower the maximum patient waiting time from 58.2 minutes to 27.0 minutes (tan, chua, yong, & wu, 2009). third factor that affecting waiting time is when certain prescriptions required intervention. based on our study, prescription that required intervention increased the waiting time by 10.0 minutes. intervention of the prescription was the main contributing factors of increased waiting time because pharmacist needs to contact the prescriber to rectify any problem before it can proceed to the next steps. kim and schepers (2003) reported that majority of the error (76%) did not reach to the patients but had significant potential to cause morbidity and mortality (kim & schepers, 2003). in addition, another study revealed that high dose errors were the most common category of interventions which constitutes for 43.6% of interventions (alderman & farmer, 2001). this shows the importance of intervention to optimize patient therapy although it will lead to an increase in waiting time. the filling process was also proven to affect the waiting time. the waiting time was reduced by 3.1 minutes if the item in the prescription was filled by pharmacist in comparison to other personnel such as prps, pharmacy assistants and students. pharmacists are challenged with keeping up on the increasing number of new drugs and literature. drug information and literature evaluation skills are crucial for building clinical knowledge and providing evidence-based recommendations. a study conducted in 2015 has shown that time taken to fill up the prescriptions were shorter when it was done by pharmacists because they are more familiar with the medications (ghaibi, ipema, & gabay, 2015). perhaps, similar exposure in term of continuing professional development (cpd) should be given to other staff as well in order to ensure they are also familiar with the medication and thus can help in reducing waiting time. the limitation of our studies include that we did not investigate the satisfaction level of patients with the current waiting time. future studies may investigate this aspect to ensure healthcare services can be improved in term of efficiency, effectiveness and patient satisfaction. according to a study conducted by fauzia et al., (2017), the patient’s satisfaction with the outpatient hospital pharmacy was influenced by the dispensing process, consultation service by pharmacist, and general satisfaction aspect where the dispensing processes includes the time for filling prescription (fauzia, fahrurazi et al. (2022) journal of pharmacy, 2(1), 1-7 page 6 setiawati, & surahman, 2017). therefore, improving and making the filling process efficient and timely is an effective method to improve service quality and improve customer satisfaction. our study did not find a significant association among other predictors like age, gender, queue number, day of visit, types of disease, medications and burden of prescriptions. based on r2 value, r2 = 18.2% indicated there are still many factors which are not considered in this study but might influence waiting time in outpatient pharmacy. however, this study still might be useful in providing insights on current level of performance to improve to even better service for the public in future. conclusion the waiting time at the outpatient pharmacy of hrpz ii indicated the acceptable range of quality services which met the patient’s satisfaction. the factors contributed to waiting time included number of medications in the prescription, number of staff working on that day, prescriptions requiring intervention and filling personnel. future studies are needed to confirm the satisfaction level of patients and further improve quality of the service. acknowledgements the authors would like to thank the director general of health malaysia for his permission to publish this article. no external organisation was involved in this study as it was self-funded by the authors. conflict of interest the authors declare that they do not have any personal conflict of interest that may arise from the research publication. references afolabi, m. o., & erhun, w. o. 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(2001). queuing theory and customer satisfaction: a review of fahrurazi et al. (2022) journal of pharmacy, 2(1), 1-7 page 7 terminology, trends, and applications to pharmacy practice. hospital pharmacy, 36(3), 275-279. sadi, b. m. a., harb, z., el-dahiyat, f., & anwar, m. (2019). improving patient waiting time: a quality initiative at a pharmacy of a public hospital in united arab emirates. international journal of healthcare management, 14(3), 1-6. sun, j., lin, q., zhao, p., zhang, q., xu, k., chen, h., . . . liu, y. (2017). reducing waiting time and raising outpatient satisfaction in a chinese public tertiary general hospital-an interrupted time series study. bmc public health, 17(1), 1-11. suss, s., bhuiyan, n., demirli, k., & batist, g. (2017). toward implementing patient flow in a cancer treatment center to reduce patient waiting time and improve efficiency. journal of oncology practice, 13(6), e530-e537. tan, w. s., chua, s. l., yong, k. w., & wu, t. s. (2009). impact of pharmacy automation on patient waiting time: an application of computer simulation. annals academy of medicine singapore, 38(6), 501. yang, j.-h., liao, y.-f., lin, w.-b., & wu, w. (2019). prescribing errors in electronic prescriptions for outpatients intercepted by pharmacists and the impact of prescribing workload on error rate in a chinese tertiary-care women and children’s hospital. bmc health services research, 19(1), 1013. yi, f., ahmad, a., said, n., manshor, t., ling, s. o. o., jamil, n., & roby, b. (2021). identification of factors leading to excessive waiting time at the pharmacy unit of health clinics in temerloh district, pahang, malaysia. malaysian journal of pharmaceutical sciences, 19, 97-111. discussion this study reported the mean (sd) waiting time of 23.0 minutes (sd = 11.0) which is lower than waiting time reported in other studies in malaysia. yi et al. (2021) reported that the waiting time in klinik kesihatan bandar mentakab and klinik kesihatan... there were four factors which had significant effect on the waiting time in our study which were 1) number of medications in the prescription, 2) number of staff, 3) prescriptions required intervention and 4) filling process. the number of medications... in our study, it was found that the number of staff working reduced the waiting time by 1.8 minutes when the staff was increased by one person. understaffing creates instability in the pharmacy operations leading to long queues and long prescription p... third factor that affecting waiting time is when certain prescriptions required intervention. based on our study, prescription that required intervention increased the waiting time by 10.0 minutes. intervention of the prescription was the main contrib... the filling process was also proven to affect the waiting time. the waiting time was reduced by 3.1 minutes if the item in the prescription was filled by pharmacist in comparison to other personnel such as prps, pharmacy assistants and students. pharm... the limitation of our studies include that we did not investigate the satisfaction level of patients with the current waiting time. future studies may investigate this aspect to ensure healthcare services can be improved in term of efficiency, effecti... introduction methodology results discussion this study reported the mean (sd) waiting time of 23.0 minutes (sd = 11.0) which is lower than waiting time reported in other studies in malaysia. yi et al. (2021) reported that the waiting time in klinik kesihatan bandar mentakab and klinik kesihatan... there were four factors which had significant effect on the waiting time in our study which were 1) number of medications in the prescription, 2) number of staff, 3) prescriptions required intervention and 4) filling process. the number of medications... in our study, it was found that the number of staff working reduced the waiting time by 1.8 minutes when the staff was increased by one person. understaffing creates instability in the pharmacy operations leading to long queues and long prescription p... third factor that affecting waiting time is when certain prescriptions required intervention. based on our study, prescription that required intervention increased the waiting time by 10.0 minutes. intervention of the prescription was the main contrib... the filling process was also proven to affect the waiting time. the waiting time was reduced by 3.1 minutes if the item in the prescription was filled by pharmacist in comparison to other personnel such as prps, pharmacy assistants and students. pharm... the limitation of our studies include that we did not investigate the satisfaction level of patients with the current waiting time. future studies may investigate this aspect to ensure healthcare services can be improved in term of efficiency, effecti... conclusion the waiting time at the outpatient pharmacy of hrpz ii indicated the acceptable range of quality services which met the patient’s satisfaction. the factors contributed to waiting time included number of medications in the prescription, number of staff... acknowledgements the authors would like to thank the director general of health malaysia for his permission to publish this article. no external organisation was involved in this study as it was self-funded by the authors. conflict of interest references barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework page 79 azmi et al. (2021) journal of pharmacy, 1(2), 79-86 assessment of medication adherence and quality of life among patients with type 2 diabetes mellitus in a tertiary hospital in kelantan, malaysia nazmi liana azmi1*, nurul aida md rosly1, tang hock chun1, anis fariha che darof1 and nor dini zuki1 abstract introduction: previous studies have reported the relationship between medication adherence and quality of life are interrelated. however, many of the results were found to be conflicting. this study aimed to assess the level and association of medication adherence and quality of life among type 2 diabetes mellitus patients in raja perempuan zainab ii hospital, kelantan, malaysia. materials and methods: a cross-sectional survey was conducted among adult type 2 diabetes mellitus patients on treatment for over 1 year using convenience sampling at outpatient. medication compliance questionnaire (mcq) and revised diabetes quality of life questionnaire (dqol) instrument were self-administered to eligible subjects. data were analysed using gnu pspp version 0.8.5 and reported for descriptive statistics as well as correlation of both parameters. results: a total of 200 patients were recruited and they were mostly at the age of 40 to 60 years old. the mean (sd) score for mcq was 26.0 (1.6) with the majority of them were non-adherent (55.0%, n=110). the mean (sd) score for overall revised dqol instrument was 25.5 (8.9) while each domain of “satisfaction”, “impact” and “worry” had mean (sd) scores of 12.0 (5.0), 7.7 (3.4) and 5.9 (2.7), respectively. the scores obtained were only approximately half of the possible range of scores for qol. there was no significant correlation between total score of medication adherence and quality of life when tested using pearson’s correlation (r=-0.083, p=0.240). independent t-test also demonstrated no significant relationship between medication adherence status and quality of life (p=0.883). conclusion: type 2 diabetes mellitus patients in our setting had unsatisfactory adherence but exhibited acceptable quality of life. we observed that both variables were not associated with one another. further research is warranted to identify potential factors affecting non-adherence to medication. article history: received: 17 january 2021 accept: 10 march 2021 published: 31 july 2021 keywords: medication adherence, quality of life, diabetes mellitus, association how to cite this article: nazmi liana azmi, nurul aida md rosly, tang hock chun, anis fariha che darof and nor dini zuki (2021). assessment of medication adherence and quality of life among patients with type 2 diabetes mellitus in a tertiary hospital in kelantan, malaysia journal of pharmacy, 1(2), 79-86. doi: 10.31436/jop.v1i2.66 authors’ affiliation: 1 department of pharmacy, raja perempuan zainab ii hospital, ministry of health malaysia, 15586 kota bharu, kelantan, malaysia *corresponding author: email address: nazmiliana@moh.gov.my phone: +6097452478 / +60179224977 fax: +6097482033 page 80 azmi et al. (2021) journal of pharmacy, 1(2), 79-86 introduction type 2 diabetes mellitus (t2dm) has become one of the most prevalent non-communicable diseases and is classified as a public health threat. the prevalence of this debilitating illness has increased dramatically in all parts of the world. the most recent estimates recorded that 463 million adults were diagnosed with t2dm in 2019, a figure that is projected to continue to escalate to 700 million in less than 3 decades (51.2%) (international diabetes federation, 2019). proper medication intake is vital in the management of t2dm which contributed to the therapeutic success (marínpeñalver et al., 2016). a positive impact on quality of life (qol) as perceived by the patient is also a crucial criterion for evaluating the effectiveness of t2dm management (hale et al., 2019). adherence to drug treatment can be defined by the extent to which patients follow the instructions of their physician or other healthcare providers (jimmy & jose, 2011). meanwhile, qol is a measurement of an individual’s functioning and well-being from various aspects of physical, emotional and social (abedini et al., 2020). these two distinct concepts have in common that they are both interrelated and important to consider when assessing the impact of treatment in healthcare at the patient level (zioga et al., 2016). the management plan should consider the patient’s present condition and lifestyle and yet he or she must work on self-management and adherence to the treatment (american diabetes association, 2003). such examples are poor medication adherence can lead to progression of uncontrolled diabetes with complications of nephropathy and retinopathy (alodhaib et al., 2021) while poor qol is suggestive of patient being not satisfied with the treatment due to its rigid schedule for insulin injection or adverse events (brod et al., 2014). together, the patient would not have received the best medical care for t2dm (american diabetes association, 2003). evidences have shown that in chronic diseases such as t2dm, patients who adhere to their treatment tend to possess better health and qol. those who routinely take their medications as instructed generally have more positive clinical outcome (farhat et al., 2019). examples of the benefits are the relationship between both variables was well-documented across studies around the globe, but many of the results were found to be conflicting (alfian et al., 2016; zioga et al., 2016). to the best of our knowledge, publications that evaluate the relationship between medication adherence and diabetes specific qol are still scarce especially in the local setting. this was because many of the investigations utilized generic tools that were not disease-specific for qol (alfian et al., 2016). therefore, we aimed to assess the level of medication adherence and diabetes specific qol as well as their association among t2dm patients in a tertiary care hospital in the state of kelantan, malaysia. materials and methods study design and population a cross-sectional survey was conducted over a fourmonth period from november 2018 until march 2019 using self-administered questionnaires. adult patients who were diagnosed with t2dm and received treatment for at least 1 year were considered eligible to be enrolled in the study. as the dominant language in kelantan is bahasa melayu, those who were unable to speak or write in bahasa melayu were excluded along with patients who had cognitive impairment and end-stage renal disease. data collection the data collection process took place at the outpatient pharmacy, raja perempuan zainab ii hospital (hrpz ii). subjects were recruited using convenience sampling when the patients came for prescription filling. they were explained about the survey and voluntarily consented to participate in the study. this survey employed two types of self-administered and validated questionnaires in bahasa melayu to measure the outcomes. the assessment of medication adherence was done using medication compliance questionnaire (mcq) that consisted of seven questions, scored with a 4-point likert scale ranging from 1 (all the time) to 4 (none of the time). respondents with a total score of 27 and above were considered as adherent to their t2dm medications (ahmad et al., 2013). as for qol, the evaluation was done using the revised diabetes quality of life questionnaire (dqol) instrument. compared to the original dqol instrument of 46 items, the revised version was validated to maintain the same three domains with a smaller number of items. altogether there were 13 questions: satisfaction (6 questions), impact (4 questions) and worry (3 questions). all items were scored with a 5-point likert scale with 1 (very satisfied) to 5 (very dissatisfied) for “satisfaction” domain as well as 1 (never) to 5 (all the time) for “impact” and “worry” domains. a higher average score indicated a poorer qol (bujang et al., 2018). permission was obtained from the authors before both instruments were distributed to the study population. single mean formula was used for the purpose of sample size calculation. the values of two-tailed α = 0.05, σ = 0.7 and d = 0.1 were entered in the equation which yielded a minimum of 189 subjects (burroughs et al., 2004). statistical analyses data analyses were carried out using gnu pspp software version 0.8.5 (gnu project, 2015). for descriptive statistics, numerical data were presented in mean and standard deviation (sd) while categorical data were expressed in frequency and percentage. the association between page 81 azmi et al. (2021) journal of pharmacy, 1(2), 79-86 medication adherence and qol was determined using pearson’s correlation and independent t-test. the significance level was set at 0.05 with the corresponding confidence level of 95%. ethical approval this research was registered with national medical research registry (nmrr-18-2991-44264) and approved by medical research and ethics committee (mrec), ministry of health malaysia. results sociodemographic characteristics in total, 200 respondents were recruited for this study. patients were mostly malay (98.0%) male (54.5%) of the age 46 to 60 years old (38.5%), married (76.5%) with secondary education (75.0%) and monthly income of less than rm1,000 (64.5%). the majority of them had been diagnosed with t2dm for over five years (69.5%) with comorbidities (97.5%), on combination oral hypoglycaemic agents (oha) (41.0%) and more than seven types of medications (67.0%) (table 1). table 1: sociodemographic and clinical characteristics of respondents (n=200) characteristics n % age 26 to 45 46 to 60 >60 58 77 65 29.0 38.5 32.5 gender female male 91 109 45.5 54.5 ethnic malay non-malay 196 4 98.0 2.0 education level primary secondary tertiary 22 150 28 11.0 75.0 14.0 marital status married 153 76.5 single 47 23.5 monthly salary (rm) <1,000 ≥1,000 129 71 64.5 35.5 duration of t2dm (years) <5 5 to 10 >10 61 84 55 30.5 42.0 27.5 t2dm treatment monotherapy combination oha combination oha and insulin 46 82 72 23.0 41.0 36.0 comorbidities no yes 5 195 2.5 97.5 number of medications <3 3 to 7 >7 18 48 134 9.0 24.0 67.0 medication adherence score the mean (sd) score for mcq was 26.0 (1.7) with the majority of patients were non-adherent (55.0%, n=110). only 90 of them managed to score a sum of 27 and higher were classified as adherent (45.0%). it was found that the main reason for non-adherence in t2dm patients was they forgot to take their medications. however, experiencing side effects or adverse drug reactions did not simply hinder the patients from taking their medications as prescribed. this could be seen in the table details whereby out of the seven questions, item q1 had the lowest mean (sd) score of 3.4 (0.7) while item q5 had the highest mean (sd) score of 3.9 (0.4) (table 2). table 2: mcq score for each item (n=200) questions range of score minmax mean (sd) q1: how often do you forget to take your medication? 1 to 4 1 to 4 3.4 (0.7) page 82 azmi et al. (2021) journal of pharmacy, 1(2), 79-86 q2: how often do you decide not to take your medication? 1 to 4 3 to 4 3.8 (0.4) q3: how often do you miss taking your medication because you feel better? 1 to 4 3 to 4 3.8 (0.4) q4: how often do you decide to take less of your medication? 1 to 4 2 to 4 3.6 (0.7) q5: how often do you stop taking your medication because you feel sick due to its effects? 1 to 4 3 to 4 3.9 (0.4) q6: how often do you forget to bring along your medication whenever you travel? 1 to 4 3 to 4 3.7 (0.5) q7: how often do you miss taking your medication because you run out of it at home? 1 to 4 3 to 4 3.8 (0.4) qol score the revised dqol instrument had 13 questions for all 3 domains. the mean (sd) score for overall revised dqol instrument was 25.5 (8.9) while each domain of “satisfaction”, “impact” and “worry” had mean (sd) scores of 12.0 (5.0), 7.7 (3.4) and 5.9 (2.7), respectively. the scores obtained were only approximately half of the possible range of scores for qol. since a higher average score would signify a poorer qol, it seemed that the disease did not badly affect the qol among t2dm patients. they were satisfied with the amount of time they spent due to t2dm, the current treatment, knowledge and life in general. apart from that, they also felt that t2dm had very seldom impact on their life and therefore were not really worried (table 3). when tested using pearson’s correlation, it was found that there was no significant correlation between total score of medication adherence and qol (r=-0.083, p=0.240) (table 4). the outcome was further confirmed by independent t-test which also demonstrated no significant association between qol and medication adherence status (p=0.883) (table 5). discussion medication adherence remains as a major hurdle for t2dm patients. over the years, the results regarding medication adherence among t2dm patients have been inconsistent. a systematic review by cramer (2004) reported that the overall adherence rate in retrospective and prospective studies was between 36 to 93%. this corroborated that many t2dm patients were not adherent to their treatment, including both oha and insulin (cramer, 2004). two more recent systematic reviews also reported that in some investigations, medication adherence rates were found to be as low as 31 to 33% (capoccia et al., 2016; odegard & capoccia, 2007). in malaysia, our findings on low medication adherence were in-line with a previous report at primary health clinics in selangor that observed non-adherence at (52.8%, n = 294) with mean (sd) score of mcq was 25.6 (2.4) (ahmad et al., 2013). another study by al-qazaz et al. (2011) among t2dm patients at penang hospital also reported low level of adherence (al-qazaz et al., 2011). however, better medication adherence rates could be seen in other researches. for an instance, 55.2% of the patients attending a public hospital in selangor were adherent (abdullah et al., 2019). another survey conducted in a tertiary hospital in malaysia found that 66% of the subjects obtained high scores in medication adherence (omar & san, 2014). the discrepancies in these findings might be attributed to the variation of age among the study population and the use of different measurement tools for adherence. page 83 azmi et al. (2021) journal of pharmacy, 1(2), 79-86 table 3: dqol score for each item (n=200) domains questions range of score min-max mean (sd) satisfaction q1: how satisfied are you with the amount of time it takes to manage your diabetes? q2: how satisfied are you with the amount of time you spend getting checkups? q3: how satisfied are you with the time it takes to determine your sugar level? q4: how satisfied are you with your current treatment? q5: how satisfied are you with your knowledge about your diabetes? q6: how satisfied are you with life in general? 1 to 5 1 to 5 1 to 5 1 to 5 1 to 5 1 to 5 1 to 4 1 to 4 1 to 5 1 to 4 1 to 5 1 to 4 2.2 (1.0) 2.0 (0.9) 2.2 (1.1) 1.9 (0.9) 1.8 (1.1) 2.0 (1.0) impact q1: how often do you feel pain associated with the treatment for your diabetes? q2: how often do you feel physically ill? q3: how often does your diabetes interfere with the family life? q4: how often do you find your diabetes limiting your social relationships and friendships? 1 to 5 1 to 5 1 to 5 1 to 5 1 to 5 1 to 5 1 to 4 1 to 4 2.0 (1.1) 2.2 (1.0) 1.7 (0.9) 1.8 (1.0) worry q1: how often do you worry about whether you will pass out? q2: how often do you worry that your body looks different because you have diabetes? q3: how often do your worry that you will get complications from your diabetes? 1 to 5 1 to 5 1 to 5 1 to 5 1 to 4 1 to 5 1.8 (1.0) 1.7 (0.9) 2.4 (1.2) summary satisfaction impact worry overall 6 to 30 4 to 20 3 to 15 13 to 65 6 to 24 4 to 17 3 to 13 13 to 41 12.0 (5.0) 7.7 (3.4) 5.9 (2.7) 25.5 (8.9) table 4: correlation between total score of medication adherence and qol variables medication adherence qol medication adherence 1.63a 0.240b qol -0.083c 8.89a asd, bp-value, ccorrelation coefficient (r). table 5: comparison of qol and medication adherence status variable mean (sd) mean difference (95% ci) t-statistic (df) p-value a qol non-adherent (n=110) adherent (n=90) 0.2 (-2.3, 2.7) 0.15 (198) 0.883 25.61 (9.11) 25.42 (8.67) page 84 azmi et al. (2021) journal of pharmacy, 1(2), 79-86 in many cases, forgetting to take the medication as instructed is the common cause for non-adherence. this statement is supported by systematic reviews of diabetes medication-taking behaviour which identified that remembering to take medication or obtaining refills was one of the several key barriers to medication adherence (capoccia et al., 2016; odegard & capoccia, 2007). other factors involved were regimen complexity, adverse events, fear of insulin injection, depression and a patient’s lack of belief in the benefits of the medication (capoccia et al., 2016; odegard & capoccia, 2007). a similar result was seen in our respondents whereby they scored the least for the question addressing forgetfulness in taking medications. it is well-known that t2dm is a challenging disease and the changes it inflicted on lifestyle will in time lead to impairment of qol. the never-ending demands of diabetes care, the symptoms of hypoglycaemia and hyperglycaemia as well as fear about or the reality of complications could influence the well-being of t2dm patients’ lives. the presence of comorbidities would further deteriorate one’s qol (jannoo et al., 2017; trikkalinou et al., 2017). however, there were patients who showed lack of concern on some aspects of qol even though they were affected. this could be due to inadequate information or a perception of lack of vulnerability. it might also signify a mean of coping strategy to reduce anxiety. other possible reasons were the absence of initial symptoms and timing, as perception of risk was related to visible signs while the complications of poor diabetes control were obvious only in the long run (pera, 2011). this could best explain why our respondents maintained an acceptable qol score despite many being diagnosed with t2dm for over 5 years with comorbidities. contradicting to other literature, no significant relation was found between medication adherence and qol among our patients. adherence to prescribed medication generally showed improvement in qol in patients with t2dm (alfian et al., 2016; khayyat et al., 2019; zioga et al., 2016). it was suggested that treatment adherence could influence qol, improve clinical condition, reduce morbidity and mortality rates as well as slow down the disease progression (asche et al., 2011). however, a systematic review reported that the analysis of the relationship between both variables had mixed results. while there were investigations which showed positive effect between qol and adherence, however, another research also failed to recognize this association (de fátima gusmai et al., 2015). some authors suggested that this inconsistency might be due to the diversity of the methods and study populations (khayyat et al., 2019; zioga et al., 2016). we opted to use the mcq as the questions assessed patients’ intentional as well as unintentional non-adherence to medication regimen and probed their reasons for non-adherence (ahmad et al., 2013). as for the revised version of dqol, it was chosen for its excellent construct with lesser number of items (bujang et al., 2018). there were a few drawbacks of this survey. firstly, the study population was conveniently sampled from a single site which did not necessary represent the actual population of malaysian living with t2dm. in addition to that, the use of self-reporting assessment made it prone to biases as the respondents might resort to more socially acceptable answers rather than being honest. also, the interpretation of the questions could be different among them and the respondents might not be able to assess themselves accurately without a proper clinical examination by a medical practitioner. finally, this study only evaluated the aspect of medication adherence and did not include other important therapies such as dietary and lifestyle, exercise, foot care, smoking and blood sugar testing. having said that, the findings of this survey is still essential to improve the medication adherence among t2dm patients in malaysia. the results can be used to assist with the planning for delivery of personalized counselling and health education which ultimately optimize their disease management. conclusion patients with t2dm had unsatisfactory medication adherence but exhibited acceptable qol. medication adherence on its own was not associated with qol. however, qol is undeniably complex and multifaceted. nevertheless, high level of medication adherence is crucial to ensure successful management of t2dm. further research is warranted to identify potential factors affecting non-adherence to medication. acknowledgements the authors wish to extend their gratitude to the director general of health malaysia for his permission to publish this work. this study did not receive any specific funding and involve no external organization. conflict of interest the authors certified that there was no conflict of interest to declare from this research publication. references abdullah, n. f., khuan, l., theng, c. a., sowtali, s. n., & juni, m. h. 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(2011). living with diabetes: quality of care and quality of life. patient preference and adherence, 5, 65–72. https://doi.org/10.2147/ppa.s16551 trikkalinou, a., papazafiropoulou, a. k., & melidonis, a. (2017). type 2 diabetes and quality of life. world journal of diabetes, 8(4), 120–129. https://doi.org/10.4239/wjd.v8.i4.120 zioga, e., kazakos, k., dimopoulos, e., koutras, c., marmara, k., marmara, e.-e., marmaras, a., & lavdaniti, m. (2016). adherence and quality of life in patients with type ii diabetes mellitus in northern greece. materia socio-medica, 28(4), 258–262. https://doi.org/10.5455/msm.2016.28.258-262 introduction materials and methods results discussion conclusion acknowledgements conflict of interest the authors certified that there was no conflict of interest to declare from this research publication. references barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework page 19 rajkumar et al. (2023) journal of pharmacy, 3(1), 19-26 in vitro antimicrobial assessment of seeds of selected medicinal plants in sri lanka gowri rajkumar1*, mihiri rangika jayasinghe1 and vinotha sanmugarajah2 abstract introduction: people are suffering from many noncommunicable diseases as a result of the covid pandemic and the stress that has followed after it. diabetes mellitus is a complex noncommunicable disease and its incidence in sri lanka is almost high. while a number of antidiabetic medications are available, herbal management of diabetes is encouraged due to its low side effects and efficacy. examining the antibacterial properties of anti-diabetic plants may be highly valued because, diabetics are chronic immunocompromised individuals who are more prone to microbial infections. to focus this aim, the present in-vitro antimicrobial assessment carried out for the seeds of selected four medicinal plants, such as syzygium cumini (l.) skeels, sinapis alba l., trigonella foenum-graecum l. and nigella sativa l. that are commonly used for diabetes management in sri lanka. materials and methods: crude ethanol extract from the seeds has been studied for their antibacterial potential against three bacterial strains such as enterococcus faecalis, staphylococcus aureus and escherichia coli by using agar well diffusion method in triplicates. the statistical analysis was performed using a one-way analysis of variance. results: the seed extract of s. cumini showed the highest value of zone of inhibitions (e. faecalis: 24.70±037, s. aureus: 16.15±1.20 and e. coli: 10.37±1.51 mm) and s. alba exhibited the lowest value of zone of inhibition (1.08±2.65, 1.08±2.65, 0 mm) for all selected pathogens respectively and which were comparable to the positive control streptomycin (e. faecalis: 25.45±1.18, s. aureus: 21.08±0.26 and e. coli: 19.37±1.35mm). conclusion: the result shows that s. cumini seed extract poses the highest antimicrobial activity in selected bacteria. therefore, this seed is potential to be further developed as an herbal antibiotic for the management of infection in diabetes in future. article history: received: 7 july 2022 accept: 3 january 2023 published: 31 january 2023 keywords: medicinal plants, antimicrobial activity, agar well diffusion method, seeds, syzygium cumini how to cite this article: gowri rajkumar, mihiri rangika jayasinghe & vinotha sanmugarajah (2023). in vitro antimicrobial assessment of seeds of selected medicinal plants in sri lanka. journal of pharmacy, 3(1), 19-26. doi: 10.31436/jop.v3i1.179 authors’ affiliation: 1 department of botany, faculty of science, university of jaffna, jaffna, 40000, sri lanka 2 unit of siddha medicine, university of jaffna, jaffna, 40000, sri lanka. *corresponding author: email address: gowrir@univ.jfn.ac.lk phone: +94773604037 mailto:gowrir@univ.jfn.ac.lk page 20 rajkumar et al. (2023) journal of pharmacy, 3(1), 19-26 introduction the number of people with diabetes is exponentially increasing world-wide and has become an important global public health problem (global report on diabetes, 2016; shehab et al., 2022). sri lanka, despite being a developing nation, has always maintained health indices on par with most developed countries. currently, sri lanka is in the midst of its worst economic crisis in history (pathmanathan & abhayaratna 2022). in sri lanka, people are suffering from many noncommunicable diseases as a result of the covid pandemic and the stress that has followed after it. that condition gets more critical as a result of the country's economic situation. diabetes mellitus is one of the most common endocrine disorders in sri lanka. 10% of the sri lankan population and 18% of the urban population are affected by it (pathmanathan & abhayaratna 2022). diabetes mellitus is characterized by hyperglycaemia and is especially classified into two types, type-i (insulin dependent diabetes) and type-ii diabetes (non-insulin dependent diabetes) (idf diabetes atlas, 2013; aoac, 2016; american diabetes association, 2019; king, 2012). the incidence of diabetes (% of the population aged 20-79) in sri lanka was 11.3% in 2021 (the world bank group, 2022). this condition may be due to the common risk factors such as genetic, environmental, different life stylishness and physical inactivity (wu et al., 2014; arawwawala, 2006) and prevalence of depression (akter & latif 2021). there is another type is gestational diabetes which mainly arises during the pregnancy (buchanan, xiang & page, 2012). the oxidative stress is a recognized pathogenic mechanism in the development and progression of diabetes which reasons owing to augmented free radical production and weakened antioxidant defences (unuofin & lebelo, 2020). a study mentioned that the antiglycation properties of herbal extracts and their complexes powerfully interrelated with their antioxidant capacity with that antioxidant and anti-glycation activities are associated strongly with phenol and flavonoid contents (babich et al., 2022). generally, antibacterial actions are facilitated by the immune-modulating and antioxidant capabilities of medicinal plants (aryal et al., 2021). antibacterial action is the most significant distinctive of medical textiles, to deliver satisfactory defence against microbes, biological fluids, and infection transmission (alihosseini, 2016). prevention of food spoilage and food intoxication pathogens is regularly attained by use of chemical preservers. plant extracts have been used to control food poisoning diseases and preserve foodstuff (mostafa et al., 2018). an extensive range of biological constituents as alkaloids, flavonoids, glycosides, terpenoids, phenols, and coumarins have been stated from different parts of the plant, which are responsible for numerous biological activities as well as antimicrobial, antioxidant, and anti-inflammatory properties (sarkar, salauddin & chakraborty, 2020; phuyal et al.,2020). while a number of antidiabetic medications are available, herbal management of diabetes is encouraged due to its low side effects and efficacy. examining the antibacterial properties of anti-diabetic plants may be highly valued because, diabetics are chronic immunocompromised individuals who are more prone to microbial infections (hegazy et al., 2021). for this purpose, this in-vitro antimicrobial assessment was performed for the ethanol extracts of four seeds of medicinal plants namely syzygium cumini (l.) skeels, sinapis alba l., trigonella foenum-graecum l. and nigella sativa l. against three bacterial strains as e. faecalis, s. aureus and e. coli. materials and methods chemicals and reagents ethanol, streptomycin, distilled water and nutrient agar were purchased from sigma-aldrich. all reagents and chemicals were of analytical grade. collection of medicinal plants and preparation of the seed extract the plant materials with seeds of s. cumini, s. alba, t. foenum graecum and n. sativa (table 1) were collected and botanically authenticated by a curator of the national herbarium center, department of national botanic garden, peradeniya, sri lanka. the fresh seeds were washed in tap water for several times to remove the soil and dust particle. then they were air dried in thoroughly at room temperature until dried and blended to form a fine powder and stored in airtight containers at room temperature until needed for analysis. fifty grams of powered materials of each seed were separately weighed and placed in 500 ml of culture bottles. as much as 150 ml of absolute ethanol was added to it and mixed well. lid of each bottle were covered with para film. the solution was kept for 5 days with occasional shaking by using shaker at 150 rpm for 15 minutes in every morning and evening. they were filtered through whatman filter paper no.1. the part of filtered content was concentrated by using rotatory evaporator (buchi, chi minh city, vietnam) at 52 0c (rajkumar, jayasinghe & sanmugarajah, 2021). crude extracts were kept at 20 0c for further analysis. test microorganisms three bacterial strains were provided by the faculty of science, university of jaffna were used for the antimicrobial tests, according to table 2. all the test strains were preserved on nutrient agar slants at 4 0c and sub-cultured on to nutrient broth for 24 hours prior to testing. these bacteria served as test pathogens for this assay. page 21 rajkumar et al. (2023) journal of pharmacy, 3(1), 19-26 assay of antimicrobial activity using agar well diffusion method about 22.68 g of nutrient agar (na) powder was dissolved in 810 ml of distilled water. then 15 ml parts of the na medium were poured into boiling tubes. medium which was contained in the boiling tubes were autoclaved at 121 0c for 20-30 minutes. then 15 ml of sterilized nutrient agar was mixed with 100 µl of bacterial suspensions inside the laminar air flow chamber. the mixture was stirred well and it was poured into sterile petri dishes separately (dwivedi et al., 2017). after the solidification the wells were punched over the agar plates using sterile cork-borer (5mm in diameter) and 15 µl of plant extracts were added to the wells separately. the plates were incubated for 24 hours at 37 0c. distilled water and streptomycin (100 µg/µl) were used as the negative and the positive control respectively. after incubation the diameter of the formed inhibitory zones formed around each well were measured (mm) in four different fixed directions and recorded. each experiment was conducted in triplicate. data analysis data were statistically analysed by one way analysis of variance and tukey’s multiple comparisons at probability value (p˂0.05) using a sas statistical package (version 9.1.3) and mean separation was performed by least significance difference. results are expressed as mean ± se and statistical significance was evaluated by anova. results this in vitro antimicrobial assay was done for four seeds of selected antidiabetic medicinal plants as s. cumini, s. alba, t. foenum-graecum and n. sativa against three selected common bacterial strains. based on figure 1, ethanolic seed extract of s. cumini exhibited the significant antimicrobial activity while the ethanolic seed extracts of s.alba represented minimum antibacterial activity against the all three bacteria as e. faecalis, e. coli and s. auereus. the results showed that significant amount of inhibition zone was obtained against all the tested bacterial strains which was comparable to the positive control streptomycin. based on figure 1, the ethanolic seeds extract of s. cumini exhibited the significant antimicrobial activity while the ethanolic seeds extract of s. alba showed minimum activity against all three bacterial strains at 37°c. there are also significant differences of antimicrobial activity among selected human pathogens. it showed that highest antimicrobial activity for e. faecalis, also it represented antimicrobial activity for both of gram negative and positive pathogens. while s.alba showed same antimicrobial activity against e. faecalis and s. aureus, inhibition zone is absent for e.coli. based on table 3, moderate antibacterial activity was showed by t. foenum-graecum and n. sativa against e. coli & e. faecalis and e. coli respectively. there is no any inhibition zone against s. aureus by seeds extract of t. foenum-graecum but it showed relative values of inhibition zones against both of e. faecalis and e. coli. there is no any inhibition zone against e. faecalis by n. sativa, and inhibition zones are represented against s. aureus and e. coli human pathogens respectively. positive and negative control are represented by streptomycin and distilled water respectively. when consider about positive and negative controls, streptomycin exhibited the highest inhibitory effect against e. faecalis and the lowest inhibitory effect against e. coli. in negative controls there were no any inhibitory zones. table 1: medicinal plant seeds tested for their antibacterial activity in the study botanical name family name common name sinhala tamil english syzygium cumini myrtaceae mahadan naval black plum sinapis alba brassicaceae aba kaduku mustard trigonella foenumgraecum fabaceae asumodhagam vendayam fenugreek nigella sativa ranunculaceae kaluduru karumjeerakam black cumin page 22 rajkumar et al. (2023) journal of pharmacy, 3(1), 19-26 table 2: pure bacterial cultures name type atcc no. enterococcus faecalis gram positive 29212 staphylococcus auereus gram positive 29213 escherichia coli gram negative 25922 variation of mean diameter of seeds extracts of selected medicinal plants against selected human pathogens at 37℃. mean followed by same letters are not significantly different by lsd at 5% level, p value <0.0001 figure 1. antimicrobial activity of selected seeds of anti-diabetic medicinal plants -10 -5 0 5 10 15 20 25 30 35 2 9 2 1 2 2 9 2 1 3 2 5 9 2 2 m e a n d ia m e te r ( m m ) bacterial strains of human pathogen syzygium cumini trigonella foenum-graecum sinapis alba nigella sativa positive control negative control page 23 rajkumar et al. (2023) journal of pharmacy, 3(1), 19-26 table 3: bacterial growth inhibition zones of selected seeds of anti-diabetic medicinal plants plant species diameter of the inhibition zones (mm) 29212 29213 25922 syzygium cumini 24.7±0.37 16.15±1.20 10.37±1.51 sinapis alba 1.08±2.65 1.08±2.65 0 trigonella foenum-graecum 10.83±0.34 0 11.7±1.97 nigella sativa 0 5.58±4.37 15.7±1.56 positive control 25.45±1.18 21.08±0.26 19.37±1.35 negative control 0 0 0 discussion based on this in vitro study the researchers found that the ethanolic seed extract of s. cumini had shown highest anti-bacterial activity against selected bacterial strains. t. foenum-graecum seed extract also shown higher antibacterial effect against some bacterial strains. but, s.alba had shown least antibacterial activity. present study results could be comparable with the previous studies which were stated that the seed phenolic extract of s.cumini showed antibacterial activity against tested bacterial strains (santos et al., 2020); the methanol fraction of ethanol extract from the seeds of s. cumini was found to have significant antibacterial activity (yadav et al., 2011; patoary et al., 2014; das, das & dharani, 2019). however, the present results of s.alba is not in line with another study which was done by the boscaro et al., 2018. that study found that the s.alba seed hydroalcoholic extract was effective against e. coli and s. aureus in disc diffusion test (boscaro et al., 2018). the hexane extract of s. alba seeds showed the highest anti-microbial activity (sujatha & mariajancyrani, 2013). sharma et al found that the methanol extract of t. foenum-graecum seeds shown maximum zone of inhibition against e. coli and staphylococcus (sharma, singh & rani 2017). another study found that the oil which was extracted from fenugreek seeds has a good antimicrobial activity against some bacteria (sara & abdalbasit, 2022). further another study found that the n. sativa seed oil had a strong antibacterial activity significantly (p<0.01) greater inhibition zone than that of gentamicin (forouzanfar, bazzaz & hosseinzadeh, 2014). bakathir and abbas informed that the n. sativa ground seeds possessed antibacterial effect against the staphylococcus (bakathir & abbas 2011). based on all of the representation of antimicrobial activity, it showed that highest antimicrobial activity is showed by s. cumini rather than other extracts, also when consider about phytochemical screening there is highest representation in seeds extracts of s. cumini. antimicrobial and antibiotic principle are highly showed by phytochemical compounds such as alkaloids, saponins, tannins, flavonoids and steroids which are known to be biologically active (nethathe & ndip, 2011; patra, 2012; mujeeb, bajpai & pathak, 2014; ali et al. 2018; pizzi, 2021; nek rahimi et al., 2022). since gram positive bacteria's cell walls are more permeable than gram negative bacteria, whose outer membrane has a lipopolysacharide layer that prevents some antibiotics and antibacterial compounds from penetrating, most plant extracts are thought to be more effective against gram positive bacteria (wintola & afolayan, 2015). among the all-selected medicinal plants, s. cumini showed in high level of antimicrobial activity. plant produces a range of chemical constituents to protect themselves from the attack of various pathogenic micro-organisms. substances can either prevent the growth of microorganism or kill them. it can be considered as resources for developing new drugs for various infectious diseases. antibiotic substances are recognized to vary in concentration in different tissues of same plant, between plants of same and different species and concentration of antibiotics in plant is determined by its environment (sushil kumar, bagchi & darokar, 1997). in seeds, reserve materials are starch, fixed oils, proteins, fixed oils, fatty acids, some proteins are known to possess good antimicrobial activity and antifungal protein (cowan, 1999). https://www.frontiersin.org/people/u/924566 https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/antibacterial-activity https://www.cabdirect.org/globalhealth/search/?q=au%3a%22sujatha%22 https://www.cabdirect.org/globalhealth/search/?q=au%3a%22mariajancyrani%22 page 24 rajkumar et al. (2023) journal of pharmacy, 3(1), 19-26 conclusion this research on the antimicrobial activity of the herbs helps to develop effective herbal remedies as antimicrobial activity to reduce the infection in diabetes. the result shows that s. cumini seeds extract poses the highest antimicrobial activity in gram positive and negative bacteria. therefore, this seed is potential to be further developed as an herbal antibiotic for the management of infection in diabetes in future. more detail study such as are fractionation and characterization of active phytochemicals which are responsible for the antimicrobial activity, as well as in-vivo activities recommended to be conducted in the future study. acknowledgements the authors greatly appreciate the financial support given by the university of jaffna research grant (grant no. urg/2021/seit/27). conflict of interest authors have no conflict of interest. references akter, n. and latif, z.a., (2021). assessment of depression and its associated factors among patients with type 2 diabetes. sri lanka journal of diabetes endocrinology and metabolism, 11(2): 14–25. doi: http://doi.org/10.4038/sjdem.v11i2.7448 aryal b., niraula p., khadayat k., adhikari b., khatri chhetri d., sapkota b. k., bhattarai b. r., aryal n., parajuli n. 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(2011). antibacterial and anticancer activity of bioactive fraction of syzygium cumini l. seeds, hayati journal of biosciences, 18 (3): 118-122. https://doi.org/10.4308/hjb.18.3.118 http://doi.org/10.4038/sjdem.v12i1.7457 https://www.cabdirect.org/globalhealth/search/?q=au%3a%22sujatha%22 https://www.cabdirect.org/globalhealth/search/?q=au%3a%22ravishankar%22 https://www.cabdirect.org/globalhealth/search/?q=au%3a%22mariajancyrani%22 https://www.ijbs.com/ https://www.ijbs.com/ https://doi.org/10.4308/hjb.18.3.118 microsoft word jop_article 135 galley word_final version.docx page 89 *corresponding author: email address: norilyani@iium.edu.my or norilyaninazar@gmail.com @gmail.com protective factors for mortality among hivpositive people who inject drugs (pwid) in malaysia aida roziana ramlan1,2, nor ilyani mohamed nazar1*, norny syafinaz ab rahman1, dzawani muhamad3, and suraya zin1 abstract introduction: people who inject drugs (pwid) have long been associated with hiv transmission in the community. in many countries, methadone maintenance therapy (mmt) programme has been regarded as a critical platform to ensure hiv-positive pwid receive appropriate antiretroviral treatment. despite treatment availability and accessibility in malaysia, mortality was extensively observed, and the protective factors are still scarcely studied in this population. this study investigated the protective factors for mortality among hiv-positive pwid in malaysia's two mainstream treatment settings. method: this was a retrospective cohort study. the data was collected from one hiv clinic of a tertiary hospital and six (6) mmt programs of primary care clinics in the district of kuantan, pahang, from 2006-2019. the patients were categorised into three different subgroups. the mortality was recorded until the end of the study period. the survival rate was analysed using one-way anova, and the associated factors were analysed using cox proportional hazard regression analysis. results: in total, 141 records were found to have met the inclusion and exclusion criteria. fifteen hiv-related mortalities were recorded throughout the study period. there were significant differences in survival years between the mmt-only group versus mmt + art and the mmt-only group versus the art-only group (p-value <0.001 and 0.003, respectively). a longer art duration was significantly associated with improved survival of pwid (hr=0.5, 95% ci=0.28-0.88). extended duration in mmt program (hr= 0.50, 95% ci=0.33 – 0.78), started on art (hr= 0.21, 95% ci=0.04 – 0.97) and received art counselling (hr= 0.13, 95% ci=0.02 – 0.85) were significantly identified as protective factors for mortality among pwid on mmt. conclusion: three factors were identified as protective factors for mortality among hiv-positive pwid in malaysia. extended retention in the mmt programme, initiation of art, and regularly receiving hiv-related counselling were the significant factors contributing to reduced mortality rates among the patients. article history: received: 8 march 2022 accepted: 14 july 2022 published: 31 july 2022 keywords: hiv-positive people who inject drugs, protective factors, mortality rate how to cite this article: ramlan, a. r., mohamed nazar, n. i., ab rahman, n. s., muhamad, d. & zin, s. (2022). protective factors for mortality among hiv-positive people who inject drugs (pwid) in malaysia journal of pharmacy, 2(2), 89-98. doi: 10.31436/jop.v2i2.135 authors’ affiliation: page 90 introduction globally, around 11 million people inject drugs, and approximately 1 in 8 (or 1.4 million) live with hiv (unodc, 2020). in malaysia, it was estimated that the number of active pwid population in 2014 was about 153,000 patients, and in the year 2017, the number steadily increased to approximately 156,000 patients. at the end of 2018, it is estimated that 87,041 people were living with hiv (plhiv) in malaysia (hiebert et al., 2020). in the initial years of encounters, pwid has been strongly associated with hiv risk transmission primarily via the sharing of injecting paraphernalia (cheong et al., 1997, rusli et al., 1995). however, the country observed gradual changes in the hiv epidemic landscape in the past decade from predominantly pwid to more sexual transmission. the proportion of sexual transmission has increased to more than 90% in 2019 (suleiman, 2019). nevertheless, it is still relevant to treat pwid with appropriate treatment or harm reduction modalities (osornprasop et al., 2018; reddon et al., 2019). methadone maintenance therapy has been regarded as a harm reduction approach to preventing hiv transmission from pwid to the community other than needle/syringe exchange programs (nsps), condom distribution, and outreach programs. highly active antiretroviral therapy (haart) is the mainstay treatment among hiv-positive pwid unless contraindicated (osornprasop et al., 2018; myers et al., 2012). the initiation of haart is based on the symptoms or cd4 counts. starting with an art regimen is highly recommended in symptomatic patients, especially those already with an opportunistic infection. in asymptomatic patients, the treatment is recommended in patients with a cd4 count of less than 350cell/ml (moh, 2011). the government has adopted methadone maintenance therapy (mmt) program since 2006 as one of the harm reduction approaches to reduce the hiv seroprevalence rates. in many countries, mmt has been regarded as one of the essential platforms to ensure that hiv-positive pwid receives appropriate antiretroviral treatment. however, abstinent patients may also enrol on the infectious disease clinic of a public hospital. people who inject drugs have an elevated risk of death, although mortality rates vary across different settings. any comprehensive approach to improving health outcomes in this group must include efforts to reduce hiv infection and other causes of death, particularly drug overdose (mathers et al., 2013). there are many predictors of all causes of mortality and premature death among pwid living with hiv aids. among the identified factors were untreated concomitant infectious diseases such as tuberculosis, hiv aids and viral hepatitis; developing acute toxicity, especially in the case of overdose; unstable house conditions or homelessness; being sex workers; polydrug users, lack of regular employment, and poor retention rate at a drug substitution treatment (davis et al., 2017.; vinh et al., 2021; zivanovic et al., 2015). whereas for protective factors, opioid substitution therapy such as mmt and initiation of haart were found to independently and significantly protect against hiv-related death, drugrelated death and death due to other causes (nosyk et al., 2015). in malaysia, despite wide treatment accessibility, mortality was still being observed, and the protective factors were still scarcely investigated among the malaysian population (ngah et al., 2019; bazazi et al., 2018; lubis et al., 2013). this study aimed to investigate the mortality rate and its protective factors among the hivpositive pwid from the two mainstream treatment settings in malaysia. methodology this study was designed as a retrospective cohort. it has received prior ethics committee approval by the medical research and ethics committee, ministry of health malaysia (nmrr-18-3067-43939). the data was collected from the available patients' records at an infectious disease (id) hiv clinic of a tertiary hospital (hospital tengku ampuan afzan or htaa) and mmt programs of six (6) primary care clinics in the district of kuantan, pahang. the timeframe for the collected data was from 2006-2019 using a data collection form. the patients' records were screened for the inclusion and exclusion criteria. the inclusion criteria include 1) hiv-positive patients diagnosed upon enrolment or at any time during mmt or id hiv clinic, 2) categorised under pwid – used to or still injecting drugs, and 3) available demographic and baseline data. the data will be excluded if there are prominent missing/incomplete data. the hivpositive pwid's records were divided into three different categories based on their antiretroviral treatment (art) / methadone maintenance therapy (mmt) programme; 1) mmt only; 2) mmt + art, and 3) art only. all demographic, baseline data, patients' progress and hivrelated mortality were obtained from the medical records and treated strictly. the flow of the data collection process is summarised in figure 1. the analysis was conducted using spss ver 24. the descriptive baseline data was analysed and tabulated. the mortality rate and years of survival were explored using one-way anova, and the associated factors were analysed using cox proportional hazard regression analysis. results in total, 141 records were found to have met the inclusion criteria and are appropriate for further analysis. there were 29 (20.6%) patients on mmt only, 41 (29.1%) were receiving mmt +art, and most of the records retrieved were those receiving art only, 71 (50.4%). table 1 summarises the baseline data collection. page 91 figure 1: flow chart of data collection process. page 92 table 1: patients' demographic characteristics. note:the statistical test used is fisher's exact test unless stated otherwise, aone-way anova, independent t-test sd: standard deviation mortality and years of survival at the end of the study period, the number of hivrelated mortality was 15 subjects (10.6%). table 2 compares mortality (death) and years of survival between groups of pwid. the number of fatalities was significant between the mmt-only group and the art-only group, with a p-value < 0.001 (fisher's exact test). the mean (sd) survival years were the shortest among the mmt-only group, which was 3.6 (sd=2.7) years. there were significant differences in survival years between the mmt only group versus mmt + art and mmt only group versus art only group with p-value <0.001 and 0.003, respectively (independent t-test). protective factors for mortality among pwid initiated on art a total of 14 independent variables were analysed with univariate cox proportional hazards regression analysis, and only duration on art was found to be significantly associated with improved survival of pwid (hr=0.5, 95% ci=0.28-0.88). the longer the patient was on art, the risk of death was found to be reduced by 50%. table 3 summarises the results. the protective factors for mortality among patients on mmt three independent variables were chosen to predict mortality among the mmt subjects in this study. all significant variables were incorporated in the multivariate analysis. the final model showed that the extended duration in mmt program (hr= 0.51, 95% ci=0.33 – 0.78), started on art (hr= 0.21, 95% ci=0.04 – 0.97) and received art counselling (hr= 0.13, 95% ci=0.02 – 0.85) were significantly identified as protective factors for mortality among pwid on mmt. the results are tabulated in table 4. discussion the mortality rate among hiv-infected pwid high mortality rates among hiv-infected people who inject drugs (pwid) have been well studied. high-risk behaviour of the pwid population who practised needle sharing, unprotected sex and substance abuse, the risk for blood-borne infections, sexually transmitted disease, and a high rate of substance overdose were among the top contributing factors identified (farhadian et al., 2021). blood-borne infections mainly include hiv, hbv and hcv. hcv was one of the common co-infection in the patients' characteristics number, n (%) of patients total 141 mmt only 29 (20.6) mmt+art 41(29.1) art only 71(50.4) p-value gender male 136 (96.5) 29 (100) 41 (100) 66 (93) 0.124 female 5 (3.5) 5 (7.0) age 0.221a mean (sd) years 37 (6.4) 37 (4.1) 34 (4.3) 38 (7.6) marital status 0.737 single 69 (48.9) 13 (44.8) 22 (53.7) 34 (47.9) married 53 (37.6) 12 (41.4) 13 (31.7) 28 (39.4) divorced 17 (12.1) 3 (10.3) 5 (12.2) 9 (12.7) widow 2 (1.4) 1 (3.5) 1 (2.4) work 0.297 yes 94 (66.7) 22 (75.9) 29 (70.7) 43 (60.6) no 47 (33.3) 7 (4.9) 12 (29.3) 28 (39.4) page 93 table 2: mortality and years of survival among pwid. afisher’s exact test. bone-way anova pwid population, and it definitely increases the mortality threshold in this population (ali et al., 2018; draper & mccance-katz, 2005; mehta et al., 2010; teoh et al., 2018). from another perspective, suboptimal access to hiv care was also found to lead to mortality in the subpopulation [des jarlais et al., 2018]. in this study, the patients who were not on art had the shortest survival rate compared to those treated with art. this result is consistent with other studies since art is expected to restore immune function and improve patients’ survival (hogg et al., 1998; manosuthi et al., 2021; trickey et al., 2017). art initiation as a protective factor for mortality among hiv-positive pwid this study showed that those patients who started with art have a protective factor towards mortality compared to those without/not started, regardless of their treatment setting (mmt or without mmt). this finding is supported by many other studies emphasising the important role of art in reducing the mortality rate among the pwid population (kitahata et al., 2009; liu et al., 2013; wood et al., 2008; zhao et al., 2017). in a study applying multivariable extended cox regression analyses of all-cause mortality, those receiving art and having undetectable vl (<50 copies/ml) had a 42% lower allcause mortality hazard among both men and women as compared to those who did not receive art (brattgård et al., 2022; shoko & chikobvu, 2019). those receiving art but having detectable vl did not have a significantly lower risk of death (croxford et al., 2017; elvstam et al., 2021). the duration also plays a significant role in predicting mortality among the hiv pwid without mmt, where the more extended the art duration resulted in better survival (bogdanić et al., 2021). the time for art treatment initiation is also crucial, and the treatment delay has been associated with increased mortality (ramlan et al., 2019). extended mmt programme retention as a protective factor for mortality among hiv-positive pwid under the mmt programme in mmt-treated pwid, it was found that other than the initiation of art, a more extended period of mmt programme retention and attending the art counselling sessions were the protective factors that have been significantly associated with improved survival and reduced mortality among patients. these findings are coherent with a study in china (zhao et al., 2013a). pwid who was not in the mmt program had a higher mortality rate than those in the mmt program, although both groups were initiated on art. findings in a few other studies supported that enrolment into the opioid-substitution program will improve access to hiv care and facilitate the early initiation of art (hogg et al., 2011; montaner & wood, 2011; liu et al., 2013; mukandavire et al., 2017; pham et al., 2017). however, from a local study, about 33% of the mmt patients defaulted to the program due to negative perceptions and poor patient satisfaction (ali et al., 2018), which needs improvement. the low mortality rate among the mmt + art group indicated that both mmt and art effectively reduced the mortality rate. regular attendance to hiv and art counselling sessions as protective factors for mortality among the hiv-positive pwid the counselling session is prescribed to improve patients’ understanding of the disease and the related treatment. the results from this study are consistent with other studies on the essential roles of counselling sessions in improving medication adherence, treatment outcomes and reducing the rate of mortality among patients (go et al., 2017; hussain et al., 2022). characteristics number, n (%) of patients total 141 mmt only 29 (20.6) mmt+art 41(29.1) art only 71(50.4) p-value death 15 8 (53.3) 5 (33.3) 2 (13.3) <0.001 a survival, mean (sd) years 5.5 (3.5) 3.6 (2.7) 6.6 (3.2) 5.6 (3.6) <0.001b page 94 table 3: the cox proportional hazards regression analysis on protective factors for mortality among pwid initiated on art. note:ref = reference group, hr = hazard ratio, *significant when p < 0.05 variables univariate analysis multivariate analysis hr hr 95% ci p hr hr 95% ci p age (years) 1.00 0.87 – 1.14 0.946 working yes no (ref) 0.31 0.07 – 1.38 0.123 married yes no (ref) 0.50 0.10 – 2.60 0.411 ever had a positive urine test yes no (ref) 0.29 0.07 – 1.31 0.108 ever been detained or prisoned yes no (ref) 0.39 0.05 – 3.21 0.380 cd4 baseline (cells/mm3) 1.00 0.99 – 1.01 0.593 cd4 post art (>6 months (cells/mm3) 0.99 0.97 – 1.01 0.388 start art from diagnosis (year) 1.14 0.96 – 1.37 0.141 start art from the eligible date (year) 1.03 1.01 – 1.05 0.001* 1.00 0.97 – 1.03 0.928 duration on art (year) 0.60 0.41 – 0.88 0.008* 0.50 0.28 – 0.88 0.016* existing oi 1.83 0.35 – 9.48 0.471 oi after art 4.05 0.78 – 20.90 0.095 hiv related counselling yes no (ref) 0.10 0.02 – 0.43 0.002* 17.19 0.92 – 320.9 0.06 rvd treatment centre hospital health clinic (ref) 0.20 0.04 – 0.92 0.038* 2.00 0.13 – 31.02 0.62 page 95 table 4: cox proportional hazards regression analysis on protective factors for mortality among pwid on mmt. note:ref = reference group, hr = hazard ratio, *significant when p < 0.05 variables univariate analysis multivariate analysis hr hr 95% ci p hr hr 95% ci p age (year) 1.20 1.06 – 1.38 0.006 working yes no (ref) 1.03 0.286 – 3.80 0.960 married yes no (ref) 0.51 0.14 – 1.84 0.301 ever had a positive urine test yes no (ref) 1.08 0.38 – 3.56 0.903 ever been detained or prisoned yes no (ref) 1.53 0.50 – 4.740 0457 duration on mmt (year) 0.60 0.46 – 0.79 <0.001 0.51 0.33 – 0.78 0.002* mmt dose (mg) 0.99 0.97 – 1.01 0.279 mmt compliance yes no (ref) 0.69 0.19 – 2.52 0.574 start art yes no (ref) 0.21 0.07 – 0.66 0.008 0.21 0.04 – 0.97 0.046* cd4 baseline (cells/mm3) 1 0.998 – 1.002 0.883 existing oi 1.592 0.528 – 4.803 0.409 art counselling yes no (ref) 0.12 0.03 – 0.56 0.007 0.13 0.02 – 0.85 0.033* page 96 conclusion three factors were identified as protective factors for mortality among hiv-positive pwid in malaysia. extended retention in the mmt programme, initiated on art and regularly receiving hiv& art-related counselling were the significant protective factors in reducing the mortality rate among the patients. acknowledgements substance use disorder research group, all pharmacists in charge of the methadone clinics in kuantan, record unit of pahang state health department. conflict of interest we would like to declare that we do not have any conflict of interest in conducting this study. references ali, n., aziz, s. a., norliza, c., & abdullah, n. 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(2021) journal of pharmacy, 1(1), 45-53 madi page 45 antimicrobial study of chloroform fraction from the leaves of entada spiralis ridl. fitri rachmadita1, erryana martati1, sharifah nurul akilah syed mohamad2 and siti zaiton mat so’ad2,* abstract introduction: entada spiralis ridl., or locally identified as sintok, contains flavonoid, saponin, tannin, and glycoside, compounds that have antifungal and antibacterial activities. this research aims to identify bioactive compounds and determine the antimicrobial activity from crude and fraction of e. spiralis extract. methods: the crude extract was prepared by macerating the leaves with chloroform, and then proceeded to fraction it by vacuum liquid chromatography with dichloromethane (dcm)/hexane (hex) (1/9) and dichloromethane (dcm)/methanol (meoh) (9/1) solvent system. disk diffusion test and microdilution assay evaluated the extracts' antimicrobial activity against s. aureus, e. coli and c. albicans. the determination of bioactive compounds was done by thin layer chromatography (tlc). determination of total phenolic (tpc) and flavonoid content (tfc) were performed by folin-ciocalteu and alcl3 colourimetric assay results: the greatest inhibition zone against c. albicans was obtained from fraction chloroform (chcl3) extract with an inhibition zone of 10.33 mm. dcm/meoh (9/1) effectively killed s. aureus and e.coli with an inhibition zone of 11.67 and 12 mm, respectively. the minimum inhibitory concentration (mic) of chcl3 crude extract were 1.563 mg/ml for both e. coli and s. aureus, and 0.781 mg/ml for c. albicans. the tlc revealed the presence of tannins, saponin, glycosides, phenol, flavonoid, triterpenoid, and aromatic compound in chcl3 crude extract. tpc of dcm/meoh (9/1), chcl3, and dcm/hex (1/9) were 50.56 ± 0.188, 51.913± 0.089, 24.16 ± 0.175 mg gae/g extract. conclusion: in conclusion, e. spiralis leaves could be a source of active antifungal and antimicrobial agents used for food preservation by using a semipolar solvent for extraction. article history: received: 5 august 2020 accepted: 5 november 2020 published: 8 january 2021 keywords: antimicrobial, chloroform, entada spiralis, fraction. how to cite this article: rachmadita, f., martati, e., mohamad, s. n. a. s. & so’ad, s. z. m. (2021). antimicrobial study of chloroform fraction from the leaves of entada spiralis ridl. journal of pharmacy, 1(1), 45-53. authors’ affiliation: 1 department of food science and technology, faculty of agricultural technology, brawijaya university, malang, east java, indonesia. 2 department of pharmaceutical chemistry, kulliyyah of pharmacy, international islamic university malaysia (iium), jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia. *corresponding author: email address: dszaiton@iium.edu.my tel: +6013 9843575 rachmadita et al. (2021) journal of pharmacy, 1(1), 45-53 madi page 46 introduction nowadays, researchers pay more attention to natural antimicrobials, such as extracts from plants, to preserve food and modern medicine. this issue is related to the increase of public concern over chemical and synthetic preservatives. about three-quarters of the world population was estimated to depend mainly on plants and plant extracts for their health care because synthetic products could cause postural hypertension, heart failure, and impotence (zhang et al., 2013). one of the potential plants is e. spiralis. ridley, (synonym, entada scheffleri), locally known as sintok, sea bean, or beluru, from leguminosae. traditionally, it is used for hair treatment and as a cleaner for some skin disease. this plant is a tropical liana or woody climber that could go up to 25 m long (national park, 2019), and it is commonly found in australia and southeast asia. a previous study showed that e. spiralis stem bark contains flavonoid, saponin, tannin, and glycoside (harun et al., 2014). these compounds have anti-inflammatory, antifungal, anti-yeast, and antibacterial activities (arabski et al., 2012). several researchers already found the potential of genus entada as an antimicrobial agent. for example, entada rheedii ethanol extract was reported to display moderate inhibitory against s. aureus and c. albicans (ram et al., 2004). on the same species, the extract of methanol and fraction of entada spiralis stem bark showed antifungal activity against t. mentagrophytes, m. gypeseum, t. mentagrophytes, s. aureus, and s. epidermis (harun et al., 2014). meanwhile, the chloroform extract of entada spiralis leaves showed flavonoid, phenol (mohammad, 2017). according to the previous result related to the potential agent of e. spiralis leaves as an antimicrobial agent, we aimed to enlarge the potential of the crude chloroform extract of e. spiralis leaves as food preservation against s. aureus, e. coli, and c. albicans. methodology maceration e. spiralis leaves were obtained from tasik chini, pahang, with voucher specimen no. kms-5228. the 900 grams of dried leaves were later milled into a fine powder and macerated with 4 l of chloroform for 48 h furthermore, the extracts were then filtered using a whatman no. 1’s filter paper. chloroform was completely evaporated using a rotary evaporator at 40 °c. once thoroughly dried, the extract was placed in a 5 l erlenmeyer and stored in a fridge at a temperature of -4 °c before further analysis. phytochemical screening test – thin layer chromatography (tlc) tlc silica gel plate 60 f254 (merck, germany) was sized into 5 x 3 cm. the 5 μl of the extract was spotted in the middle bottom of the tlc plate. the dcm/hex (9/1) and dcm/meoh (9/1) solvent system solutions were used as mobile phases that were already tested from the preliminary research. the developing stage was conducted in a covered tlc developing chamber. the solvent was evaporated in a fume hood at 29 °c. some colouring agents and wavelength, such as vanillin, dragendroff’s, fecl3, uv 365 nm, uv 254 nm, iodine pearl, and concentrated h2so4 was given onto the different developed plates to determine several bioactive compounds such as saponin, terpenoid, alkaloid, flavonoid, phenol, tannin, aromatic compound, and conjugated compound by rf values calculations. fractionation of extract by vacuum liquid chromatography (vlc) in the present study, a modified method from mohammad (2017) was performed. it conducted a dilution of 10 grams of crude extract into 25 ml of chloroform solvent. the activated silica gel 60 pf254 (merck, germany) through a 24 h heating at 80 °c was dipped into the extract solution. sample and silica gel were kept agitated in a hotplate stirrer (lab tech, korea) at 70 °c until was mixed gently. the silica gel was loaded into the vlc column until the solvent front reached 5 cm of the column height. hexane was utilized to rinse the column by pressed the layer with aspirator a-1000s (eyela, japan). the silica gel was stabilized by setting it down overnight in the vlc column. ten (10) grams of the crude extract was submitted into vlc, eluted using a gradient system of dcm/hex (9/1) and dcm/meoh (9/1) as much as 1 l for each. the solvent system's use depended on the result of rf value in preliminary research by using tlc. furthermore, every 200 ml of the fraction, or based on the uv lamp band result, was separated respectively in erlenmeyer flask. the fractions were tested with the tlc profile to identify the specific compounds. the extract for each solvent system was combined and evaporated in the rotary evaporator ika hb10 basic (buchi, switzerland) to dryness at 40 °c. disk diffusion agar, kirby-bauer method the antimicrobial activity was conducted for crude (chcl3) and fractional (dcm/hex (9/1), and dcm/meoh (9/1)) extracts using a modified method from harun et al. (2014) was performed in the present study. a 1600 mg/ml stock solution was formulated by dissolving 1.6 g of sample in 1 ml of the specific solvent system depend on the polarity of the fractions. the serial two-fold dilution was done to make the several concentrations ranged from 12.5 mg/ml 1600 mg/ml. the 20 ml/μl of rachmadita et al. (2021) journal of pharmacy, 1(1), 45-53 madi page 47 each concentration was dipped onto paper discs (whatman aa disc, 6 mm, united states of america) by using a micro-pipette. the discs were evaporated at the laminar airflow cabinet (erla cmp series, malaysia) until the solvent was evaporated completely. all discs were stored at -5 °c in chiller lc-213ld (law chain, taiwan) for further analysis. the positive control of antifungal, gram-negative bacteria and gram-positive respectively employed 100 μl nystatin (oxoid, united kingdom), 10 μl ampicillin (oxoid, united kingdom), and 10 μl gentamicin (oxoid, united kingdom). the chloroform, dcm/hex (9/1), and dcm/meoh (9/1) (merck, germany) was used as a negative control. the pure culture of e. coli, s. aureus, and the fungal strain c. albicans were collected from the basic medicine science of international islamic university of malaysia. bacterial and fungi were incubated in 24 h at 37 °c in mueller hilton-agar (oxoid, united kingdom) and saboraud dextrose agar (becton, united states of america). the od of bacteria was 0.1 at 600 nm uv-vis spectrophotometer (secomam, united states of america). thus, the standard inoculation of bacteria was standardized at 1.5 x 106cell/ml and was swabbed on an agar plate using a sterile cotton bud. the sterile paper discs were aseptically transferred onto the inoculated agar plates' surfaces and were submitted to 24 h of incubation at 37 ℃. the effect of the antimicrobial agent was indicated through a clear zone. the determination of this assay was conducted in triplicate. minimum inhibitory concentration (mic), by broth microdilution assay the present study modified a method by harun et al. (2014) and mohamad (2012) by diluting the microbial stock into the mueller hilton-broth (oxoid, united kingdom) to an absorbance of 0.11 at 600 nm for bacteria and saboraud dextrose broth (becton, united states of america) to the absorbance of 0.6 at 450 nm for fungi. the 1 ml inoculum was added to 100 ml of sterile broth and diluted until a reached number of od 105 cells/ml for fungi and 1.5 x 106 4cells/ml for bacteria. the first row of the 96 well microtiter plate (trueline, united states of america) was filled with 180 μl of 1/100 diluted microbial solution and 100 μl for the rest. therefore, the 20 μl of two-fold serial dilution 50 mg/ml 0.39 mg/ml concentration extract was diluted with dmso. the microplates were incubated at 37 ℃ for 24 h. the 20 ul 0.5% (w/v) of mtt (3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyl tetrazolium bromide (life technologies, united states of america) and phosphate buffered saline solution (base, singapore) was put to each well and re-incubation for 2 h for fungi and bacteria. the yellow color indicated the inhibition of microbial growth; meanwhile, the dark blue indicates microorganisms' presence. minimum bactericidal concentrations (mbc) and fungicidal concentrations (mfc) the mbc and mfc were used for each concentration on the solvent system of all microorganisms tested. the mbc and mfc pursued the lowest concentration by calculating fewer than three colonies to achieve approximately 99% inhibition growth. 100 μl of the mixture from mic that showed the positive result was incubated on sda (for fungi) and mha (for bacteria) at 37 ℃ for 24 h. total phenolic content (tpc) the folin-ciocalteu assay was performed to identify the amount of tpc in e. spiralis leaves based on mohamad’s method (2012). 50 μl of the extract was formulated from 2 mg/ml with the original solvent. the standard of gallic acid solution (7.8 -1000 μl/ml) was diluted into 100 μl of methanol, where the blank was using methanol. the sample was diluted with a ratio of 4:1 (water: folinciocalteu phenol reagent) and 50 μl of 1 m sodium carbonate (na2co3) solution in water. 25 μl sample or standard combined with 100 μl folin ciocalteu phenol reagent were filled into a 96-well plate and incubate for 5 min at 37 ℃. 75 μl na2co3 was added and was kept from light for 1 h. the result's absorbance was analyzed at 765 nm against blank by a multi-detection microplate reader (infinite m200 nanoquant, switzerland). results phytochemical screening of crude extract the phytochemical screening results of e. spiralis crude extract by tlc can be seen in table 1. crude extract showed several bioactive compounds such as; terpene, steroid, terpenoid, phenol, tannin, saponin, flavonoid, and aromatic compound, as shown in figure 1. antimicrobial activity assay by kirby-bauer test the chloroform crude extract strongly inhibited the e. coli with an inhibition zone of 10.67 mm in table 2, followed by c. albican and s. aureus with an inhibition zone of 10.33 and 9.67 mm, respectively. moreover, the fraction dcm/meoh (9/1) extract was strongly inhibited the e. coli with a 12.00 mm inhibition zone in table 3, followed by c. albicans and s. aureus with an inhibition zone of 9.00 and 11.67 mm, respectively. among all microorganisms that were used, e coli was found to be the most susceptible, whereas s. aureus and c. albicans were less susceptible to all extracts. rachmadita et al. (2021) journal of pharmacy, 1(1), 45-53 madi page 48 table 1: tlc analysis of entada spiralis crude extract reagent sprayer spot on tlc plate total spot color rf compound nature 7 yellow 0.1125 neoxanthin yellow 0.1625 violaxanthin dark yellow 0.2 lutein blue green 0.275 chlorophyll b grey 0.6875 anthocyanin dark green 0.75 pheophytin a yellow 0.9375 carotene uv254 light 7 grey 0.1125 aromatic compound grey 0.1625 dark grey 0.2 dark grey 0.275 dark grey 0.6875 dark grey 0.75 dark grey 0.9375 sulphuric acid 3 green 0.25 tertepenes yellow 0.5 steroid pink 0.6875 steroid iodine 3 brown 0.25 aromatic and organic compound brown 0.5 dark brown 0.6875 uv365 light 1 fluoresence 0.6875 terpenoid fecl3 2 green 0.1125 phenol green 0.357 tannin vanillin 3 dark purple 0.175 saponin green 0.2625 furastanol violet 0.5375 spirostanol alcl3 1 orange 0.6155 flavonoid rachmadita et al. (2021) journal of pharmacy, 1(1), 45-53 madi page 49 figure 1: tlc result of e. spiralis leaves chloroform extract with several conditions; (a) nature, (b) uv254 light, (c) sprayed by dragendorff reagent, (d) h2so4, (e) sprayed by iodine, (f) uv365 light, (g) sprayed by fecl3, (h) sprayed by vanillin reagent, (i) sprayed by alcl3 rachmadita et al. (2021) journal of pharmacy, 1(1), 45-53 madi page 50 table 2: in vitro antifungal and antimicrobial activity of fraction and crude extract of e. spiralis leaves extract concentration (mg/ml) inhibition zone (mm)a ec sa ca dcm/hex 12.5 25 50 100 200 6.67 ± 0.577c negative control dcm/hex chloroform 12.5 7.33 ± 1.155bc 7.17 ± 0.288b 25 7.67 ± 0.577bc 7.33 ± 0.577b 50 7.67 ± 0.577bc 7.67 ± 1,443ab 100 8.33 ± 0.577abc 7.77 ± 1,328ab 200 9.00 ± 1ab 7.67 ± 0.577ab negative control chloroform dcm/meoh 12.5 8.33 ± 0.577abc 7.33 ± 1,527b 6.33 ± 0.577bc 25 8.67 ± 0.577ab 7.67 ± 0,527ab 6.67 ± 0.577bc 50 9.00 ± 1ab 8.33 ± 0,577ab 7.0 ± 0b 100 9.00 ± 0ab 9.167 ± 0,763a 7.0 ± 0b 200 9.67 ± 0.577a 9.00 ± 1a 7.0 ± 0b negative control dcm/meoh nystatin 100 24.0 ± 1 ampicillin 10 24.33 ± 1.527 gentamicin 10 32.67 ± 0.577 no activity; ec; e. coli, ca ; c. albicans, sa ; s. aureus; ±, standard deviation (sd); a mean of triplicates. statistical significance was determined using anova. differences were analyzed significant (p<0.05) on the same microbes. the different notation means significantly different values. table 3: in vitro antimicrobial and antifungal activity of dcm:meoh fraction and crude extract of e. spiralis leaves extract concentration (mg/ml) inhibition zone (mm)a ec sa ca chloroform 400 9.33 ± 0.577b 7.67 ± 0.577b 8.67 ± 0,577ab 800 10.33 ± 1.154ab 8.833 ± 1,607ab 8.67 ± 1,155ab 1600 10.67 ± 1.154ab 9.667 ± 2,081ab 10.33 ± 2,309a negative control chloroform dcm / meoh 400 9.00 ± 1b 8.00 ± 0b 7.33 ± 0,577b 800 11.33 ± 0.577a 9.67 ± 0,577ab 8.33 ± 1,155ab 1600 12.00 ± 1a 11.67 ± 2,887a 9.00 ± 0ab negative control dcm/meoh nystatin 100 24.0 ± 1 ampicillin 10 24.33 ± 1.527 gentamicin 10 32.67 ± 0.577 no activity; ec; e. coli, ca; c. albicans, sa; s. aureus; ±, standard deviation (sd); a mean of three replicates statistical significance was determined using annova. differences were analyzed significant (p<0.05) on the same microbes. the different notation means significantly different values. rachmadita et al. (2021) journal of pharmacy, 1(1), 45-53 madi page 51 determination of mic, mfc and mbc the mic results of the positive result fractions against bacteria and fungi are presented in table 4. the chloroform crude extract showed the mic value of 1.563 mg/ml against both e. coli and s. aureus, and 0.781 mg/ml against c. albicans. meanwhile, fraction dcm/meoh (9/1) extract showed the mic value of 1.563 mg/ml against e. coli and 3.125 mg/ml against both s. aureus and c. albicans. according to the result of minimum bactericidal concentration (mbc), the chloroform extract had the lowest mbc result against e. coli, s. aureus, with 0.39 and 0.781 mg/ml compared to fraction dcm/meoh (9/1) with mbc results of 0.781 and 1.563 mg/ml and positive control 0.39 mg/ml for gentamicin (μg/ml) and 0.39 mg/ml for ampicillin (μg/ml). on the other hand, chloroform extract also gave lower mfc result (0.781 mg/ml) compared to dcm/meoh (9/1) with an mfc result of 1.563 mg/ml, but still fell behind the positive control nystatin (μg/ml) with an mfc result of 0.39 mg/ml. in conclusion, the chloroform extract has a promising role as an antimicrobial agent against significant microorganisms, especially bacteria. table 4: minimum inhibitory concentration, minimum fungicidal (mfc) and bactericidal concentration (mg/ml) of e. spiralis crude extract extract mbc mfc mic ec sa ca ec sa ca chloroform crude extract 0.390 0.781 0.781 1.563 1.563 0.781 fraction (dcm /meoh) 0.781 1.563 1.563 0.781 3.125 3.125 gentamicin (μg/ml) 0.390 0.390 nystatin (μg/ml) 0.390 0.390 ampicillin (μg/ml) 0.390 0.390 total phenolic content total phenolic content of e. spiralis leaves extracted by chloroform, fraction dcm/meoh (9/1), and fraction dcm/hex (1/9) were 50.56 ± 0.089, 51.913± 0.188, 24.16 ± 0.175 the concentration of phenolic was in terms of equivalent (mg gae/g of the dry weight of extract), separately. discussion in the kirby-bauer method, the restraint zone's size expressed the compound's competences wherein the highest the zone, the more powerful the compound. higher active compounds from the extracts could cause the higher inhibition zones recognized at s higher concentration of all extracts. fractionation in several plant extracts brought about improved movement, yet others brought about the loss of the action. for instance, in an extract of fraction dcm/hex (1/9), the inhibition was only detected at a concentration of 200 mg/ml against e. coli with zone inhibition of 7. since the chcl3(100%) and dcm/meoh (9/1) extract showed promising anti-dermatophytes and antibacterial activity, thus it was chosen to undergo fractionation and was further assayed to investigate the effectiveness of the fractions, except dcm/hex (1/9). the greatest inhibition zone against c. albicans was obtained from fraction chloroform (chcl3) extract with an inhibition zone of 10.33 mm. dcm/meoh (9/1) effectively killed s. aureus and e. coli with an inhibition zone of 11.67 and 12 mm, respectively. those results were determined by the polarity of the solvent's polarity that may be affected by the bioactive compound after the fractionation process and the chemical nature of its bioactive constituents. the antifungal and antibacterial potency of several plants was related to secondary metabolites established in some fractionations of the unrefined concentrate, permits the appropriation of bio-active compounds into solvents as per their polarity. the current investigation's perception showed that the antifungal and antibacterial activity of the fraction dcm/meoh (9/1) and chcl3(100%) extract of e.spiralis leaves maybe showed that the active compound was either modestly nonpolar or polar. this may fill into the natural dissolvable that can be utilized in removing dynamic elements of e.spiralis leaves. the phytochemical compound can be isolated. for example, the related movement against contagious and antibacterial action may be associated with the nearness of phytochemicals, such as alkaloid, flavonoid, saponin, sterol, and tannins in the individual portion that previously controlled by tlc profile in past study. the inhibitions were supported by microdilution assay in which the chloroform crude extract gave the highest mic value of 1.563 mg/ml against e. coli, 1.563 mg/ml against s. aureus, 0.781 mg/ml against c. albicans. as mentioned before, the polarity of the fraction will affect the compound that, in turn, will affect the efficiency of antimicrobial activity. for an extract of fraction, dcm/hex (1/9) has nonpolar properties. the nonpolar solution contains only a few bioactive compounds such as; flavonoid, sterol, phenol, alkaloid (widyawati, 2014), terpenoid (liu et al., 2011), and diterpenoid (hidayat, 2014) that dissolved by hexane solvent. meanwhile, in rachmadita et al. (2021) journal of pharmacy, 1(1), 45-53 madi page 52 semipolar polar extract of fraction dcm/meoh (9/1) has more bioactive compounds such as sterol, phenol, flavonoid, and alkaloid (widyawati, 2014). in semipolar polar extract of dcm/meoh (9/1) may also have polar properties such as alkaloid, phenolic, saponin, carotenoid, tannin, amino acid, and glycoside (elfirta, 2018). terpenoids, tannins, phenolics, and flavonoids are secondary metabolites compounds that naturally exist in several types of plants. the secondary metabolites realized with pharmacological significance are commonly known as bioactive compounds (swabha, 2018). these bioactive compounds will increase lymphocyte activity, such as macrophage cells, especially compounds such as phenolics and its derivatives. phenolics play several important functions in plants as an immunomodulator and antioxidant. they give opposition against microbes and predators, other than that phenolics protect cellular membranes and tissues from lipid peroxidation, and fix dna by electron transfer responses (shankar et al., 2007). flavonoids, particularly catechins, have been known for their antimicrobial activity in gram-positive and gramnegative microorganisms (tsuchiya 2015). catechins will have collaborate with the cell layer of microbes by authoritative to the lipid bilayer and by inactivating or hindering the union of intracellular and extracellular compounds (reygaert, 2014). tannins also fill as guards against microorganisms. their antimicrobial activity method might be identified with their capacity to idle microbial adhesin, cell envelope transport protein, catalyst several enzymes, that properly known as astringency (bobbarala, 2012). they are fit for restricting metal particles related to the development of microscopic organisms. in this manner, by consolidating with ca2+ particles engaged with gram-negative bacteria's structure, dense tannins influence the bacterial wall cell, disrupting the retention of minor components fundamental for bacterial development (joseph, 2016). conclusion the chcl3(100%) extract of e. spiralis uncovered several bioactive compounds such as tannins, saponin, glycosides, phenol, flavonoid, triterpenoid, and aromatic compound through thin layer chromatography (tlc). the highest zone inhibition zone for e. coli was obtained from dcm/meoh (9/1) extract with a diameter of 12.00 ± 1 mm. the highest inhibition zone for s. aureus was obtained from dcm/meoh (9/1) extract with a diameter of 11.67 ± 2.887 mm. the greatest inhibition zone for c. albicans was obtained from chcl3(100%) extract with a diameter of 10.33 ± 2.309 mm. the inhibitions were supported by microdilution assay in which the chloroform crude extract gave the highest mic value of 1.563 mg/ml against e. coli, 1.563 mg/ml against s. aureus, 0.781 mg/ml against c. albicans. this study suggests that the leaf extract of entada spiralis has great potential to become a natural preservative for foods, replacing chemical preservatives. a future study is needed to distinguish the isolated specific bioactive compounds that can be done by utilizing an lc-ms or gc-ms examination. acknowledgements the authors would like to express gratitude to the ministry of science and technology for sf grant no:0050055 for the financial support, kulliyyah of pharmacy, international islamic university malaysia (iium), kuantan, pahang, malaysia, pn. norhayati abdullah. tasik cini, pahang for the supplier of entada spiralis ridl leaves, and faculty of agricultural technology, university of brawijaya, indonesia for organizing the research join the program. conflict of interest authors have no conflicts of interest with this publication. references arabski, m. aneta, w.c., czerwonka, g., anna, l., & kaca, w. 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(2021) journal of pharmacy, 1(1), 27-33. page 27 prevalence, factors and cost comparison due to potentially inappropriate medications (pims) of elderly outpatients in a state hospital in malaysia. wei chern ang1,2*, nur syafiqah zulkepli2, nur safinaz mukhtar2,3 and nur atikah zulkefli2 abstract introduction: malaysia will be a full aging nation by 2030. the elderly (aged ≥65 years old) population often has multiple comorbidities, which increases the risk of polypharmacy and potentially inappropriate medications (pims). this study aims to investigate the prevalence, factors associated with pims among elderly outpatients, and its burden of direct pharmacotherapy cost to the ministry of health malaysia. materials and method: a cross-sectional study involving clinic prescriptions among the elderly with more than one-month prescribing duration received from a tertiary hospital specialist clinic pharmacy from march to april 2017. patient identifiers were screened using the pharmacy information system (phis) by including prescriptions from other clinics while excluding multiple visits and duplicate prescriptions. patients were categorised as pim group and non-pim groups using beers criteria 2015. logistic regression analysis was conducted to examine the factors associated with pims. the median monthly prescription cost was compared between pim and non-pim groups by mannwhitney test. results: among 472 patients, 39.4% of patients had at least one pim while 60.6% of patients did not receive any pim. the number of medications prescribed was an independent risk factor contributing to pims (or:2.04; 95% ci:1.40, 2.97). the median monthly prescription cost for the pim group was myr 29.50 (≈usd 7.53) which was not statistically significant (p=0.735) compared with the non-pim group which was myr 28.50 (≈usd 7.28). conclusion: pim was frequently prescribed in our setting with the number of medications as the only factor. however, the prescribing of pim did not add nor reduce the direct cost of pharmacotherapy. article history: received: 1 september 2020 accepted: 1 december 2020 published: 8 january 2021 keywords: potentially inappropriate medication, cost, elderly, outpatient, patient safety. how to cite this article: ang, w. c., zulkepli, n. s., mukhtar, n. s. & zulkefli, n. a. (2021). prevalence, factors and cost comparison due to potentially inappropriate medications (pims) of elderly outpatients in a state hospital in malaysia. journal of pharmacy, 1(1), 27-33. authors’ affiliation: 1 clinical research centre, hospital tuanku fauziah, ministry of health malaysia, jalan tun abdul razak, 01000 kangar, perlis, malaysia 2 department of pharmacy, hospital tuanku fauziah, ministry of health malaysia, jalan tun abdul razak, 01000 kangar, perlis, malaysia 3 university health centre, universiti utara malaysia, 06010 sintok, kedah, malaysia *corresponding author: email address: wei.ang.1990@gmail.com tel:+604 9738413 tel:+609 ang et al. (2021) journal of pharmacy, 1(1), 27-33. page 28 introduction population ageing is a phenomenon in which people live a longer life and takes place nearly in almost all countries in the world. the reduction in mortality led to an increase in population survival rate, thus increasing the proportion of elderly. the united nations takes 60 years old and above as age reference for geriatrics. however, in most developing countries, the age of 65 is used as a cut-off as this is the age at which citizens are eligible for elderly social security payments (guzmán, pawliczko, & beales, 2012). in malaysia, the proportion of elderly aged 65 years and over had increased to 5.1% according to the national population and housing census 2010, as compared to 3.9% in 2000, with a projection of increment of 0.1% per year (hairi, bulgiba, cumming, naganathan & mudla, 2010). according to a projection by the united nation, malaysia will achieve the status of a full ageing nation by 2030 when 15% of the population is classified as senior citizens (hairi et al., 2010; abd mutalib, ismail & miskiman, 2020). along with ageing, the body functions deteriorate and subsequently lead to multi-morbidities. to manage the symptoms and/or to treat these multi-morbidities, there is a higher risk of the usage of many medicines known as polypharmacy (mortazavi et al., 2016). polypharmacy is defined as the use of a large number of medications, commonly considered as five or more (mortazavi et al., 2016). in a study in a university hospital in malaysia, polypharmacy was further worsened with an increment in age, morbidity, usage of over-the-counter (otc) drugs, female and the use of cardiovascular, endocrine and musculoskeletal drugs (senik, 2006). it has been stated that patients using two drugs experience a 13% higher risk of drug-drug interactions (ddis) and/or adverse drug reactions (adrs), which increase to 38% when four drugs were taken and to 82% when taking seven or more drugs at the same time (gallagher, barry & o'mahony, 2007). furthermore, ddi and/or adr were frequently misinterpreted as the onset of another medical condition in those older patients. an example of adr is extrapyramidal symptoms (eps) induced by metoclopramide could be misdiagnosed as the onset of parkinson's disease, although this misdiagnosis would be less likely in young patients as parkinson's disease is uncommon in a younger population (kalisch, caughey, roughead & gilbert, 2011). this could lead to prescribing cascade among elderly in which additional drug(s) is being prescribed to treat the ddi and/or adr of another drug(s) which then cause polypharmacy (hilmer, 2008). hence, potentially inappropriate medications (pims) are more likely to be prescribed in this age group. close monitoring and rational pharmacotherapy are needed when prescribing to this vulnerable group either in terms of dose, frequency or duration. pims can be defined as “medications in which the risks outweigh benefits where there is a safer or more effective alternative therapy for the same conditions” (galli, reis, & andrzejevski, 2016; hefner et al., 2015). this definition of pims has been applied in various settings using a list of explicit criteria, such as the screening tool of older people's prescriptions (stopp) criteria (o'mahony et al., 2015) and beers criteria (fick et al.,2019). beers criteria were first compiled by dr mark h. beers, a geriatrician based on a consensus panel of experts by delphi method on 1991, initially focused exclusively on nursing home residents. the function of the criteria is to identify potentially high-risk medications used by older people. due to factors such as local prescribing practises and formularies, these instruments differ in their ability to distinguish pims in various healthcare settings. there is a systematic review on comparing applicability and sensitivity of stopp and 2002 version of beers criteria. six studies obtained were investigated and concluded that stopp is more sensitive than the 2002 beers criteria (hill‐taylor et al., 2013). however, on a more recent study shows that the 2012 version of beers criteria identified more pims compared to stopp criteria (oliveira et al., 2015). the updated version includes drugs that should be avoided or should have their dose adjusted based on individual kidney functions and selected drug-drug interactions. it has been used widely in geriatric clinical care, education, research and in the development of quality indicators (radcliff et al., 2015). with a long history and its development in 1991, beers criteria were frequently updated in 1997, 2003, 2012, 2015 and most recently in 2019 by the american geriatrics society (ags) (fick et al.,2019). in contrary, stopp criteria are only in version 2 in 2015 after its development in 2008 (o'mahony et al., 2015). pims used in the elderly is associated with an increased risk of ddis and/or adrs (galli et al., 2016; hefner et al., 2015). a systematic review conducted by xing et al. (2019) in investigating associations between pims exposure and adverse events, such as adrs, hospitalisation, and mortality. despite no significant association between mortality and pims, a statistically significant correlation between adrs and hospitalisations with pims was found in the combined study. a previous study shows that pims such as non-steroidal antiinflammatory drugs (nsaids) had been associated with adverse outcomes and increase the cost of hospitalisation (galli et al., 2016). thus, it is crucial to identify the pims to reduce ddi and/or adr in older patients. by concept analysis, deprescribing may lead to cost reduction in terms of reduced medications, reduced hospitalisation and improved adherence as having less medication to monitor (page, clifford, potter, & etherton‐beer, 2018). in the health economics of potentially inappropriate medication (hepime) study among elderly aged 65 years ang et al. (2021) journal of pharmacy, 1(1), 27-33. page 29 old and above in germany, by controlling for the number of prescribed medicines, the gap in overall healthcare expenses between pim and non-pim groups was € 401 (≈myr 1877.25, as of 2017) in a 3-month period. there is a lack of published study in malaysia that investigates pims in a malaysian public hospital and incorporating prescription cost analysis among elderly outpatients. hence, this study aims to determine the prevalence of pims by using beers criteria 2015 and the factors associated with pims. this study also compares pharmacotherapy cost associated with pims and nonpims. methods this study was conducted in a tertiary hospital in malaysia, hospital tuanku fauziah, the only hospital in the state of perlis at the time of the study which serves a population of 252,000 during our period of study in 2017 (department of statistics malaysia, 2017). this hospital did not have in-house nor visiting geriatrician. a retrospective study was conducted among clinic outpatients aged more than 65 years old. all clinic prescriptions (prescribed for at least 4 weeks of treatment and must be taken regularly) were collected in a specialist clinic pharmacy from 1st march 2017 to 15th april 2017. the exclusion criteria were referral repeats prescriptions for patients to get the next refills in other healthcare facilities, prescriptions for intravenous and external preparation. the patients’ identifiers were further screened using the pharmacy information system (phis, pharmaniaga®, shah alam, malaysia) to include visits to other specialist outpatient clinics. if there were duplicate prescriptions of the same patients, the latest data were considered. the number of medications is defined as the number of types of medication prescribed for the patient using the latest prescriptions and considering all current clinic visits. the beers 2015 criteria were used to identify and assess any inappropriate prescribing. any medication categorised under pim was checked whether it was appropriate for that patient using the phis system on history of medication taking and the patient medical record kept in the respective clinic. if the pim were appropriate, it would be removed as pim. for example, for proton pump inhibitors, they would be screened if the patient were on prolonged corticosteroids or nsaid use, or diagnosed with erosive esophagitis, barrett’s esophagitis, pathological hypersecretory condition. two researchers examined patients’ medications for pims used independently and any discrepancies were resolved by consensus from all researchers. the existence of polypharmacy was analysed as one of the independent variables: patients were subjected to polypharmacy when they received more than 5 medications. as pill burden is not in the scope of our study, the number of medications was calculated as the number of active ingredients for any combination drug (multiple active ingredients in a single dosage form) prescribed. patients were divided into two groups either pim (prescribed with at least one pim) or non-pim (was not prescribed with any pim) groups. prior data indicated that the proportion of outpatients prescribed with at least one pim (pim group) was 0.276 (lim et al., 2016). by considering type i probability error and precision both to be valued at 0.05, we needed to study 308 samples. the data were analysed using ibm spss statistics for windows version 20.0 (ibm corp, armonk, ny). for descriptive analysis, categorical data were presented as frequencies and percentage while numerical data were presented as mean and standard deviation (sd) or median and interquartile range (iqr). for inferential analysis, binomial logistic regression was used to study the covariates on the prescribing of pim. prescriptions cost comparison was analysed using mann-whitney tests as the data were non-parametric. p-values of less than 0.05 were considered as statistically significant. this study was registered with the national medical research registry (nmrr-17-2668-36550) and was approved by the medical research ethics committee (mrec) malaysia. results a total of 472 outpatients were analysed in this study. 186 (39.4%) patients were prescribed with at least one pim while 286 (60.6%) patients were not prescribed with any pim. the mean age of patients in the pim group was 73.8 (6.94) while for the non-pim group was 73.0 (6.31). table 1 summarises patient characteristics into pim and non-pim groups. table 1: patient characteristics (n=472) characteristics pim (n=186) n (%) non-pim (n=286) n (%) age (years) 65-70 82 (44.1) 127 (44.4) 71-75 38 (20.4) 72 (25.2) >75 66 (35.5) 87 (30.4) gender male 95 (51.1) 162 (56.6) female 91 (48.9) 124 (43.4) race malay 144 (77.4) 216 (75.5) non-malay 42 (22.6) 70 (24.5) polypharmacy no 69 (37.1) 156 (54.5) yes 117 (62.9) 130 (45.5) number of clinic visits 1 181 (97.3) 276 (96.5) > 1 5 (2.7) 10 (3.5) ang et al. (2021) journal of pharmacy, 1(1), 27-33. page 30 among the 472 geriatric patients, the most prescribed pims were diuretic, which was prescribed to 74 (15.7 %) patients followed by short and immediate-acting benzodiazepine to 55 (11.7%) patients and proton pump inhibitors to 54 (11.4%) patients, as shown in table 2. a total of 278 pims was prescribed to 472 geriatric patients, which turns up to be 0.6 pims/geriatric patient. there is an average of 1.5 pims prescribed to 186 pim patients. table 2: types of potentially inappropriate medication (pim) (n=278) prescribed to sample population (n=472) pim prescribed pim frequency (%) diuretic 74 (15.7) short and immediate-acting benzodiazepine 55 (11.7) proton pump inhibitor (ppi) 54 (11.4) selective serotonin reuptake inhibitor (ssri) 29 (6.1) antipsychotic 21 (4.4) peripheral α1 blocker 20 (4.2) chlorpheniramine 14 (3.0) digoxin 5 (1.1) amitriptyline 5 (1.1) ticlopidine 1 (0.2) table 3: factors associated with prescribing of potentially inappropriate medication (pim) by logistic regression factors odd ratio p-value age 1.02 (0.99-1.05) 0.150 age group 65-70 1.00 (ref.) 71-75 0.88(0.54-1.42) 0.591 >75 1.06 (0.69-1.61) 0.803 gender male 1.00 (ref.) female 1.25 (0.86-1.81) 0.236 race malay 1.00 (ref.) non-malay 0.90 (0.58-1.39) 0.636 polypharmacy no 1.00 (ref.) yes 2.04 (1.40-2.97) < 0.001 number of clinic visits 1 1.00 (ref.) > 1 1.31 (0.44-3.90) 0.626 factors being investigated were age, gender, races, the existence of polypharmacy and numbers of visit in different clinics. the number of medications was the only significant covariate (p<0.001): patients subjected to polypharmacy had 2.04 higher odd of having pims compared to patients who were not (table 3). the median pharmacotherapy cost for pim group, myr 29.50 (≈usd 7.53, as of 2017), was not statistically significant (p=0.735) compared to the non-pim group which was myr 28.50 (≈usd 7.28). due to this insignificance, cost of unneeded pim was not further calculated. discussions in our study, the prevalence of being prescribed pim is 39.4%. based on the malaysian elders longitudinal research (melor) cohort study among urban community-dwelling older adults in malaysia, the prevalence of pim was 31.8% (lim et al., 2017). a study in new zealand regarding the prevalence of pims among elderly showed that the rate of at least one pim being prescribed was 42.7% (nishtala, bagge, campbell, & tordoff, 2014). in a previous study from tertiary care hospital in india, showed that 29.2% of patients did have at least one pims (shah, joshi, christian, patel & malhotra, 2016). a study conducted in a university medical centre in seoul, korea, among the 25810 outpatients, 7132 (27.6%) did have at least one pim (lim et al., 2016). however, the difference in the prevalence of at least one pim might be due to the difference in study settings, availability of medications, and prescribing pattern (abdulah et al., 2018). the most prescribed pims in our setting were diuretic (15.7 %), short and immediate-acting benzodiazepine (11.7%) and ppi (11.4%). a study in india also found that the most commonly found pims was spironolactone (15.7%) and benzodiazepine (6.4%), while a study in japan found out that the most common pims were histamine-2 (h2) blocker (20.5%) followed by benzodiazepines (11.4%) (akazawa, imai, igarashi, & tsutani (2010). however, a study in korea stated that the most prescribed pims were benzodiazepines, specifically alprazolam (11.2%) followed by clonazepam (10.8%) (lim et al., 2016). based on table 4 from beers criteria, diuretics are classified as pim use with caution due to worsening or cause a syndrome of inappropriate antidiuretic hormone secretion of hyponatraemia. all benzodiazepines were classified as pims which increases the risk of cognitive impairment, delirium, falls, fractures and motor vehicles crash in older adults while protonpump inhibitors which are classified as pim, which increases the risk of clostridium difficile infection, bone loss and fractures. previous studies have reported an increased risk of pims with age, gender, multiple medications and number of co-morbidities. a study from new zealand showed that older age, being female and european were associated with increased risk of pims. in addition, reported from a study in brazil, increasing in age and being female contributed to increasing the risk of pims. in contrast, a study from india and japan found that age and sex did not contribute to pims. a study from korea found that an increasing number of medications and prescribing doctors were associated ang et al. (2021) journal of pharmacy, 1(1), 27-33. page 31 with pim use. a systemic meta-analysis illustrated that only polypharmacy is positively associated with pim use among the elderly, which supports the finding of our study (santos et al., 2015). we observed a close association between pims and polypharmacy. this proved that polypharmacy is a factor strongly associated with pims as patients received a high number of medications tend to be prescribed with pim. furthermore, this study also found out that older age and increased number of comorbidities were associated with increased medication use (lim et al., 2017). hence, the result reflects the need for extra monitoring and precautions from all healthcare professionals towards elderly patients who are on polypharmacy. in public hospitals, malaysian citizens pay a nominal fee of myr 5 (≈usd 1.24) for each specialist consultation visit which the cost includes the supply and refill of medication from the pharmacy (jaafar, 2013). in our study, we only compared the median cost of medication per month based on acquisition cost, which is the direct pharmacotherapy cost on the ministry of health malaysia’s budget. the median cost for the pim group was not significantly different as to the non-pim group. there might not be many alternatives in the formulary. other studies on prescription cost analysis were based on mean monthly prescription expenditure (as paid by patients). in an indian study, they identified that cost of therapy per month in the pim group, usd 29.40 (≈myr 118.06) was higher (p < 0.01) than the non-pim group, usd 19.80 (≈myr 79.51) (shah, joshi, christian, patel, & malhotra, 2016). in the study conducted in germany, statutory health insurance covering 1/3 german population: cost for pim patients, € 118.37 (≈myr 554.14) was higher (p<0.001) than the non-pim group, € 91.76 (≈myr 429.57) (heider et al., 2017). there were several limitations to our study. we did not know the exact indication for each medication that was prescribed to the elderly population. furthermore, drugs, herbal medicine or supplements from other facilities were not considered. we did not know the exact outcomes of patients caused by pims. conclusion this study shows that approximately one in three patients (38.9%) received at least one pims, with the common pims prescribed were diuretics, short and immediate-acting benzodiazepines and ppis. our study also shows that polypharmacy was the only covariate that affects prescribing of pim(s). the prescribing of pim did not affect the direct cost of pharmacotherapy in our setting. pharmacists should conduct periodic medication reviews among elderly with polypharmacy, in collaboration with prescribers. acknowledgements the authors would like to thank the director-general of health, malaysia for his permission to publish this research. we would also like to extend our appreciation to a’tia hashim (pharmaceutical services programme, ministry of health malaysia), nor azlina bakar (perlis state health department), othman warijo as the hospital director, hospital tuanku fauziah (htf), nik mah nik mat (department of pharmacy, htf) and also khairul shakir ab rahman (clinical research centre, htf) for the continuous support in this study. conflict of interest the authors declare no conflict of interest nor receive any external funding. references abd mutalib, z. a., ismail, m. f., & miskiman, n. 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(2016). prevalence of potentially inappropriate medications and prescription cost ang et al. (2021) journal of pharmacy, 1(1), 27-33. page 33 analysis among older cardiac patients in an outpatient department of a tertiary care hospital in india. journal of basic and clinical pharmacy, 7(4), 110-115. doi: 10.4103/0976-0105.189434 xing, x. x., zhu, c., liang, h. y., wang, k., chu, y. q., zhao, l. b., jiang, d. c., … yan, s. y. (2019). associations between potentially inappropriate medications and adverse health outcomes in the elderly: a systematic review and meta-analysis. annals of pharmacotherapy, 53(10), 1005–1019. doi: 10.1177/1060028019853069 barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework khaairuddin et al. (2022) journal of pharmacy, 2(2), 99-106 page 99 tablet splitting practice among patients in kemaman, terengganu: an exploratory study on practical issues and their association with medication adherence. nurul khalilah khairuddin1, kwek cheng hau2 and najihah mohd muzafar shah3* abstract introduction: the practice of tablet cutting is influenced by tablet shape and size, splitting technique, device used and human competency. difficulties upon tablet cutting could affect patients' medication adherence, resulting in overdose or under-dose therapy. this study aimed to; 1) evaluate patients' practice in tablet cutting, 2) identify problems associated with tablet cutting, and 3) assess the association between tablet cutting related difficulties with medications adherence. method: a cross-sectional survey was conducted among patients and caregivers aged over 18 years old, with prescription written at least one medication required to be split. data were obtained from eight health clinics across the kemaman district from july to september 2020. trained pharmacists conducted face-to-face structured interviews. this study used a validated questionnaire consisting of three sections: the sociodemographic, practice of tablet cutting and medication adherence. descriptive and non-parametric tests (spssv.26) were used for analysis with p<0.05 set as statistically significant. results: the study recorded 383 returned questionnaires with a response rate of 95.8%. the most common technique used was kitchen knife (40.5%) followed by hand breaking (35.8%), scissors (12.8%) and tablet cutter (7.3%). 64.2% perceived they achieved an equal half split after cutting their medications. 25.3% claimed medication to be ruined or broken after splitting. 12.8% took the whole tablet instead of following the required dose because of splitting difficulty and 25.1% discarded parts of their tablets when splitting did not result in equal half based on their perspective. there was no association between practical problems of tablet cutting with medication adherence. conclusion: in conclusion, knife is the most favourable tablet splitting technique but there is no consensus on the best technique for tablet splitting. in order to overcome problems encounter by patient on tablet splitting, healthcare providers should make every effort to use commercially available oral tablet if available. however, if tablet splitting is still necessary, counselling by pharmacist is compulsory to ensure quality of medication and to optimize patient’s adherence as well as medication therapy. article history: received: 7 april 2022 accepted: 24 june 2022 published: 31 july 2022 keywords: tablet cutting, splitting technique, medication splitting, medication adherence how to cite this article: khairuddin, n. k., kwek, c. h. & muzafar shah, n. m. tablet splitting practice among patients in kemaman, terengganu: an exploratory study on practical issues and their association with medication adherence. journal of pharmacy, 2 (2)., 99-106 doi: 10.31436/jop.v2i2.141 authors’ affiliation: 1 klinik kesihatan kerteh, kemaman, terengganu, malaysia. 2 klinik kesihatan batu 2.5, kemaman, terengganu, malaysia. 3 klinik kesihatan seri bandi, kemaman, terengganu, malaysia. *corresponding author: email address: najihahmuz@gmail.com khaairuddin et al. (2022) journal of pharmacy, 2(2), 99-106 page 100 introduction tablet-cutting is an act of splitting a whole tablet into half or quarter, which is a common medical practice in clinical settings. almost 25% of all drugs administered in primary care have their dose altered through cutting medications practice (chaudhri et al, 2019). the goals of tablet cutting are to ease administration to those having difficulty in swallowing the whole tablet (marquis et al, 2013), decrease drug-related costs (mosena & merwe, 2009) and deliver the desired dose, which is often less than the available manufactured strength (arnet & hersberger, 2010). most health care settings allow tablet cutting when the expected dosage strength is not commercially available for those admitted to the hospital or treated in primary health clinics (grissinger, 2010). many tools are used to cut medications, potentially producing varied dose weight or uniformity. the accuracy of tablet cutting may be affected by tablet shape, size, technique, or device used (verrue et al, 2010). however, certain medications such as sustainedrelease preparations and enteric-coated tablets are not expected to be associated with dose alterations in clinical efficacy as these formulation are not meant to be cut (wright, 2002). prior literature showed no identified standards to determine content uniformity of tablet fragments after they have been halved (freeman et al, 2012). product information available on drug package leaflets only include information on the suitability of the specific tablet to be cut. (bosworth et al, 2017). moreover, hospitals often have their own drug formularies and accompanying drug information, often in conflict, leading to confusion (chaudhri et al, 2019). patients' confusion about the correct dose was the leading type of error contributing to a significant prevalence of medication errors in malaysia (samsiah et al, 2016). cutting medications was the additional task to the regimen complexity that will increase the risk of medication errors and adverse drug reactions (denneboom et al, 2005). high prevalence of medication errors in malaysia justified the need for a more effective interventional approach (akram et al, 2018). therefore, it is timely to review current interventions and routine clinical practices such as high alert medications and tablet cutting practice. thus, our study aimed to evaluate patients' practice in tablet cutting, identify problems associated with tablet cutting, and assess the association between tablet cutting related difficulties with medications adherence. methodology study design a cross-sectional investigation was conducted among conveniently selected participants from several kemaman district health clinics: kerteh, kemasik, kijal, seri bandi, batu 2.5, air putih, cheneh and kuala kemaman from july to september 2020. study population four hundred surveys were performed on follow-up patients' ages above 18 years old with at least one medication that needed to be cut. the questionnaires were distributed in outpatient pharmacy, government health clinics of kemaman district. patients with cognitive problem, newly started on medication requiring tablet splitting, caregivers not involved with medication administration, and those who did not agree to participate were excluded. six trained pharmacists conducted face-toface structured interviews using the validated questionnaire. sample size the calculated sample size using raosoft calculator was 377 with a margin of error of 5%, confidence interval of 95%, response rate of 50%, and maximum population size of 20,000. the calculated sample size which is 377 was then rounded up to 400 in order to minimize the dropout rate. dropout included incomplete returned questionnaire. study instruments the questionnaire consisted of three sections. the first part covered patients' sociodemographic data (7 items). this included patients’ living situation whether patients’ lived alone, with family or friends or in a foster care. foster care means patients have been placed into a group home or private home. then, patients’ household income were categorized to b40 (monthly income below rm4849), m40 (monthly income from rm4850 to rm10,959) and t20 (monthly income more than rm10,960) based on the household income and basic amenities survey report 2019, department of statistics malaysia. the following part is close ended questions consisted of the patient's tablet cutting practice and problems associated with tablet cutting (10 items). finally, the third part (12 items) inquired patient's medication adherence concerning tablet cutting using 4 point likert scale. the second part of the questionnaire, particularly for tablet cutting practice and their associated problems, was adapted from a previous study done by gharaibeh et al. (2018). forward and backward translation was done to suit the native population's language. translation procedure of the questionnaire was forward translated into malay with the help of one bilingual expert who is a senior lecturer in university malaya. subsequently, the instrument was back translated into english by one additional bilingual expert who works as a senior lecturer in universiti teknologi mara (uitm). the language experts compared the original english instrument with the back-translated english instrument and edited to obtain the matched malay version. following minor adjustments, a final english version was khaairuddin et al. (2022) journal of pharmacy, 2(2), 99-106 page 101 used to reevaluate the malay versions. this section consists of 10 close ended questions. the validation process was facilitated by family medicine specialists and senior pharmacists of the kemaman district. each reviewer received an evaluation kit comprising a cover letter, demographic information sheet, and translated instruments prior to review. experts reviewed the draft of the ten items to ensure its consistency with the conceptual framework. the four dimensions were (a) item consistency to the content area, (b) item wording clarity, (c) perceived item difficulty, and (d) whether (and why) they thought the item should be included in a revised version of the test. face validity determined the instrument's appropriateness to evaluate the construct of interest. face validity did not refer to whether the instrument measured what it measured. instead, it assessed whether it was superficially valid to the examinees. in this study, face validity was completed via a standardized form to evaluate the malay version of the tablet cutting practice and their associated problems questionnaire. ten adult patients were conveniently selected from klinik kesihatan kerteh and completed the face validity form. they evaluated the overall features of the instrument and whether sentences were clear, concise, easy to understand, and free from typographical errors. after further discussion, the final malay versions of the translated ten items were ready to be used in the study. for patient medication adherence, 12 items validated questionnaire, malaysian medication adherence assessment tool (mymaat), was used. this questionnaire is in dual language, malay and english; no translation was done. scoring less than 54, indicate moderate and poor adherence while scoring more or equal than 54 indicate good adherence. ethical approval the study was done with the approval from medical research & ethics committee (mrec), ministry of malaysia (nmrr no: nmrr-20-251-52820). data collection procedure participants that meet the inclusion and exclusion criteria were approached while waiting for their prescription to be filled in the pharmacy. a set of questionnaires was given to the agreed participants, and then collected after dispensing process for data analysis. data analysis statistical analysis was done using spss version 26.0 (ibm spss statistics for windows, armonk, new york, usa). data were analyzed using descriptive and nonparametric tests (mann whitney and kruskal-wallis test). a p-value less than 0.05 was set as statistically significant. results a total of 383 completed questionnaires were received with a response rate of 95.8%. sociodemographic characteristics were summarized in table 1. table 1: sociodemographic characteristics of participants (n=383). demographic n (%) age 18-60 years 206 (53.8) more than 60 years 177 (46.2) gender male 191 (49.9) female 192 (50.1) marital status married 359 (93.7) divorced/widowed 17 (4.4) single 7 (1.8) living situation live alone 13 (3.4) live with family or friends 367 (95.8) foster care or group home 3 (0.8) education primary school 111 (29.0) secondary school 210 (54.8) tertiary education 47 (12.3) no education 15 (3.9) employment status employed 95 (24.8) self-employed 49 (12.8) unemployed 183 (47.8) retired 56 (14.6) household monthly income b40 344 (89.8) m40 36 (9.4) t20 3 (0.8) khaairuddin et al. (2022) journal of pharmacy, 2(2), 99-106 page 102 the most common medication taken by participants that need to be split are simvastatin 40mg (60.8%) followed by acetylsalicylic acid 300mg (20.6%) and perindopril 4mg (10.7%). refer table 2. table 2: most common medication that need to be split (n=383). active ingredient strength brand name scored n (%) allopurinol 300 mg pharmaniaga allopurinol® yes 18 (4.7) acetylsalicylic acid 300 mg millispirin® yes 79 (20.6) atenolol 100 mg pharmaniaga atenolol® yes 30 (7.8) bisoprolol 2.5 mg bisocor® yes 13 (3.4) frusemide 40 mg pharmaniaga frusemide® yes 11 (2.9) gliclazide 80 mg diamitex® yes 16 (4.2) hydrochlorothiazide 25 mg apo-hydro® yes 12 (3.1) levothyroxine 100 mcg thyrosit® yes 7 (1.8) metoprolol 100 mg betawin® yes 21 (5.5) perindopril 4 mg provinace® yes 41 (10.7) propranolol 40 mg corbeta® yes 6 (1.6) simvastatin 40 mg simvor yes 233 (60.8) others 25 (6.53) table 3 showed that most (n=374, 97.7%) participants split their medications based on prescription instructions. the most common technique used was kitchen knife (n=155, 40.5%) followed by hand breaking (n=137, 35.8%), scissors (n=49, 12.8%) and tablet cutter (n=28, 7.3%). nearly two-thirds (n=246, 64.2%) were confident of achieving equal half upon cutting their medications. table 3: practice of tablet cutting among participants (n=383). variables n (%) reasons for tablet cutting following the prescribed dose 374 (97.7) save cost 6 (1.6) difficulty in swallowing 3 (0.8) tablet cutting routine split medications once for the whole month and keep in a pill container 103 (26.9) split medications every time prior administration 280 (73.1) person who cut the medication in half self 359 (93.7) caregiver 24 (6.3) khaairuddin et al. (2022) journal of pharmacy, 2(2), 99-106 page 103 accuracy perception to get equal half split confident of achieving equal half 246 (64.2) confident not achieving equal half 98 (24.6) not sure to achieve equal half 39 (10.2) technique used for tablet s cutting hands 137 (35.8) tablet cutter 28 (7.3) scissors 49 (12.8) others 14 (3.7) table 4 showed most of the participants (62.1%) had no issue regarding tablet cutting. however, more that 20% of participants agreed tablet cutting are too troublesome and would destroy the medications. majority of participants (n=223, 58.2%) claimed no problem arises due to tablet cutting practice. only 25% of participants (n=97, 25.3%) claimed medication to be ruined or broken after splitting it into half. only a tenth of participants (n=49, 12.8%) took the whole tablet instead of following the required dose because of splitting difficulty. 25% of participants (n=96, 25.1%) discarded parts of their tablets when splitting did not result in equal parts from their perspective. table 4: problems associated with tablet cutting (n=383). variable n (%) problems encountered associated with tablet splitting* take quite a long time to cut 20 (5.2) too troublesome 95 (24.8) unstable or shaky hands 14 (3.7) medication ruined or broken after cutting 97 (25.3) medication size too small 34 (8.9) medication has no score line 7 (1.8) no problem 223 (58.2) patients' response due to difficulty of cutting medications take the whole tablet 49 (12.8) discard all parts that inaccurately cut 96 (25.1) use both parts 238 (62.1) multiple answer question* khaairuddin et al. (2022) journal of pharmacy, 2(2), 99-106 page 104 as in figure 1, 83% of participants have good adherence despite have to split the medications. figure 1: medication adherence among participants (n=383). a statistical test was conducted to determine whether there is a difference in mymaat scores between those without and with splitting problems. the result indicated no statistical significant difference between both groups (table 5). table 5: comparison of median mymaat scores between those with and without splitting problems. group (n) median (iqr) z-statistica p-value with splitting problems (160) 56 (3) -0.775 0.438 no problems (223) 56 (6) -0.775 0.438 amann-whitney test was applied discussion current study found that most patients would use a knife (n=155, 40.5%) to cut their medications. in contrast, prior evidence showed that more than 60% (68.1%) split their medications without any tool or device (akram et al, 2018). another study by gharaibeh et al (2018) reported that the majority of patients used hands (63.5%), apart from other techniques such as kitchen knives (14.3%) and tablet splitter (9%). patients educational status and population idiosyncrasies might associated with different preferred techniques observed in this study. previous study done in jordan and germany were parallel with current study, where the most common reason for tablet splitting was to follow the prescribed dose, save cost, and ease swallowing difficulties (gharaibeh et al, 2018; quinzler, szecsenyi, & haefeli, 2007). although the food and drug administration (2013) advised splitting tablets just before ingestion, the result showed 26.9% of the respondents split their medications once for the whole one month and kept them in a pill container. however, this practice was not recommended because heat, humidity, and moisture content may affect the medications quality & stability which resulting in the possibility of overdose or under-dose therapy. studies done by novartis (2015) and astrazeneca (2019) found no medications was cut by respondents that were not scored. however, the presence of a score line can be misleading, indicating that tablet s splitting is allowed to obtain two equal doses; instead, it is to overcome swallowing difficulties. therefore, pharmacists need to refer to the medications package insert before dispensing medications to patients and educating other healthcare professionals regarding which medications are suitable for tablet splitting. medication adherence 83% good adherence moderate to poor 17% shirazi et al. (2022) journal of pharmacy, 2(1), 16-30 page 105 more than 60% (n=246, 64.2%) perceived they achieved an equal half split after cutting their medications. regardless of the patients claiming they would get an accurate cut using any common method, a previous study showed that obtaining an accurate dose from a split tablet is uncertain (clark, 2002). thus, there is no agreement on the best technique for splitting tablets (eserian et al, 2018). preceding studies recommended avoiding tablet splitting due to inaccuracy (habib et al, 2014). although the likelihood of under or overdosage using tablet halves remained a clinical concern, a study by ashrafpour et al (2018) showed no significant impact of the underlying disease and its complications on tablet splitting accuracy. nevertheless, health care professionals, patients and caregivers should understand that tablet splitting do cause dosing inaccuracies and could potentially contribute to medication errors. our study recorded that nearly half of the participants claimed problems arose due to tablet cutting practice. when splitting did not result in equal parts from their perspective, 25% of participants discarded parts of their tablets, leading to increased healthcare costs. thus, pharmacists have a vital role to educate the patients and planning a strategies with other healthcare professionals to overcome the splitting difficulties and reduce the possibility of drug wastage. there was no association between problem of tablet cutting with medication adherence; meaning that splitting medications was not cause patients to skip their doses. instead, patients might have been more adherent, probably because of increased awareness and sense of responsibility to split the tablet. previous study by gee et al (2002) reported that less than 25% of participants stated that they missed more doses in a month, with most patients being compliant with the tablet-splitting practice. another study by gharaibeh (2018) reported that only 25% of study participants sometimes skipped their doses due to tablet splitting difficulties. a systematic review by the canadian agency for drugs and technologies in health (2015) confirmed tablet splitting had no impact on adherence or compliance with medications. limitation the study was conducted in public setting hence the result may not be generalized to other private settings across malaysia due to differences in medication brand procurement. conclusion in conclusion, knife is the most favourable tablet splitting technique but there is no consensus on the best technique for tablet splitting. in order to overcome problems encounter by patient on tablet splitting, healthcare providers should make every effort to use commercially available oral tablet if available. however, if tablet splitting is still necessary, counselling by pharmacist is compulsory to ensure quality of medication and to optimize patient’s adherence as well as medication therapy. acknowledgements we thank the director-general of health malaysia for permission to publish this paper. we would like to thank the department head of pejabat kesihatan daerah kemaman, dr mohd anuar bin abd rahman and the pharmacy unit head of pejabat kesihatan daerah kemaman, puan normawar binti ibrahim for their approval for us to conduct this research. our deepest gratitude extends to cik mazlina mukhtar who offer sincere insight and expertise in this research since the very beginning. this research would not have been possible without our colleagues from pharmacy unit for giving precious contributions towards the completion of this research. conflict of interest the authors declare that there is no conflict of interest. references akram, a., moawia, a.t., zain, a.s., & zainab, a. 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(2002). tablet crushing is a widespread practice but it is not safe and may not be legal. pharmaceutical journal; 2 introduction tablet-cutting is an act of splitting a whole tablet into half or quarter, which is a common medical practice in clinical settings. almost 25% of all drugs administered in primary care have their dose altered through cutting medications practice (chau... most health care settings allow tablet cutting when the expected dosage strength is not commercially available for those admitted to the hospital or treated in primary health clinics (grissinger, 2010). many tools are used to cut medications, potentia... prior literature showed no identified standards to determine content uniformity of tablet fragments after they have been halved (freeman et al, 2012). product information available on drug package leaflets only include information on the suitability o... thus, our study aimed to evaluate patients' practice in tablet cutting, identify problems associated with tablet cutting, and assess the association between tablet cutting related difficulties with medications adherence. methodology study design a cross-sectional investigation was conducted among conveniently selected participants from several kemaman district health clinics: kerteh, kemasik, kijal, seri bandi, batu 2.5, air putih, cheneh and kuala kemaman from july to september 2020. study population four hundred surveys were performed on follow-up patients' ages above 18 years old with at least one medication that needed to be cut. the questionnaires were distributed in outpatient pharmacy, government health clinics of kemaman district. patients ... sample size the calculated sample size using raosoft calculator was 377 with a margin of error of 5%, confidence interval of 95%, response rate of 50%, and maximum population size of 20,000. the calculated sample size which is 377 was then rounded up to 400 in or... study instruments the questionnaire consisted of three sections. the first part covered patients' sociodemographic data (7 items). this included patients’ living situation whether patients’ lived alone, with family or friends or in a foster care. foster care means pati... the second part of the questionnaire, particularly for tablet cutting practice and their associated problems, was adapted from a previous study done by gharaibeh et al. (2018). forward and backward translation was done to suit the native population's ... the validation process was facilitated by family medicine specialists and senior pharmacists of the kemaman district. each reviewer received an evaluation kit comprising a cover letter, demographic information sheet, and translated instruments prior t... face validity determined the instrument's appropriateness to evaluate the construct of interest. face validity did not refer to whether the instrument measured what it measured. instead, it assessed whether it was superficially valid to the examinees... for patient medication adherence, 12 items validated questionnaire, malaysian medication adherence assessment tool (mymaat), was used. this questionnaire is in dual language, malay and english; no translation was done. scoring less than 54, indicate ... ethical approval the study was done with the approval from medical research & ethics committee (mrec), ministry of malaysia (nmrr no: nmrr-20-251-52820). data collection procedure participants that meet the inclusion and exclusion criteria were approached while waiting for their prescription to be filled in the pharmacy. a set of questionnaires was given to the agreed participants, and then collected after dispensing process f... data analysis statistical analysis was done using spss version 26.0 (ibm spss statistics for windows, armonk, new york, usa). data were analyzed using descriptive and non-parametric tests (mann whitney and kruskal-wallis test). a p-value less than 0.05 was set as s... results table 3: practice of tablet cutting among participants (n=383). table 5: comparison of median mymaat scores between those with and without splitting problems. discussion current study found that most patients would use a knife (n=155, 40.5%) to cut their medications. in contrast, prior evidence showed that more than 60% (68.1%) split their medications without any tool or device (akram et al, 2018). another study by ... previous study done in jordan and germany were parallel with current study, where the most common reason for tablet splitting was to follow the prescribed dose, save cost, and ease swallowing difficulties (gharaibeh et al, 2018; quinzler, szecsenyi, &... studies done by novartis (2015) and astrazeneca (2019) found no medications was cut by respondents that were not scored. however, the presence of a score line can be misleading, indicating that tablet s splitting is allowed to obtain two equal doses; ... limitation conclusion acknowledgements barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework sudarman & haris (2023) journal of pharmacy, 3(1), 9-18 page 9 spectrophotometric simultaneous analytical method validation to determine isoniazid and pyridoxine in pure and 3d printed tablet forms nur suhaila sudarman1, muhammad salahuddin haris1,2,* abstract introduction: isoniazid (inh) is the anti-tuberculosis drugs being used to counter tuberculosis since 1952. patients on inh should be given daily prophylactic pyridoxine (pyr) with 10-50 mg/day to prevent the development of isoniazid-induced neuropathy. within the framework of this research, the uv-vis spectrophotometer is used to quantify simultaneously the drug content of inh and pyr. methods: the standard curve for both inh and pyr were plotted using the concentration of 5 µg/ml, 10 µg/ml, 15 µg/ml, 20 µg/ml, 25 µg/ml, and 30 µg/ml and tablets were analysed using simultaneous equation method. the proposed method was validated by analytical method validation for the linearity, specificity, accuracy, intermediate precision, limit of detection (lod), and limit of quantification (loq). results: a regression equation of inh standard and sample were found to be y = 0.0279x + 0.0637 and y = 0.0280x + 0.0522 obtained from the calibration curve and linear with correlation coefficient (r2) values of 0.9950 and 0.9964, respectively. a regression equation of pyr standard and sample were y= 0.0267x + 0.0723 and y = 0.0259x + 0.0806 and to be linear with r2 values of 0.9981 and 0.9962, respectively. the result of accuracy obeyed the accepted criteria of percentage recovery in between 98% to 102%. the method exhibited intermediate precision as demonstrated by relative standard deviation <2%. the lod and loq of inh were 0.166 µg/ml and 0.5018 µg/ml while the lod and loq of pyr were 0.122 µg/ml and 0.371 µg/ml, respectively in the pure form. in tablet dosage form, the lod and loq of inh were 0.071 µg/ml and 0.215 µg/m while lod and loq of pyr give the result 0.124 µg/ml and 0.375 µg/ml, respectively. conclusion: this spectrophotometric simultaneous analytical method validation for inh and pyr was successfully conducted with the notion to spearhead the development of inh and pyr in a single dosage form to improve compliance among tuberculosis patients. article history: received: 12 may 2022 accepted: 11 october 2022 published: 31 january 2023 keywords: isoniazid, pyridoxine, uv-vis spectrophotometer, simultaneous equation method, analytical method validation how to cite this article: sudarman, n. s. & haris, m. s. spectrophotometric simultaneous analytical method validation to determine isoniazid and pyridoxine in pure and 3d printed tablet forms. journal of pharmacy, 3(1)., 9-18 doi: 10.31436/jop.v3i1.176 authors’ affiliation: 1 department of pharmaceutical technology, kulliyyah of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, 25200 kuantan, malaysia. 2 ikop pharma sdn. bhd., jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia. *corresponding author: email address: solah@iium.edu.my sudarman & haris (2023) journal of pharmacy, 3(1), 9-18 page 10 introduction isoniazid (inh) is a highly effective treatment for m. tuberculosis that the world health organization recommends (who). it serves as the main ingredient of several fixed-dose combination tablets, each of which contains two or more anti-tb drugs and has been in use since 1952 to treat tuberculosis. in addition to extending the tablet's shelf life, antioxidants and inh cocrystals may lessen oxidative stress in tb patients receiving therapy (mashhadi et al., 2021). pyridoxine (pyr) species are immediately inactivated by inh metabolites. in people with high-risk conditions, pyr deficiency can result in neurologic adverse effects such peripheral neuropathy. the clinical practice guidelines state that daily prophylactic pyridoxine (vitamin b6) administration to inh patients with 10 to 50 mg/day is recommended to avoid the onset of isoniazid-induced neuropathy. pyridoxine (pyr) is a water-soluble vitamin that aids in the metabolism of carbohydrates, lipids, and amino acids. this vitamin has a significant impact on the metabolism of nitrogen-containing compounds such serotonin, dopamine, norepinephrine, gamma-aminobutyric acid (gaba), and the component of haemoglobin. in addition to encouraging the development of red blood cells, pyridoxine aids in the balance of salt and potassium. table 1 shows the physicochemical properties of both inh and pyr, respectively (wishart et al., 2018). the uv-vis spectrophotometer's fundamental principle is the absorption of light by a sample. when utilising a uv-vis spectrophotometer, the purity of the inh and pyr samples may be measured based on how much light and its wavelength are absorbed by the samples. a sample solution is placed in a cuvette, and an ultravioletvisible (uv-vis) spectrophotometer analyses the light's intensity as it passes through the solution and compares it to the light's intensity before the sample. a uv-vis spectrophotometer's primary components are a light source, a sample holder, a dispersive device to separate the light's various wavelengths, and an appropriate detector. the visual depiction of the uv-vis spectrum in general is the absorbance as a function of wavelength. within the framework of this research, the uv-vis spectrophotometer is used to quantify simultaneously the drug content in the pure form of inh and pyr by measuring all the absorbance values for each concentration at determined wavelengths which are 263 nm of inh and 290 nm of pyr then will be calculated in the simultaneous equation method. according to beer’s lambert law, it states that absorbance is proportional to concentration. so, this research study uses two types of modes of the uv-vis spectrophotometer. the first one is the photometric mode. the photometric mode can help define the known wavelengths of inh and pyr by measuring the absorbance at a single wavelength or at multiple wavelengths. the second type is a spectrum mode. the spectrum mode may obtain sample spectra using wavelength scanning thus resulting in a peak wavelength of each inh and pyr as required. the uv-vis spectrophotometer interprets the data analysis to provide the necessary information and can subsequently obtain the results. table 1: physicochemical properties of inh and pyr properties inh pyr chemical structure chemical name pyridine-4-carbohydrazid 4,5-bis(hydroxymethyl)-2-methylpyridin-3-ol molecular formula c6h7n3o c8h11no3 molecular weight 137.14 g/mol 169.18 g/mol melting point 171.4 °c 159 °c to 162 °c solubility (at 25 °c) 1.4 x 105 mg/l 2.2 x 105 mg/l log p -0.70 -0.77 half-life 0.5 to 1.6 hours for fast acetylators 2 to 5 hours for slow acetylators 15 to 20 days sudarman & haris (2023) journal of pharmacy, 3(1), 9-18 page 11 materials inh standard (99.7% purity), pyr standard (99.9% purity), inh and pyr analytical grade powders were purchased from sigma-aldrich (darmstadt, germany). inh 300 mg 3d printed tablet and pyr 10 mg tablet were used for analysis purpose. distilled water was used as a solvent in this experiment. methodology 1. preparation of standard solution accurately 10 mg of inh and 10 mg of pyr standard were separately transferred into individual 100 ml volumetric flasks, then dissolved appropriately with distilled water and diluted up to the mark with distilled water to give solutions containing 100 µg/ml of inh and 100 µg/ml of pyr (figure 1). 2. preparation of calibration curve the calibration curve was prepared by using the stock solution to produce six different concentrations of inh and pyr standard which are 5 µg/ml, 10 µg/ml, 15 µg/ml, 20 µg/ml, 25 µg/ml, and 30 µg/ml (figure 1). the absorbance of each concentration was acquired at the λ max using a fixed wavelength measurement mode. the calibration curve representing concentration versus absorbance was plotted. 3. determination of wavelength of maximum absorbance (λ max) a solution containing 15 µg/ml of inh and 15 µg/ml of pyr was scanned separately using full output mode with medium scanning speed for a whole range of dual wavelengths by using a uv-visible spectrophotometer (shimadzu, kyoto, japan) ranging from 400 – 200 nm with distilled water as blank. after acquiring the spectrum, the maximum absorbance was identified. figure 1. preparation of stock and calibration curve solutions. sudarman et al. (2023) journal of pharmacy, 3(1), 9-18 page 12 4. simultaneous equation method this method of analysis is based on the absorption of inh and pyr at the wavelength maximum of each other (tilinca et al., 2017). two wavelengths selected for the development of simultaneous equations were 263 nm and 290 nm which were lambda maximum of inh and pyr respectively. the absorbances of inh and pyr measured at selected wavelengths (tilinca et al., 2017). absorptivity values were calculated. the concentrations of both the drugs in mixture can be calculated by using following equations 1 and 2: 𝑪𝒙 = 𝑨𝟐𝒂𝒚𝟏−𝑨 𝟏𝒂𝒚𝟐 𝒂𝒙𝟐𝒂𝒚𝟏−𝒂𝒙𝟏𝒂𝒚𝟐 eq. 1 𝑪𝒚 = 𝑨𝟏𝒂𝒙𝟐−𝑨 𝟐𝒂𝒙𝟏 𝒂𝒙𝟐𝒂𝒚𝟏−𝒂𝒙𝟏𝒂𝒚𝟐 eq. 2 where, a1 and a2 are absorbances of mixture at 263 nm and 290 nm, respectively. ax1 and ax2 are the absorptivity of inh at 263 nm and 290 nm, respectively. ay1 and ay2 are the absorptivity of pyr at 263 nm and 290 nm, respectively. cx and cy are concentrations of inh and pyr, respectively. 5. application of the proposed method for the determination of inh and pyr in tablets the 3d-printed tablet containing 300 mg inh and 10 mg pyr was analysed by this method. an amount equivalent to 10 mg inh and 10 mg pyr of the selected tablet was weighed and dissolved in 100 ml distilled water to obtain a stock solution containing 100 µg/ml standard solution. the solution was then filtered through whatman filter paper. inh and pyr were diluted appropriately. the absorbance of the resulting solutions was measured at 263 nm and 290 nm. the concentration of inh and pyr in the sample solution was calculated using the equation constructed from the calibration curve of each drug.values were substituted in the respective formula to obtain concentrations. 6. analytical method validation (amv) the main objective of performing analytical method validation is to demonstrate that the analytical method which is a uv-vis spectrophotometer is suitable and adequate for its intended purpose (patil, patil, chalikwar, surana, & firke, 2019). the validation of the developed method was carried out in terms of specificity, linearity, accuracy, precision, intermediate precision, the limit of detection (lod), and limit of quantification (loq). it was validated according to the international conference on harmonization guidelines. 6.1 specificity specificity is its ability to detect and differentiate the analyte of interest in the presence of other substances, including its related substances to guarantee character of an analyte (patil et al., 2019). the specificity of the direct spectrophotometric method was assessed by comparing the spectrum obtained from the solvent system alone (placebo), which is distilled water, and of standard inh, pyr solution in the diluent. 6.2 linearity and standard curve in order to find the line that best fits a provided set of data, the linearity was established. this allowed for a visual representation of the relationship between the data points (patil et al., 2019). the linearity of this method was established using six different calibration standards. standard inh and pyr were tested at six known concentrations using a pre-determined wavelength. every concentration's absorbance was recorded. the linearity was determined by plotting six concentrations (x-axis) of inh and pyr standard and sample against absorbance (yaxis). the equation of y = mx + c and the r2 was developed. 6.3 accuracy the degree to which test results agree with the genuine value, or how closely the outcomes of the method correspond with the true value, is known as accuracy. in order to minimise potential operating errors, it is often established on samples of the material to be analysed that have been produced with quantitative accuracy. (patil et al., 2019). accuracy should be established across the specified range of the analytical procedure. to ascertain the accuracy of the proposed methods, recovery studies were carried at three different levels which are 80%, 100% and 120% were subjected to the determined wavelength (nm) in which 263 nm of inh and 290 nm of pyr. the percentage recovery should be in between 98% and 102% to meet the acceptance criteria. 6.4 intermediate precision precision is how close individual measurements are to each other. intermediate precision is a part of precision in which the method is tested on multiple days, instruments, and analysts to measure of the ruggedness of the method’s reliability when performed in different environments (patil et al., 2019). the intraday precision of inh and pyr was checked by assay the sample solution on same day at an interval of one hour for three hours and interday precision was carried out by estimating the correspondence responses on three different days with different preparations. according to this study, the solutions may be sudarman et al. (2023) journal of pharmacy, 3(1), 9-18 page 13 analysed within 48–72 hours without negatively affecting the drug's chemical stability when urea is present. the wavelength was applied to each concentration in triplicates, and the mean and standard deviation were then calculated. to achieve the acceptance standards, the accuracy percentage of the relative standard deviation (rsd) value must be less than 2.0%. 6.5 limit of detection (lod) and limit of quantification (loq) the lowest concentration of an analyte in a sample that can be identified and measured with suitable precision and accuracy under the specified test conditions was used for the evaluation of lod and loq. the following equations 3 and 4 describe the precise calculations to estimate lod and loq, respectively: lod = (3.3 x sd)/m eq. 3 loq = (10.0 x sd)/m eq. 4 sd or standard deviation in the formula was referring to the standard deviation of the absorbance values of the blank and 𝑚 is the slope of the standard curve constructed previously (ismail et al., 2016). all readings for lod and loq were conducted in triplicates. results 1. specificity the identification of wavelength with maximum absorbance is needed for quantitative uv analysis. the specificity should not be tested without any blank or matrix spectrum because it does not give any reading or specified wavelength of drug content. the standard solution of inh and pyr with concentration of 15 µg/ml1 was separately scanned in the range of 200-400 nm. the result showed that the λmax was determined for each drug. the λmax inh and pyr were found to be 263 nm with the absorbance is 0.519 and 290 nm with the absorbance is 0.422, respectively as shown in figure 2a and figure 2b. after scanned both drugs separately, then overlap the spectras and obtained the isosbestic wavelength at 280 nm as λmax of common absorbance as shown in figure 2c. isosbestic wavelength is used when two substances of equimolar concentration show the same absorbance at particular wavelength and by using isosbestic wavelength it may record the absorbance of formulation or multi wavelength photometric mode of uv-vis spectrophotometer. figure 2. a) uv–vis spectra of standard inh (λ:263 nm). and pyr (λ:290 nm). c) uv-vis overlaid spectra of both standards (isosbestic λ:280 nm). sudarman et al. (2023) journal of pharmacy, 3(1), 9-18 page 14 2. linearity and calibration curve the linearity was confirmed using the absorbance values at a constant set wavelength, which is 263 nm, and the direct percentage relationship between the concentration of standard inh and sample inh. in the meantime, the linearity was established using the absorbance values at a set established wavelength, which is 290 nm, and the direct percentage relation between the concentration of standard pyr and sample pyr. six known concentrations of both standard and sample of inh and pyr were prepared namely are 5 µg/ml, 10 µg/ml, 15 µg/ml, 20 µg/ml, 25 µg/ml, and 30 µg/ml were subjected to 263 nm and 290 nm for inh and pyr to get the absorbance values for each sample. the calibration of the developed uv-vis spectrophotometer method was in linear form with the equation and the r2 of standard inh is y = 0.0279x + 0.0637 and 0.9950 respectively while the equation and the r2 of standard pyr is y = 0.0267x + 0.0723 and 0.9981 respectively. in addition, the calibration of the developed uv-vis spectrophotometer method was in linear form with the equation and the r2 of sample inh is y = 0.0280x + 0.0522 and 0.9964 respectively while the equation and the r2 of sample pyr is y = 0.0259x + 0.0806 and 0.9962, respectively. 3. accuracy in pure form, to ascertain the accuracy of the proposed methods, recovery studies were carried at three different levels which are 80%, 100% and 120% were subjected to the determined wavelength (nm) in which 263 nm of inh and 290 nm of pyr. three samples were prepared for each level, and their absorbance was quantified using a uv-vis spectrophotometer. the concentrations of inh and pyr in the sample solution were determined using an equation derived from the calibration curves of the respective drugs. to acquire concentrations, absorbance values were substituted in the applicable formula. following that, the concentration was subtracted with 6 ppm, which is stated in the linearity of standard curve for the six concentrations created, and the result was represented as the obtained value. the actual value was determined using the crossmultiplication approach based on the different levels (80%, 100%, 120%), and the actual value was the fixed value for both inh and pyr. then, the percentage recovery calculation was followed by using eq. 5. % recovery = 𝐎𝐛𝐭𝐚𝐢𝐧 𝐯𝐚𝐥𝐮𝐞 𝐀𝐜𝐭𝐮𝐚𝐥 𝐯𝐚𝐥𝐮𝐞 × 𝟏𝟎𝟎 eq. 5 futhermore, in 3d printed tablet dosage form, the accuracy was developed by prepared six samples for each different levels which are 80%, 100%, and 120% and the absorbance was recorded to obtained the concentration of the sample. the simultaneous equation method was utilised by applying the absorbance and absorptivity values in the calculation in order to get the true concentration and then dividing it with the initial concentration. next, it was multiplied by 100 and the result represents the percentage recovery. overall, the percentage recovery should be in between 98% and 102% to meet the acceptance criteria, and all these accuracy results of inh and pyr for both pure form and 3d printed tablet dosage form were tabulated in table 2 and table 3, respectively. table 2: results of accuracy for the simultaneous determination of inh and pyr in pure form drug level of accuracy recovery (%) rsd (%) inh 80 100 120 98.13 98.17 99.17 0.17 0.16 0.36 80 100 120 98.13 98.17 99.17 0.17 0.16 0.36 80 100 120 98.13 98.17 99.17 0.17 0.16 0.36 pyr 80 100 120 99.74 100.33 102.36 0.56 0.15 0.23 80 100 120 99.74 100.33 102.36 0.56 0.15 0.23 80 100 120 99.74 100.33 102.36 0.56 0.15 0.23 sudarman et al. (2023) journal of pharmacy, 3(1), 9-18 page 15 table 3: results of accuracy for the simultaneous determination of inh and pyr in 3d printed tablet dosage form drug label claim sample (mg) actual (mg) accuracy (%) sd rsd (%) inh 80 100 120 8 10 12 8.16 9.91 11.86 101.96 99.00 98.83 0.0010 0.0030 0.0006 0.40 1.17 0.27 80 100 120 8 10 12 8.16 9.91 11.86 101.96 99.00 98.83 0.0010 0.0030 0.0006 0.40 1.17 0.27 80 100 120 8 10 12 8.16 9.91 11.86 101.96 99.00 98.83 0.0010 0.0030 0.0006 0.40 1.17 0.27 pyr 80 100 120 4 5 6 4.02 4.97 5.99 100.52 99.50 99.87 0.0010 0.0017 0.0020 0.14 0.23 0.42 80 100 120 4 5 6 4.02 4.97 5.99 100.52 99.50 99.87 0.0010 0.0017 0.0020 0.14 0.23 0.42 80 100 120 4 5 6 4.02 4.97 5.99 100.52 99.50 99.87 0.0010 0.0017 0.0020 0.14 0.23 0.42 4. immediate precision the intraday precision study of inh and pyr in pure form was carried out by estimating the correspondence responses three times on the same day with concentrations of 5 g/ml, 10 g/ml, and 15 g/ml, and the interday precision study of inh and pyr was carried out by estimating the correspondence responses three times the next day with different preparations of 5 g/ml, 10 g/ml, and 15 g/ml had been recorded in table 4. meanwhile, the method's intermediate precision was assessed in 3d printed tablet dosage form by assaying the sample solution on the same day at one-hour intervals (intraday precision) for three hours and on three distinct days (interday precision), as shown in table 5. according to this study, the solutions may be analysed within 48-72 hours without affecting the chemical stability of the drug in the presence of urea. overall, the percentage of rsd for each concentration of simultaneous inh and pyr that was subjected to the specified wavelengths of 263 nm and 290 nm was less than 2%, which passed the acceptance requirements. 5. limit of detection (lod) and limit of quantification (loq) the limit of detection represents the lowest concentration of analyte that can be reliably detected. meanwhile, the limit of quantification represents the lowest concentration of analyte that can be analysed and quantified. the lod and loq of inh were found to be 0.166 µg/ml and 0.5018 µg/ml while the lod and loq of pyr were found to be 0.122 µg/ml and 0.371 µg/ml respectively in pure form as shown in table 6. besides that, the lod and loq of inh were obtained to be 0.071 µg/ml and 0.215 µg/ml respectively but however, the lod and loq of pyr were obtained to be 0.124 µg/ml and 0.375 µg/ml respectively in 3d printed tablet dosage form as presented also in table 6. 6. assay of 3d printed tablet dosage form the optimized method was successfully applied for the simultaneous determination of inh and pyr in the 3d printed tablet dosage forms, containing 300 mg inh and 10 mg pyr. six samples were tested using a uv-vis spectrophotometer and the absorbance of each sample was measured in triplicate at specific wavelengths of 263 nm and 290 nm. the mean absorbance and absorptivity of the samples were used and the amount found of tablets was calculated using the simultaneous equation method. satisfactory results were obtained for each compound as the found amounts were in good agreement with the amount taken as indicated in table 7. sudarman et al. (2023) journal of pharmacy, 3(1), 9-18 page 16 table 4: results of intraday and interday precision for the simultaneous quantification of inh and pyr in pure form analyte (µg/ml) sd rsd (%) intraday (n=6) inh pyr inh pyr 5 0.0006 0.0006 0.29 0.22 10 0.0010 0.0020 0.31 0.50 15 0.0006 0.0006 0.13 0.13 interday (n=18) inh pyr inh pyr 5 0.0030 0.0006 0.37 0.31 10 0.0030 0.0006 0.54 0.26 15 0.0006 0.0010 0.07 0.33 table 5: results of intraday and interday precision for the simultaneous quantification of inh and pyr in 3d printed tablet dosage form parameters drug label claim sample (mg) actual (mg) sd rsd (%) intraday (n=3) inh 300 10 9.91 0.0030 1.17 pyr 10 5 4.97 0.0017 0.23 interday (n=3) inh 300 10 10.11 0.003 0.56 pyr 100 5 4.92 0.004 0.65 table 6: lod and loq data of the uv-vis spectrophotometer method for the simultaneous determination of inh and pyr in pure and 3d printed tablet forms. drug type lod (µg/ml) loq (µg/ml) inh pure 0.1660 0.5018 3d-printed tablet 0.0710 0.2150 pyr pure 0.1220 0.3710 3d-printed tablet 0.1240 0.3750 table 7: assay results of inh and pyr determination in tablet dosage form drug label claim sample (mg) actual (mg) accuracy (%) sd rsd (%) inh 300 10 9.78 98.0 0.0006 0.61 pyr 10 10 10.01 100.1 0.0010 0.23 discussion the proposed method that used to determine simultaneous of inh and pyr in this study was the simultaneous equation method or also known as vierordt’s method. the importance of the study of the simultaneous equation method was that it allows the analysis of multicomponent drugs using different analytical techniques such as spectrophotometer, chromatography and electrophoresis. however, this study focuses more on the use of uv-vis spectrophotometer because it is an applicable method and most scientific work has been done using this technique. the reason why the author chose vierord's method is that it has many advantages. for instance, this method can save time and cost effective since the absorption measurement were obtained with ease, the process was fast, and simple. vierordt’s method also had its shortcoming such as the lambda max of two drugs should be reasonable different, there must no chemical interaction between the absorbing components, and they should obey beers law at their wavelength maximum if used in uv-vis spectrophotometer. based on the result, all the validation parameters that validate the proposed method showed it was specific, linear, accurate, precise, and sensitive. it could be consistent with the other published article, where the author (tilinca et al., 2017) also obtained the same result in the simultaneous determination of inh and rifampicin (rif) using the same method. the assay part indicated that found amount of inh (148.84 mg) and sudarman et al. (2023) journal of pharmacy, 3(1), 9-18 page 17 rif (297.68 mg) were in good agreement with the declared amount of inh (150 mg) and rif (300 mg). the correlation coefficient (r2) was more than 0.99, the specificity showed that 263 nm (ʎmax for inh) and 338 nm (ʎmax for rif), percentage recovery in the range of 98%102%, the rsd of an intermediate precision also less than 2%, and lod of inh (2.60 µg/ml) and rif (3.50 µg/ml) were showed always lesser than lowest concentration in the standard curve but the loq result of inh was (8.58 µg/ml) and pyr (11.70 µg/ml). in addition, there had one article entitle simultaneous estimation of salbutamol sulphate (sal) and ambroxol hcl (amb) from their combined dosage form by uv-vis spectrophotometer using the simultaneous equation method also discussed the same methods (panchale, gulhane, manwar, & bakal, 2020). as a result, all the validation parameters proved that the proposed method was specific, linear, accurate, precise, and sensitive. the specificity showed had two different maximum wavelength present which are 242 nm for sal and 272 nm for amb. the r2 of the linearity in the calibration curve showed that more than 0.99 and the percentage recovery’s result still in the range between 98% and 102%. moreover, the rsd of intermediate precision give less than 2% in which meet the acceptance criteria and the lod and loq result were 0.95 µg/ml and 0.18375 µg/ml respectively for both sal and amb. finally, the author recommends in the future, the methods can be employed for routine analysis in simultaneous determination of another combination drugs and also quality control analysis. conclusion in a sample that contains of two absorbing drugs like inh and pyr in which each of them absorbs at a maximum wavelength different from the other, it may be possible to determine both drugs in the pure form and 3d printed tablet dosage form by the technique of simultaneous equation method. to conclude, the described method was validated in accordance with the international conference on harmonisation guidelines and give a specific, linear, accurate, precise, and sensitive results for the simultaneous determination of inh and pyr from pure form and 3d printed tablet dosage form. as mentioned in the discussion part, all the results got in this work were acceptable and corresponded with the results of other published articles. hence, the suggested approach may be directly used to quantify inh and pyr simultaneously. conflict of interest the authors declare that there is no conflict of interest. references attala, k., & elsonbaty, a. 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(2023) formulation and evaluation of tridax procumbens (l.) l. herbal soaps.docx page 1 formulation and evaluation of tridax procumbens (l.) l. herbal soaps m. v. sudharani1*, a. c. kullayappa1, c. dheeraj1, k. bhaskar naik1, m. vandana1, p. jamalbi1 and v. sravani1 *corresponding author: email address: mvsudharanireddy@gmail.com authors’ affiliation: 1 department of pharmaceutics, sri krishnadevaraya university college of pharmaceutical sciences (skucops), anantapur, 515003, andhrapradesh, india. abstract introduction: traditional medicine is an important source of potential therapeutic compounds. tridax procumbens has great importance in traditional medicine because of its good antibacterial and antifungal properties. the current research aims to formulate herbal soap using methanolic extract of t. procumbens leaves and evaluate its physicochemical properties. method: the herbal soaps are formulated using t. procumbens leaf extract. the leaves were extracted by the soxhlet extraction method using methanol as solvent. the plant extract was evaluated for phyto constituents like saponins, phenols, alkaloids, flavonoids, tannins and steroids. four different formulations are formulated with varying doses of plant extract and ingredients. the physical parameters like colour, odour, appearance and evaluation parameters like ph, moisture content, % alcohol insoluble matter, foam height, foam retention and % free alkali are evaluated for four formulated soaps. results: the plant extract consists of phyto constituents like saponins, phenols, alkaloids, flavonoids, tannins and steroids. all four formulations had good appearance, uniform colour and odour. conclusion: among four soap formulations, f1 soap had the least number of impurities whereas f3 soap had stable foam. the evaluation parameters are in the limits prescribed by bureau of indian standards. hence formulated soaps can further be standardised and used. article history: received: 12 april 2022 accepted: 11 october 2022 published: 31 january 2023 keywords: tridax procumbens, herbal soap, anti-microbial, skin diseases, evaluation tests how to cite this article: sudharani, m. v., kullayappa, a. c., dheeraj, c., bhaskar naik, k., vandana, m., jamalbi, p., & sravani, v. (2023) formulation and evaluation of tridax procumbens (l.) herbal soaps. journal of pharmacy, 3(1), 1-8 doi: 10.31436/jop.v3i1.134 page 2 introduction skin is the first line of defence in the human body. the skin constitutes 15% of the total body weight, as it is the largest organ. it protects against physical, biological and chemical attacks and plays an important role in thermoregulation by preventing the loss of excess water from the body (kolarsick et al., 2011). crores of bacteria, fungi, and viruses reside on our skin and constitute the skin microbiota. these act as a physical barrier to prevent the invasion of pathogens. when the balance between these symbiotic bacteria and pathogens is disturbed, it may result in skin infections (allyson et al., 2018). for many years skin problems are common ailments that are affecting humans (akuaden et al., 2019). common causes of skin infections are the invasion of the skin by pathogenic microorganisms. so, any substance with antimicrobial properties, which either kills the microorganism or inhibits the growth of the microorganism is essential for treating skin infections. nature has been a major source of herbs with immense antimicrobial potential to treat mild to severe types of skin diseases. many ancient medical systems like ayurveda, siddha and unani systems of medicine have explored the use of several herbal preparations for treating skin infections (christudas et al., 2012). according to the world health organisation reports, traditional medicine is fulfilling the primary health needs of 80% of the population. the dependency on traditional medicine is more in developing countries. the increasing desire to investigate new potential drugs in natural sources has led us to create some amazing medicines (policepatel & manikrao, 2013). herbal formulations with antifungal and antibacterial activities can be prepared from various parts of the plant like stem, leaves, roots, bark, flower, or fruit for skincare. these medicines can be applied topically or administered orally. for topical administration, these medicines are formulated in the form of cream, lotion, gel, soap, sap, or ointment (kareru et al., 2010). herbal soaps are one of the highly used formulations for skincare and for treating skin diseases. soap is a surface-active agent and it is chemically the alkali metal salt of long-chain fatty acids. when a fat or oil containing triglycerides is reacted with alkali, soap is formed by a reaction called saponification reaction (akuaden et al., 2019). generally, soaps are prepared by the melt and pour method, hot press method and cold press method. oils like coconut oil, palm kernel oil, olive oil, castor oil, sunflower oil, rice bran oil and soybean oil among others are used for soap preparation. the quality of soap depends on the type of oil used, type of alkali used, its hardness, foam height, moisture content, and total fatty matter. (sindhu et al., 2019). herbal soaps incorporated with herbal extracts should show significant antimicrobial activity, provide conditioning to the skin, have good foam, fragrance and are gentle on the skin. one of the potential plants with antimicrobial properties is tridax procumbens (l.) l (t. procumbens). it is generally called coat buttons in english, jayanthi veda in sanskrit, balapaku or gaddi chamanthi in telugu. it belongs to the family asteraceae. t. procumbens is a widely spread herb, covered with hair (jain, 2012). it is widely spread in india up to 2400 m above sea level even though it is primarily native to tropical regions of south and north america (vinod & nagaraju, 2015). t. procumbens has great importance in traditional medicine. it is used to treat colds, inflammation, anaemia and liver diseases in central america. in guatemala, it is used as an antibacterial, antifungal, and antiviral agent and plays an important role in the treatment of vaginitis, stomach pain, diarrhoea, mucosal inflammations, and skin infections. in india, leaf juice is used to treat wounds and bleeding. gastrointestinal and respiratory infections, high blood pressure, and diabetes are some of the ailments for which t. procumbens is used significantly. the whole plant is used for the treatment of protozoal infections, like malaria, leishmaniasis and dysentery in guatemala (beck et al., 2018). in the west african sub-region and tropical region of the world, the leaves of the plant are used as a remedy for conjunctivitis by traditional practitioners and the native people (pandey & tripathi, 2014). the methanolic extract of the leaves consists of phytoconstituents like alkaloids, flavonoids, phenols, saponins, steroids, and tannins (kushwaha et al., 2018). the plant is rich in minerals like iron, copper, zinc, sodium and other trace minerals like phosphorus, potassium, selenium, calcium and magnesium (vinod& nagaraju, 2015). phytoconstituents like luteolin, kaempferol, apigenin, catechins, myricetin, biochanin a, baicalein, quercetin, phenolic acids, vanillic acid, akuammidine, are found to be responsible for the antibacterial and antimicrobial activity of t. procumbens plant leaves (ikewuchi et al., 2015). many studies have proved the antibacterial and antifungal potential of t. procumbens against human skin pathogens. according to policegoudra et al., (2014) the methanolic extract has shown high antifungal activity against human skin pathogens like microsporum fulvum, m. gypseum, trichophyton mentagrophytes, t. rubrum, trichosporon beigelii. bharathi et al. (2012) reported the significant antibacterial activity of t. procumbens against staphylococcus aureus, klebsiella pneumoniae, salmonella typhi and escherichia coli. according to jain et al. (2014), methanolic extract of t. procumbens leaf exhibited a significant zone of inhibition against both fusarium oxysporum and trichoderma reesei. kale & dhake, (2013) investigated the antimicrobial activity of t. procumbens against five bacterial pathogens s. aureus, e. page 3 coli, k. pneumoniae, proteus vulgaris and pseudomonas aeruginosa and reported that the methanolic extract had shown effective antimicrobial activity. t. procumbens also exhibited anti-malarial activity when tested against chloroquine resistant plasmodium falciparum using 3h – hypoxanthine assay. the results show that along with rbc protective effects the extracts of t. procumbens also inhibited the growth of the chloroquine resistant p. falciparum parasites (appiah-opong et al., 2011). the leaves exhibited anti-arthritic activity in complete freud’s adjuvant (cfa) induced arthritis in rats. it reduced the swelling of the rat paws and migration of leukocytes into the inflamed area (jain et al., 2012). the aerial parts had shown hepatoprotective activity against d galactosamine/lipopolysaccharide(d-galn/lps) induced hepatitis in rats. the hepatoprotective action may be mediated through the inhibition of udp-sugar derivatives, enhancement of glycoprotein biosynthesis and stabilisation of cell membrane and inhibition of lipid accumulation by its hypolipidemic property. the hepatoprotective property of the extract may be attributed to the presence of flavonoids (vilwanathan et al., 2005). the ethanolic extract of leaves had shown wound healing properties in streptozotocin induced diabetic and nondiabetic laboratory animals. in the excision model, animals treated with 2.5 % and 5% w/w plant extract showed significant results in wound contraction, epithelization period, and wound index (shrivastav et al., 2020). the hydro alcoholic extract of t. procumbens exhibited cardioprotective activity against isoproterenol induced myocardial infarcted rats (vadivelan et al., 2004). the acetone and ethanol extract of t. procumbens show anti-cancer activity on a549 (human lung cancer cell line), hep g2 (human liver carcinoma cell line) when tested using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (mtt) assay, and trypan blue dye exclusion assay method (vishnu priya & srinivasa rao, 2015). the t. procumbens also exhibited immunomodulatory activity (tiwari et al., 2004), anti-hyperuricemia and antioxidant (andriana et al., 2019). the leaves exhibited anti-inflammatory and analgesic activity when tested with formalin, acetic acid and cfa induced pain models in male c57bl6/j mice and male sprague‐dawley rats (prabhu et al., 2011). tridax procumbens leaves have good antimicrobial properties. this antimicrobial potential may help in treating the skin infections caused by certain types of microorganisms. so this study involves formulation and evaluation of herbal soap using tridax procumbens leaves methanolic extract. methodology collection of materials the t. procumbens plants were collected from the herbal garden of sri krishnadevaraya university college of pharmaceutical sciences, ananthapuramu, andhra pradesh, india. the collected plant was authenticated as t. procumbens (herbarium number 57417) by botanist prof b. ravi prasad rao. the leaves were separated from the plant material. the collected leaves were washed with water and shade dried for 5 days and ground into a fine powder using a mixer grinder. the glycerin soap base is brought from ghanshyam enterprises. vitamin e oil is purchased from a local pharmacy. coconut oil, tulasi oil, and honey of different brands were purchased from the local market. preparation of plant extract the dried leaf powder was used for the extraction of phytoconstituents. the powdered plant leaves were stored in an airtight container and the powder was extracted using methanol as solvent by the soxhlet extraction method. t. procumbens dried leaf powder is weighed accurately and packed in a filter paper. the solid matrix is kept in the soxhlet evaporator and the solvent is heated in the process of reflux. to 40 g of dried leaf powder 400 ml of methanol is used in the extraction process. continuous extraction was done and solvent was transferred into the reservoir from the chamber. this process is continued for 8hrs and the extract is collected and concentrated using a hot water bath. final concentrated extract is used in the formulation. formulation of herbal soap the glassware is sterilised by dry heat sterilisation technique. 100g of glycerin soap base was weighed and melted. the glycerin soap base is prepared using coconut oil and sodium hydroxide, it is alcohol-free. in another beaker plant extract (as per formulation design), vitamin e oil and other ingredients aloe vera gel, coconut oil, honey (as per the formulation design) were mixed until all the ingredients dissolve completely. 1ml of tulasi essential oil is added for the fragrance to the mixture. finally, the plant extract mixture is incorporated into the melted soap base. this mixture is poured into moulds and allowed to solidify at room temperature. four formulations were prepared. the formulation design for soaps were given in table 1. phytochemical analysis of plant extract 1. test for flavonoids alkaline reagent test: few drops of 10 % naoh solution were added to 2 3 ml of extract in a test tube. formation of intense yellow colour that becomes colourless in addition to dilute hcl indicates presence of flavonoids (shah & hossain, 2014). page 4 table 1: formulation designs for soaps. ingredients f1 f2 f3 f4 plant extract 0.5g 1.0g 1.5g 2.0g soap base 100g 100g 100g 100g vitamin e 400mg 400mg 400mg 400mg tulasi oil 0.1 ml 0.1ml 0.1 ml 0.1 ml aloe vera gel 10g honey 5g coconut oil 10g total weight 105.9g 111.4g 101.9g 112.4g (means that the ingredient is not used in the formulation) 2. test for phenols 0.5 ml of alcoholic ferric chloride (fecl3) solution was added to 2 ml of extract. formation of intense bluish black colour in addition of fecl3 solution indicates presence of phenols (kushwaha et al., 2019). 3. test for tannins gelatin test: gelatin solution was prepared by dissolving gelatin powder in water by heating using a water bath. to this gelatin solution 2 ml extract was added. presence of tannins is indicated by formation of white precipitate (kushwaha et al., 2019). 4. test for alkaloids iodine test: few drops of dilute iodine solution is added into 3 ml of test solution. formation of blue colour which disappears on boiling and reappears on cooling indicates presence of alkaloids (kushwaha et al., 2019). 5. test for saponins foam test: the extract was diluted with 20 ml of distilled water and shaken for 15 min in a graduated cylinder. formation of foam layer indicates presence of saponins (hossain et al., 2013). 6. test for steroids 2 ml acetic anhydride was added to 0.5 g of methanol extract. to this 2 ml h2so4 was added. colour change from violet to blue indicates presence of steroids (kushwaha et al., 2019). evaluation tests 1. examination of physical properties of formulated soap colour and clarity were checked by eye against white background and odour is observed. these properties were examined in all the four formulations 2. determination of ph 5 g of soap is dissolved in 100 ml of water. the ph of the soap solution was determined using a digital ph metre (systronics digital ph metre mk vi). ph for four formulations was determined separately. 3. determination of percentage free alkali 10g of sample soap was weighed using digital weighing balance (essae weighing balance, modelds-852g) and taken into a beaker, 150 ml of purified water was added and boiled for 30 minutes under reflux in a water bath (sisco water bath). the volume was made up to 250 ml in a beaker. 1ml of phenolphthalein indicator was added. it was titrated immediately with 0.1 m hcl until the solution turns colourless (mohammed haneefa et al., 2019). 4. determination of foam height 0.5 g soap sample was dispersed in 25 ml purified water. it is transmitted into a 100 ml measuring cylinder and volume was made up to 50 ml with water. 25 strokes were given. it is allowed to stand till the aqueous volume is measured up to 50 ml. foam height above the aqueous volume was measured (ahmed et al., 2021). 5. determination of foam retention 1% soap solution was prepared. 25 ml of 1% soap solution was taken in a 100 ml graduated measuring cylinder. the cylinder was covered and shaken 10 times. the time taken for the foam to disappear was recorded (ahmed et al., 2021). 6. determination of alcohol insoluble matter alcohol insoluble matter comprises most of the alkaline salts, such as talc, carbonates, borates, silicates and phosphates, as well as sulphates and starch, which are insoluble in alcohol under the test conditions. 5g of soap page 5 sample was taken in a conical flask to which 50 ml of warm ethanol was added and it was shaken vigorously until the sample was dissolved completely. the solution was filtered through a tared filter paper along with 20 ml warm ethanol and dried at 1050 c for 1 hour. the weight of the dried paper was noted. (mohammed haneefa et al., 2019). % 𝐀𝐥𝐜𝐨𝐡𝐨𝐥 𝐢𝐧𝐬𝐨𝐥𝐮𝐛𝐥𝐞 𝐦𝐚𝐭𝐭𝐞𝐫 = 𝐖𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐫𝐞𝐬𝐢𝐝𝐮𝐞 × 𝟏𝟎𝟎 𝐖𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐬𝐚𝐦𝐩𝐥𝐞 (𝟏) 6. moisture content the moisture content is used to estimate the percentage of water present in the soap. to estimate the moisture content 5g of soap was weighed and noted as wet weight or initial weight. using a hot air oven sample was dried at 100 to 1150c for one hour. the sample was cooled, weighed. this weight is recorded as the dry weight of the sample. moisture content was determined using the below formula. (mohammed haneefa et al., 2019). % 𝐦𝐨𝐢𝐬𝐭𝐮𝐫𝐞 𝐜𝐨𝐧𝐭𝐞𝐧𝐭 = 𝐈𝐧𝐢𝐭𝐢𝐚𝐥 − 𝐅𝐢𝐧𝐚𝐥 𝐰𝐞𝐢𝐠𝐡𝐭 𝐅𝐢𝐧𝐚𝐥 𝐰𝐞𝐢𝐠𝐡𝐭 × 𝟏𝟎𝟎 (𝟐) results the formulated soaps were examined for physical parameters like colour, clarity, and odour. evaluation parameters like ph, percentage free alkali, foam height, foam retention, and alcohol insoluble matter, percentage of moisture content. the results for phytochemical analysis were given in table 2, while the results of evaluation studies for the four formulations f1, f2, f3, f4 are given in table 3. table 2: phytochemical analysis of t. procumbens phytochemical analysis observation results test for flavonoids (alkaline reagent test) dark yellow colour was formed + test for phenol bluish black colour was observed + test for tannins (gelatin test) white precipitate was formed + test for alkaloids (iodine test) blue colour is observed and disappeared on heating + test for saponins (foam test) foam was generated on shaking + table 3: evaluation parameters for formulations evaluation parameters f1 f2 f3 f4 colour dark green #006400 dark green #006400 dark green #006400 dark green #006400 clarity good good good good odour tulasi odour tulasi odour tulasi odour tulasi odour ph 8.9 8.7 9.3 8.19 percentage free alkali 0.68% 1.28% 1% 0.88% foam height 35ml 36ml 53ml 5ml foam retention time 13 min 100min 110 min 33 min % alcohol insoluble matter 4% 16% 9.5% 10% moisture content 18% 28.4% 18.8% 23% discussion the methanolic extract of t. procumbens consists of saponins, alkaloids, tannins, phenols, flavonoids and steroids. all the soaps have a dark green colour, good clarity, and good odour. all the soaps have ph in the range of 8-9.1. so the soaps are a little basic in nature. percentage free alkali refers to the free or excess unreacted base (alkali) present in the formulation. these free bases cause irritation on human skin. among four soaps f2 has a high percentage of free alkali, f1, f3 and f4 are within the range of 0.6 1. f1 soap has the least percentage of free alkali i.e, 0.68% when compared to other formulations. matter insoluble in alcohol is a parameter used to determine the purity of soap (vivian et al., 2014). according to the bureau of indian standards, 10% of page 6 alcohol insoluble matter is the limit for toilet soaps of grade 2 and grade 3 (is 2888: 2004). f1, f3, f4 have 4%, 9.5%, 10% respectively. among all four soaps f1 has the least amount of alcohol insoluble matter i,e 4 % indicating it has the least number of impurities in it than other formulations. moisture content indicates the presence of moisture in the sample; if it is high the formulation will get deteriorated easily. high moisture content means it releases water, which reacts with unsaponified fat in the soap and causes hydrolysis of soap releasing free fatty acids and glycerol (vivian et al., 2014). among the four soaps f1, f3 have low moisture content compared to f2 and f4. foam height and foam retention time determine the foaming activity and cleaning efficacy of the soap. among the four formulations f1, f2, f3 showed good foam heights in the range of 35 to 53 ml. all four soaps had a relatively long period of foam retention. f3 soap has foam for a longer period i.e, 110 minutes. all the four soaps showed stable foam for more than 5 minutes. all four samples had shown good foaming properties. conclusion in this present study, four herbal soaps were prepared using tridax procumbens leaves extract with varying quantities of ingredients and drug. all the formulated soaps had good appearance, uniform colour, good odour. f1 has the least content of free alkali (0.68%). as free bases in soap cause irritation, these should be as low as possible. when compared to other soaps f1 has least making it good when compared to other soaps. it has least amount of matter insoluble in alcohol (4%), which is very less compared to other formulations and it is also in the acceptable limit (10%) of bis. the moisture content of the f1 soap is also less compared to other soaps, which is another parameter, which makes it better than others. f1 had shown stable foam for more than 5 min. f3 has shown stable foam than other formulations. but based on these considerations it can be concluded that f1 soap had shown better results than other soaps. naturally tridax procumbens leaves have good antimicrobial properties. the other ingredients like coconut oil, aloe vera gel, vitamin e oil, tulasi oil used were proven to be dermatologically safe and helps in providing additional benefits to skin like moisturising effect, and conditions skin. so, the potential use of the formulated soaps in treating skin infections can further be explored. herbal drugs like t. procumbens can be formulated in the form of soaps. but f1 had shown the best results among all for evaluation studies. it has good foaming property and the least number of impurities as per the standards of the bureau of indian standards. the potential use of the formulated soaps in treating skin infections can further be explored. herbal drugs like t. procumbens can be formulated in the form of toilet soaps. acknowledgements we are thankful to sri krishnadevaraya university college of pharmaceutical sciences for providing all the facilities for this study. conflict of interest the authors declare no conflict of interest. references akuaden, n.j., chindo, i.y., & ogboji, j. 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(2022) journal of pharmacy, 2(2), 59-68 page 59 wound contraction and epithelisation effects of acrostichum aureum l. in rabbits hendy putra herman1, deny susanti2,*, shahbudin saad3, muhammad taher4,5,* and norazsida ramli6 abstract introduction: rhizomes paste from acrostichum aureum linné was used traditionally by malays ethnic groups in malaysia for wound healing treatment. to evaluate wound healing properties of aqueous and ethanolic extract of a. aureum on rabbits. method: there were four treatments namely aqueous extracts of rhizomes, leaves and stems a. aureum with low and high dose (5 % and 10 %). there were four rabbits in three treatment groups with each rabbit were inflicted with excisional wounds on their back near the neck with 6 mm in diameter. topical treatment was applied once daily until complete healing with solcoseryl jelly served as positive control group and blank aqua cream served as negative control group. the percentage period of epithelisation and wound contraction were measured every 3 days interval. at day 15, all healed wound specimens were biopsied and stained with masson’s trichrome staining for histopathological study. results and discussion: from the results, all extracts from a. aureum possessed tannins and total tannin content showed that ethanolic extracts had higher total tannin content compared to aqueous extracts. based on percentage wound contraction and epithelisation period, the treatment with 5 % aqueous extracts of leaves a. aureum was the most effective wound healing agent in enhancing higher percentage wound contraction, rapid epithelisation period, producing more collagens and fibroblasts proliferation. conclusion: it was suggested that wound healing properties of rhizomes and leaves a. aureum was contributed by its high total tannin content. this finding would be able to justify its traditional claim as wound healing treatment by malays communities in malaysia. article history: received: 3 january 2022 accepted: 22 july 2022 published: 31 july 2022 keywords: excisional wound, mangrove, wound healing, tannin content. how to cite this article: herman, h. p., susanti, d., saad, s., taher, m., & ramli, n. wound contraction and epithelisation effects of acrostichum aureum l. in rabbits. journal of pharmacy, 2(2), 59-68. doi: 10.31436/jop.v2i2.121 authors’ affiliation: 1 universiti teknologi petronas, 32610 seri iskandar, perak darul ridzuan, malaysia. 2 department of chemistry, kulliyyah of science, international islamic university malaysia, 25200 kuantan, pahang, malaysia. 3 institute of oceanography and maritime studies, kulliyyah of science, international islamic university malaysia, 25200, kuantan, pahang, malaysia. 4 department of pharmaceutical technology, kulliyyah of pharmacy, international islamic university malaysia, 25200 kuantan, pahang, malaysia. 5 pharmaceutics and translational research group, kulliyyah of pharmacy, international islamic university malaysia, 25200 kuantan, pahang, malaysia 6 department of biomedical science, kulliyyah of allied health science, international islamic university malaysia, 25200, kuantan, pahang, malaysia. *corresponding author: email address: deny@iium.edu.my; mtaher@iium.edu.my mailto:deny@iium.edu.my herman et al. (2022) journal of pharmacy, 2(2), 59-68 page 60 introduction malaysia is one of the twelve megadiversity countries in the world, with 1100 species of ferns and fern allies found in its rainforests and mangroves (ministry of science, environment, and technology, 1998). from these large families, pteridaceae is among prominent family found worldwide with 50 genera and 950 species (smith et al., 2008). example of ferns in pteridaceae family that used ethnomedicinally is acrostichum aureum (bandaranayake, 1998). acrostichum spp. is a large ferns that can grow up to 4 m tall with not more than 30 leaflets. giesen et al., (2006) described that a. aureum and a. speciosum sharing the same habitat, one vital characteristic that could differentiate them is blunt leaflet and elongate-pointed leaflet, respectively. these ferns are found in malaysia, indonesia, thailand, singapore, and india (kathiresan & rajendran, 2005). the methanol extract of a. aureum was reported to have phenolic sulfates which are active against sk-lu-1, hepg2, and mcf7 cell lines (mint et al., 2021). wound occurs when the continuity of tissues is interrupted. goss (1992) described that wound healing process primarily involves in three stages, namely proliferation, migration, and differentiation of cells. since people in rural area still depend on medicinal plants for wound healing treatment, it is a great advantage to further analyse its properties scientifically in order to justify its traditional claim. malays communities, which are the majority ethnic group in multi-racial malaysia, have been reported to use the rhizomes of a. aureum for wound healing treatment (bandaranayake, 1999; mannan & maridass, 2008; hossan et al., 2010). other traditional uses of this plant in this region are treatment cloudy urination and sexual stimulant in bangladesh (rahmatullah et al., 2010), anti-inflammation, malaria treatment, and antidote to poison victims in vietnam (hong & san, 1993; hout et al., 2006). the water extract of a. aureum was reported to have gastroprotective effect of ethanol-induced gastric ulcer in rats (wu et al., 2019). the phenolic compound, (+)-pinoresinol-4-o-sulfate showed a moderate activity against sk-lu-1, hepg2, and mcf7 cell lines (thi minh et al., 2022). various medicinal plants found in malaysian virgin rainforests and mangroves offer huge natural resources for a novel medicinal product. in this study, the wound healing property of a. aureum as was evaluated by in vivo model and it serves as a basis for development of the plant-based products in malaysia. based on previous preliminary study of wound scratch assay on nih/3t3 fibroblasts cell line (herman et al., 2013), there were three types of crude extracts to be tested in vivo which were aqueous extracts of rhizomes, leaves, and stems of a. aureum. materials and methods plant material the whole samples (rhizomes, stems, and leaves) of a. aureum were obtained from matang mangroves, perak in february 2011. the plant was authenticated by dr. shahbudin saad from the department of biotechnology, kulliyyah of science with herbarium specimen (mt 10119) was kept in the herbarium kulliyyah of pharmacy, international islamic university malaysia. ethanolic extracts the leaves, rhizomes and stems of the plants were dried in the oven at 30 °c for 7 days. then, the samples were powdered using a mechanical grinder and 200 g of samples were for extraction purposes. each sample was extracted with ethanol (90%) using a soxhlet extractor and dried under reduced pressure to give dark brown colour to yield rhizomes (5.27 %), stems (6.90 %) and leaves (4.05 %) ethanol extracts. the extracts were stored in a 4 °c chiller until further use. aqueous extracts the aqueous extract was prepared by maceration. 50 g of finely ground plants was macerated in 1000 ml of distilled water overnight in a 40 °c water bath. the distilled water was added in ratio 1: 20 (mahmood & phipps, 2006). after cooling, the extract was filtered by whatman no. 1 filter paper and filter tunnel. the whole process was repeated three times to obtain a maximal extraction. then, it was subject to a freeze drying process to produce yellowish powder extracts. yield of extract for a. aureum’s rhizomes was 6.44 %, stems (9.64 %) and leaves (6.76 %). the extracts were stored in a 4 °c chiller for further testing. total tannin content the extract (0.1 ml) was added with 7.5 ml of distilled water. then 0.5 ml of folin-ciocalteu phenol reagent, 1 ml of 35 % na2co3 solution and 10 ml of distilled water were added. the mixture was mixed well and stored for 30 min at room temperature. the absorbance was measured at 725 nm with a uv/visible spectrophotometer. a set of standard solutions of tannic acid was read against the blank. the results were expressed as tannic acid in mg/g of extract. total tannin content was measured as mg of tannic acid equivalent per gram using the equation which was obtained from a standard tannic acid calibration curve (haile and kang, 2019). excisional wound study 12 male, new zealand rabbits (2.5+/0.5 kg)) were herman et al. (2022) journal of pharmacy, 2(2), 59-68 page 61 purchased from a supplier in seri kembangan, selangor and were acclimatized for least two weeks in an animal room. they were fed on a standard pellet diet (bendera®) with fresh carrots and water ad libitum throughout the experiment. for each group, there were four individual rabbits used. this study was approved by iium research ethics committee (irec) meeting no 3/2011 on 5th december 2011. preparation of wound this animal study was used to monitor periods of epithelisation and wound contraction. the excisional wounds were inflicted according to previous methods (ravishankar et al., 2018; xie et al. (2002). each rabbit was anaesthetized intraperitoneally using a cocktail of ketamine/ xylazine (22 and 2.5 mg/kg, respectively). the drugs prevent any movement of the animals at least for 2 hours after the administration of the anaesthetic solution. first of all, the hair on that wounding area was removed by shaving the dorsal back of the rabbit. then, the circular wound on the dorsal interscapular region of each animal sized 6 mm biopsy was left open. all wounds were treated once daily until the wound completely healed. the healing progress in the wound area was monitored with transparent graph paper with an accuracy of 1/20 mm. wound areas were also monitored with a camera on day 0, 3, 6, 9, 12, and 15. topical wound application aqua cream was purchased from upha pharmaceutical manufacturing (m) sdn containing white soft paraffin 12 % w/w, emulsifying wax 8 % w/w, and liquid paraffin 8 % w/w. each extract was homogenized with 1 g of aqua cream according to the designed concentration. solcoseryl® jelly was purchased from legacy pharmaceuticals switzerland gmbh. for treatment 1 group, wounds were treated with positive, negative control, 5 % and 10 % of aqueous extracts of rhizomes a. aureum. for treatment 2 group, wounds were treated with 5 % and 10 % of aqueous extracts of stems a. aureum and control while for treatment 3 group, wounds were treated with 5 % and 10 % of aqueous extracts of leaves a. aureum and control. for negative control, 1 g of blank aqua cream was applied and for positive control, 1 g of solcoseryl® jelly was applied once daily. the wound contraction was evaluated as follows (herman et al., 2013): wound contraction (%) = (wdo-wdt) x 100 wdo where: wdo = the wound diameter on day zero wdt = the wound diameter on day t histopathological study at the last day of the experiment (day 15), the crosssectional of full-thickness skin specimens from each group were histopathologically evaluated. skin samples were fixed in bouin’s solution before being processed and blocked with paraffin and then sectioned into 5 μm sections and stained with modified masson’s trichrome staining (mt). the method of staining was done following from suvik & effendy (2012). firstly, the skin tissue slides was deparaffinised by submerging into three series of absolute xylene followed by 100 %, 95 %, 90 %, 80 %, and 70 % of ethanol for 4 minutes, respectively. the slides were soaked in warmed bouin’s solution for 45 minutes at 60 °c and the slides were rinsed in running tap water until yellow colour in samples disappeared. then, in order to differentiate nuclei, slides were immersed in modified weigert’s haematoxylin for 8 minutes, then washed in running tap water for 2 minutes. to stain erythrocytes and cytoplasms, slides were submerged in acid fuchsin for 5 minutes, then again washed with running tap water for 2 minutes. next, the slides were treated with a solution of phosphomolybidic acid for another 10 mins and immediately submerged into a methyl blue solution for 5 minutes for staining. after that, slides were washed in running tap water for 2 minutes and lastly treated with 1 % acetic acid solution for 1 minute. finally, slides were dehydrated into 70 %, 80 %, 95 %, and 99 % ethanol for 1 minute each percentage. before evaluation, slides were dipped into absolute xylene for 1 minute and mounted with cover slip using dpx mounting. for evaluation purposes, the tissues were examined by light microscope and graded subjectively in terms of fibroblast proliferation, collagen formation, angiogenesis, and re-epithelisation process. for collagen formation, it would be graded into none, scant, moderate, or abundant. effective wound healing treatment would result in abundance of collagen. secondly, for epithelisation it would be graded into none, partial, thin complete, and mature complete epithelized cells. a good wound healing process would result in thick and mature epithelized cells as no more dead tissue would be accumulated on the healed wound. lastly, fibroblasts proliferation also would be graded accordingly based on their proliferation rate; dominant fibroblasts showed a good wound healing process. statistical analysis statistical analysis was conducted with the statistical herman et al. (2022) journal of pharmacy, 2(2), 59-68 page 62 package for the social sciences (spss) version 16. data were expressed as the mean ± s.d. significant differences between the treated groups and the control was determined by the one-way anova test, followed by post-hoc tukey’s test, at a level of p-value < 0.05 is considered as statistically significant. results and discussion phytochemicals screening showed that all ethanolic and aqueous extracts from rhizomes, leaves, and stems a. aureum contained tannins. therefore, total tannin content was done to analyze its amount and from the results, it showed that ethanolic extracts have higher total tannin content than aqueous extracts (table 1). table 1: total tannin content of the extracts of a. aureum. type of extract average absorbance at 725 nm total tannin content etoh rhizomes a. aureum 2.14 ± 0.28 118.56 etoh stems a. aureum 3.04 ± 1.28 168.56 etoh leaves a. aureum 1.85 ± 0.01 102.44 aqueous rhizomes a. aureum 0.93 ± 0.08 51.33 aqueous stems a. aureum 1.20 ± 0.46 66.33 aqueous leaves a. aureum 1.25 ± 0.11 69.11 wound contraction was measured as a percentage of the reduction in the wounded area. it was measured every three days until day 15. from the daily wound observation (figure 1), the wound healing process occurred normally without any signs of inflammations or microbial infections that could render a normal wound healing process. on day 9, the wounds treated with 5 % and 10 % aqueous extracts of leaves of a. aureum have been fully healed compared with negative control (68.75 %). this is statistically significant (p < 0.05) and showed that leaves also could enhance the wound healing process. percentage wound contraction (%) of different extracts on exsicional wound in rabbits for three interval days. on day 9, treatment with 5 % and 10 % aqueous extracts of leaves of a. aureum shows statistically significant percentage wound contraction compared to negative control (figure 2). the epithelisation period was measured as the number of days required for the falling of the eschar (deadtissue remnants) without any residual raw wound. the results (figure 3) showed that three extracts have significant effect on epithelisation period which were 10 % aqueous extract of rhizomes a. aureum, 5 % and 10 % aqueous extract of leaves a. aureum compared to negative control. these findings were essential as a faster epithelisation period means that the treatment could enhance the wound healing process compared to normal healing without any treatment. malaysia is a promising natural resource for medicinal plants. some plants have been studied for wound healing properties such as plantago major (mahmood & phipps, 2006) and rafflesia hasseltii (mahmood et al., 2009). therefore, this study aims to add more depth in malaysian medicinal plants used for wound healing treatment. due to lack of published reports on their wound healing properties, this present study will justify the medicinal use of a. aureum rhizome extracts in wound healing. the phytochemicals screening was done for all aqueous and ethanolic extracts of a. aureum. based on our result, all extracts of a. aureum possessed tannins. the finding of tannins presence in all extracts was in conformance with previous study done by bandaranayake (2002) and hemayet et al. (2012). since the target compound in wound healing study was tannins, total tannin content was done to further analyse the quantitative amount of tannins in each part and extract from a. aureum. from the result, the highest total tannin content could be found in ethanolic extract of stems a. aureum (168.56 mg tae/ g dry extract) while the lowest was found in aqueous extract of rhizomes a. aureum which was only 51.33 mg tae/ g dry extract. tannins were known to have astringent properties that would draw and contract tissues together by precipitating protein (getie et al., 2002). ong (2004) has reported that tannins could be used to reduce inflammation, stop bleeding and heal wounds as a topical application. tannins have been targeted for years by researchers around the world for wound healing study. a study by agyare et al. (2011) has successfully isolated bioactive compounds from tannins such as furosin and geraniin that increased the proliferation rate of fibroblasts and stimulated collagen synthesis. besides, tannins also were reported to have an angiogenic effect or promote the formation of new blood vessels in wound areas (li et al., 2011). theoretically, angiogenesis starts when cell contact relaxes and outer pericytes layer is disrupted. subsequently, there will be a migration and proliferation of endothelial cells that lead to the formation of new blood vessels (moon et al., 1999). herman et al. (2022) journal of pharmacy, 2(2), 59-68 page 63 aa: a. aureum, a: solcoseryl jelly; b: 5 % rhizomes aa; c: aqua cream; d: 10 % rhizomes aa; e: control; f: 5 % stems aa; g: 10 % stems aa; i: 5 % leaves aa, j: 10 % leaves aa. figure 1: photographic observation of wound contraction every three interval days from day 0 to day 15. there were no signs of inflammations and bacterial infection throughout the experiment. . day 0 3 6 9 12 15 t re at m en t 1 t re at m en t 2 t re at m en t 3 a b c d e f e e e i j g herman et al. (2022) journal of pharmacy, 2(2), 59-68 page 64 . figure 2. percentage of wound contraction (%) of different extracts on excisional wound in rabbits for three-day interval. on day 9, treatment with 5% aqueous extracts of leaves of a. aureum and 10% aqueous extracts of leaves of a. aureum showed statistically significant percentage wound contraction compared to the negative control (positive control: solcoseryl jelly; negative control: aqua cream), *: p < 0.05 figure 3: epithelisation period vs treatment group in which 10 % rhizomes a. aureum, 5 % and 10 % leaves a. aureum showed significant faster epithelisation period compared to negative control. aa: a. aureum; *: p < 0.05. (positive control: salcoseryl jelly; negative control: aqua cream). herman et al. (2022) journal of pharmacy, 2(2), 59-68 page 65 figure 4: histopathological findings showed that treatment with 10 % aqueous extract of rhizomes a. aureum (plate a) produced complete epithelisation, moderate amount of collagens and fibroblasts while treatment with 5 % aqueous extract of leaves a. aureum (plate b) produced complete epithelisation, moderate amount of collagens and predominat fibroblasts. plate c was positive treatment with solcoseryl® jelly while plate d was negative treatment with aqua cream. herman et al. (2022) journal of pharmacy, 2(2), 59-68 page 66 based on the result, on day 9, treatment with 5 % and 10 % aqueous extracts of leaves of a. aureum shows statistically significant percentage wound contraction compared to negative control. meanwhile, epithelisation period was reduced in three groups which were 10 % aqueous extract of rhizomes a. aureum, 5 % and 10 % aqueous extract of leaves a. aureum compared to negative control (10.75 ± 0.96 days). the fastest epithelisation period among them was treatment with 5 % aqueous extract of leaves a. aureum (8.00 ± 0.00 days) followed by 10 % aqueous extract of leaves a. aureum (9 days) and 10 % aqueous extract of rhizomes a. aureum (10 days). different concentration has given different epithelisation period thus it was important to study both low and high dose to obtain the best concentration (figure 4). fibroblasts proliferation was one of the parameters measured in the histopathological study. from the photomicrographs, the appearance of fibroblasts could be easily recognised by large nucleoli indicating active protein synthesis, extensive and purple-stained cytoplasm, and appearance of the granular (young et al., 2006). of all photomicrographs, the most abundant fibroblasts could be seen in skin specimens treated with 5 % and 10 % of aqueous extract of leaves a. aureum. this is solid evidence of their effectiveness as a wound healing agent compared to negative and positive control. based on the scoring method of histopathological slide, it has shown that aqua cream as a negative control asserted minimal effect to the skin regeneration process with poor proliferation of collagen, fibroblast, and macrophage. it was different when the wound was treated with 10 % aqueous extract of rhizomes and leaves a. aureum that showed better and more production of collagens, fibroblasts, and macrophages. the epithelial cells also were completely formed and it demonstrated that the wound has undergone complete recovery and regeneration of wounded cells. collagens as the largest component of the extracellular matrix are responsible for more tensile strength and support. type 1 collagen is the most abundant collagen normally enhancing wound healing process by promoting keratinocyte attachment and migration (ling et al., 2011). this is similar with a study done by ionita et al. (2009) that an intense collagen fibre was found throughout the dermal thickness due to the fibroblasts stimulation enhanced by the extract treatment. besides, another parameter to be observed is a sign of inflammation in the dermis layer because a good treatment would prevent inflammation (moghbel et al., 2005). therefore, the aqueous extract of leaves and rhizomes a. aureum have been concluded to be a powerful wound healing agent through high percentage wound contraction, rapid epithelisation period, and increasing of fibroblasts and collagen proliferation and building faster epithelised cells. conclusion this study has proven the wound healing properties of aqueous extract of rhizomes and leaves a. aureum by accelerating wound contraction and epithelisation period significantly compared to negative and positive control. this finding provided significant support for the use of medicinal plants among the malays people. further isolation of pure compounds responsible for wound healing properties need to be done before this plant could be commercialized as a new wound healing agent in malaysia. conflict of interest the authors declare no conflict of interests. references agyare c, lechtenberg m, deters a, petereit f, hensel a. 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(2002). the effect of moist exposed burn ointment on maintaining a physiological moist environment in treating burn wound. ann burns fire disasters, 15(3), 102-124. young b, lowe js, stevens a, heath jw, (2006). wheater’s functional histology; a text and colour atlas. philadephia: churchill livingstone. introduction malaysia is one of the twelve megadiversity countries in the world, with 1100 species of ferns and fern allies found in its rainforests and mangroves (ministry of science, environment, and technology, 1998). from these large families, pteridaceae is ... wound occurs when the continuity of tissues is interrupted. goss (1992) described that wound healing process primarily involves in three stages, namely proliferation, migration, and differentiation of cells. since people in rural area still depend on... various medicinal plants found in malaysian virgin rainforests and mangroves offer huge natural resources for a novel medicinal product. in this study, the wound healing property of a. aureum as was evaluated by in vivo model and it serves as a basis ... materials and methods the whole samples (rhizomes, stems, and leaves) of a. aureum were obtained from matang mangroves, perak in february 2011. the plant was authenticated by dr. shahbudin saad from the department of biotechnology, kulliyyah of science with herbarium speci... ethanolic extracts the leaves, rhizomes and stems of the plants were dried in the oven at 30 c for 7 days. then, the samples were powdered using a mechanical grinder and 200 g of samples were for extraction purposes. each sample was extracted with ethanol (90%) using a... aqueous extracts aqua cream was purchased from upha pharmaceutical manufacturing (m) sdn containing white soft paraffin 12 % w/w, emulsifying wax 8 % w/w, and liquid paraffin 8 % w/w. each extract was homogenized with 1 g of aqua cream according to the designed concen... for treatment 1 group, wounds were treated with positive, negative control, 5 % and 10 % of aqueous extracts of rhizomes a. aureum. for treatment 2 group, wounds were treated with 5 % and 10 % of aqueous extracts of stems a. aureum and control while f... wound contraction (%) = (wdo-wdt) x 100 wdo where: wdo = the wound diameter on day zero wdt = the wound diameter on day t histopathological study at the last day of the experiment (day 15), the cross-sectional of full-thickness skin specimens from each group were histopathologically evaluated. skin samples were fixed in bouin’s solution before being processed and blocked with paraffin and then ... the slides were soaked in warmed bouin’s solution for 45 minutes at 60 c and the slides were rinsed in running tap water until yellow colour in samples disappeared. then, in order to differentiate nuclei, slides were immersed in modified weigert’s ha... for evaluation purposes, the tissues were examined by light microscope and graded subjectively in terms of fibroblast proliferation, collagen formation, angiogenesis, and re-epithelisation process. for collagen formation, it would be graded into none,... statistical analysis statistical analysis was conducted with the statistical package for the social sciences (spss) version 16. data were expressed as the mean ± s.d. significant differences between the treated groups and the control was determined by the one-way anova te... conclusion this study has proven the wound healing properties of aqueous extract of rhizomes and leaves a. aureum by accelerating wound contraction and epithelisation period significantly compared to negative and positive control. this finding provided significa... conflict of interest the authors declare no conflict of interests. references conclusion this study has proven the wound healing properties of aqueous extract of rhizomes and leaves a. aureum by accelerating wound contraction and epithelisation period significantly compared to negative and positive control. this finding provided significa... conflict of interest the authors declare no conflict of interests. references barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework abu hanifah et al. (2021) journal of pharmacy, 1(2), 97-105 page 97 religion, spirituality and patient counselling: a scoping review nabihah abu hanifah1*, husna kauthar ramlan1, adhwa diyanah mohammad za’aim1, anis suzanna nor azmi1, faizah hanani ahmad azam1, noor amirah zahari1, nur aisyah maslil1 and syahrir zaini1 abstract introduction: religion and spirituality (r/s) have immense potential in influencing the effectiveness of patient counselling in the healthcare setting. although substantial literature has studied the relation of religion, spirituality and health, a limited number of studies have investigated the effect of religious/spiritual practices in patient counselling. this study aims to review available qualitative research on the outcome of practising religion and spirituality in patient counselling through scoping review. method: this scoping review collects the studies published from the year 2010 until 2020 written in english that were retrieved from pubmed and scopus databases. additional articles were retrieved from the google scholar through manual search. synonyms and varied spelling were included in the search keywords to account for differences in spelling and word use in the united states and the united kingdom. this review focus on the article’s presence with religion, spirituality, and therapeutic components. the methodology of this review was based on joanna briggs institute (jbi). the abstract from 1162 articles retrieved from database search was screened and unrelated paper were excluded. remaining 34 papers went through full-text screening and ten articles were selected. the thematic analysis was employed as the analytic method. results: five main themes that were produced from the thematic analysis of included studies which are “conventional medical counselling”, “association of religion and spirituality with medical counselling”, “components of spiritual counselling”, “challenges in applying spiritual care,” and “recommendations to spiritual counselling”. conclusion: the results from this scoping review can give some idea to health practitioners in improving the quality of patient counselling in healthcare setting. by incorporating religion and spirituality aspects in the patient counselling, it can improve patient’s understanding and adherence to their medication. this will result in achieving the desired pharmacotherapy outcomes and treatment goals. article history: received: 14 february 2021 accepted: 14 june 2021 published: 31 july 2021 keywords: religion, spiritual, medication counselling, patient counselling, adherence. how to cite this article: abu hanifah, n., ramlan, h. k., mohammad za’aim, a. d., nor azmi, a. s., ahmad azam, f. h. zahari, n. a. maslil, n. a. & zaini, s. (2021). religion, spirituality, and patient counselling: a scoping review. journal of pharmacy, 1(2), 97-105. doi: 10.31436/jop.v1i2.71 authors’ affiliation: 1 department of pharmacy practice, kulliyyah of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia. *corresponding author: email address: nbihahabuzai@gmail.com abu hanifah et al. (2021) journal of pharmacy, 1(2), 97-105 page 98 introduction researchers depict that religion and spirituality are among the vital components in medical treatment and services especially to those with severe disease (puchalski, 2007). the association of religiosity and spirituality will lead to significant medical services where it can improve patient care and outcome. there are concerns regarding religion and spirituality in clinical practice where a patient's belief may interfere with the medical decision which will influence the compliance of the treatment, mostly among patients who have a serious medical illness. the prevalence of drugrelated problems is a growing concern as it interferes with the treatment goals of medication therapy. medication nonadherence causes serious issues in the pharmacotherapy of the disease especially in treating chronic illness. untreated and uncontrolled disease will cause patients to have a risk of developing complications with a severe condition. thus, proper patient counselling practice is crucial in achieving desired pharmacotherapy outcomes considering the patients’ clinical needs. patient counselling can be defined as the process of providing information, advice and assistance to help patients to use their medications appropriately on direction of use, advice on side effects, storage, diet and lifestyle modifications (pilnick, 2003). there are studies found that medication non-adherence has been associated with lifestyle and health behaviour of the patients (gombojav et al., 2011). as religion and spirituality are the main components in influencing lifestyle and health behaviour of an individual, association of both in medication counselling will have a major role in affecting the patient's medication adherence. non-adherence due to spiritual belief may be resolved if all health care professionals including pharmacists are attentive to the patient’s spiritual and religious needs. it can be done by considering the religion or spiritual intervention in the medication counselling (koenig, 2012). it is crucial for medical practitioners to be concerned and includes the aspect of religion and spirituality in the medication counselling in order to optimize medication goals (kretchy et al., 2013). although the association of religion and spirituality has been broadly studied, the incorporation of these aspects in clinical practice has yet to be done. thus, mental health professionals such as psychologists, counsellors, clinicians and therapists have a lack of the ability to assess a patient’s spiritual belief. despite the abundant studies on the outcome of religiosity and spirituality in health, research in medication counselling on this issue is insubstantial. the finding of religious belief and spirituality association in health and their impact on medication counselling has been overlooked. in order to rationalize and justify the results found in the literature research, this study aimed to conduct a scoping review to study how religion and spirituality could affect patient counselling in various healthcare settings, including hospital, clinic, community pharmacy and other related facilities by anticipating some positive and negative effects of the religion and spirituality in patient counselling. the health outcomes could be either the disease progression gets worse or better. the objectives of this study are to determine the components of religion and spirituality-based counselling and to investigate the methods to improve the protocol for religion and spiritual-based patient counselling. this is important in order to provide effective counselling to the patient to improve the acceptance of the patient to undergo the treatment or receive the regimen medication. besides, this study aim is also to identify the challenges to have religion and spirituality in patient counselling. methodology ethic committee approval is not applicable as there is no involvement of human and animals. the keywords were searched through two online databases which are pubmed and scopus. important keywords were listed out to gather as much as possible relevant studies related to our review. synonyms and variable spelling were included in the keywords to cover different spelling and word usage in united states and united kingdom. the keywords are shown in (table 1). the results from the database searching were limited to studies from 2010 until 2020 only. besides database searching, manual search was done by retrieving articles from google scholar platform. table 1: search keywords for database searching religion; religious; religiosity; religiousness; god; prayer and spiritual; spirituality and medication counselling; medication review; patient counselling; drug counselling; discharge counselling; adherence counselling; counseling journals were the only source being included in this review. various types of research papers were included except for quantitative papers. presence of religion, spirituality and counselling components were extremely important as they are our focus for this scoping review. only english papers were selected for this review. in the stage of full-text screening, stringent criteria were made to make sure their content matches with our study interest. the studies should include the impact of religion and spirituality on patients’ health conditions. healthcare setting was important as the purpose of this study is to correlate religion, spirituality with patient counselling. the counselling was restricted to those provided by healthcare providers. only published and full-text papers were included in this review. abu hanifah et al. (2021) journal of pharmacy, 1(2), 97-105 page 99 this review was guided by the joanna briggs institute (jbi) methodology for jbi scoping review (peters et al., 2020). the database search resulted in 311 abstracts from pubmed and 851 abstracts from scopus. the total abstracts retrieved from both online databases are 1162 abstracts. three articles were obtained from manual search through google scholar platform. duplications were removed using mendeley software resulting in 999 abstracts left for title and abstract screening. 988 articles were excluded from the screening. the reasons were either the titles and abstracts were not in english, quantitative studies or does not have religion, spiritual and components. remaining 34 papers went through full-text screening to assess their eligibility. both screening stages were done based on the inclusion criteria that have been decided to ensure only relevant studies were included in our review. two authors, fhaa and naz were responsible for screening the articles. any disagreement was resolved by a third author, sz. after the meticulous selection process, ten articles were selected to be integrated into our review (figure 1). the analysis approach used in this review is thematic analysis (braun & clarke, 2006). then, analytical themes were created by analysing and comprehending all themes from the included studies. the generated themes are stated in table 2. results conventional medical counselling patient counselling or also known as patient education is a famous intervention by nurses in medical settings intended for many treatment purposes. patient education is found to be very important in managing the symptoms like nausea and vomiting experienced by patients throughout the cancer treatment course. this counselling also can provide more information to patients and caregivers regarding the pharmacology and non-pharmacologic therapy that can minimize the fatigue due to cancer therapy (sadruddin et al., 2017). counselling also can help the patient emotionally by managing the psychosocial issue related with the deadly nature of cancer and its anticancer therapy which can result in misery, fear, excessive worry, sleep apnea, anxiety as well as decreasing quality of life. thus, this counselling can minimize distress in patients with cancer by enhancing selfregulation which subsequently can lessen the emotional reactions and improving functional reactions (sadruddin et al., 2017). other than that, the healthcare professionals must educate the patients regarding their condition from the medical perspective and how medication can improve the condition. they must motivate the patient to feel more responsible in taking care of their health and giving the patient a hope to recover and change. however, patients’ compliance towards pharmacotherapy is enhanced with the goal of reducing symptom not recovery (oji, 2010). association of r/s with medical counselling religious and spirituality, as portrayed by daher et al. (2015) is believed to be the factors that can influence the psychology of a patient. religion, as well as spirituality, frequently play a crucial role in an individual's ability to pursue or seek care (daher et al., 2015). when the religious aspect was incorporated in seeking the treatment, the patients were more concerned about the medicinal ingredients. according to individual religiosity, stronger adherence and compliance towards the drug regime occur when all the ingredients contained in medicine are permissible according to their religious teaching. this will result in a better health outcome. most religious people will stop taking their medication when they have discovered a prohibited ingredient in their medication (daher et al., 2015). however, when religion and spirituality counselling is associated with the treatments, some negative consequences could happen. for example, religious beliefs can adversely affect the health of an individual by discouraging the patient to discontinue their treatment. this is because they tend to seek religious-based treatment (mueller, plevak, & rummans, 2001). this resulted in an increasing request for medication discontinuation and a higher possibility for medication non-adherence (mueller, plevak, & rummans, 2001). components of spiritual counselling components that may help the spiritual counselling to be effective on the patient include humour, spirituality and guided imaginary. one study has been reported in a research paper, humour promotes neurological and physiological benefits in the human body by muscular relaxing and increased breathing and blood circulation; it also enhances the immunity by reducing the stress hormone cortisol and boosting the levels of natural killer cells, t-lymphocytes and endorphins (sadruddin et al., 2017). the authors from that study also reported that laughter could encourage feelings of satisfaction and well-being in patients, allowing them to get away from difficult circumstances thus improve engagement in treatment, encourage open conversation between patients and health professionals, reduce anxiety and relieve stress (sadruddin et al., 2017). spiritual counselling could counsel patients on their spiritual practices to make them have better psychological well-being, as in general, life-threatening diseases bear anxiety connected to death and dying. therefore, they give rise to philosophical questions and also look for the essence and intent of life. according to the study reported by sadruddin et al. (2017), during the cancer cycle, the increase of these feelings has led to greater adoption of spiritual practices in cancer patient care. furthermore, religious traditions and rituals also assist patients to deal with lifethreatening conditions but depending on the individual and abu hanifah et al. (2021) journal of pharmacy, 1(2), 97-105 page 100 figure 1: selection process for included studies abu hanifah et al. (2021) journal of pharmacy, 1(2), 97-105 page 101 table 2: generated themes from thematic analysis main themes sample quotation conventional medical counselling “counselling provides emotional support to address the psychosocial problems associated with the life-threatening nature of cancer and its treatment, which often present as distress, fear, excessive worry, sleep difficulties and anxiety and reduce quality of life.” (p.15) “physicians, pharmacists, and nurses may collaboratively participate in medication therapy selection, provide counseling on proper medication use and adherence, monitor side effects, and assist with mechanisms to access free or discounted medications and services.” (p.7) association of religion and spirituality with medical counselling “religiosity and spirituality have been shown to be associated with decreased levels of anxiety /depression and increased levels of happiness and well-being.” (p.2) “we may thus use a variety of spiritually informed therapeutic tools that can greatly facilitate the patient’s coping ability, thus enhancing wellbeing and recovery.” (p.58) “finally, it is well known that unhealthy belief systems (eg, religious fanaticism and cults) can adversely affect health.” (p.1230) components of spiritual counselling “... found that humorous comments or jokes by fellow patients reduced anxiety” (p.16) “guided imagery is one of the most commonly used mind-diversion activities. it is based on the understanding that the mind and body are connected, and that the mind holds the power to influence the body” (p.16-17) “this also helps to divert a patient’s attention from physical or psychological symptoms. this activity helps to replace negative symptoms such as pain or anxiety with positive thoughts and emotions, including hope, peace and joy” (p.17) challenges in applying spiritual care “some authors, however, claim that the religious and spiritual concerns of patients are private and that physicians should not inquire about them.” (p.7) “some authors suggest that physicians ignore patient spirituality because they may not have the knowledge or skills to engage religiously diverse patients in meaningful discussions about their spiritual needs without offending them.” (p.7) “their reliance on divine revelation for these purposes implies that there is a high level of subjectivity in their work...can make collaboration problematic.” (p.9) recommendations to spiritual counselling “pharmacists are trained to communicate effectively so that they are able to elicit and address patient beliefs, particularly with regards to medication use.” (p.4) “a database of medications that are halal or kosher or vegetarian would make it so much easier for everyone.” (p.13) “the application of quran verses, zikir, application of sunnah, appreciation concept and treatment process are different from one centre to another.” (p.3) abu hanifah et al. (2021) journal of pharmacy, 1(2), 97-105 page 102 their society, context, family and personal values, the use of spirituality in their life vary greatly (sadruddin et al., 2017). the last component that can be implemented in the spiritual counselling is guided imagery. one of the most widely used activities for mind-diversion is directed visualization. it is based on the knowledge that the mind and body are interconnected and that the mind could affect the body (sadruddin et al., 2017). the power of imagery is incorporated based on an individual is explained by natural imagery that stimulates feelings of relaxation and a feeling of well-being. in the initial step of guided imagery, gradual muscle relaxing movements are used. muscles are rhythmically stressed and relaxed in various areas of the body, which helps to relieve tension in those parts (sadruddin et al., 2017). this practice helps substitute optimistic feelings and emotions, including hope, peace and pleasure, for depressive symptoms such as pain or anxiety. moreover, by focusing on a patient's concentration on imaginary circumstances that are calming and peaceful, such as a stroll by the seashore, optimistic thoughts are stimulated (sadruddin et al., 2017). challenges in applying spiritual care spiritual care can be a fundamental component when treating patients. this is due to the multiple evidence reported the positive health outcomes towards patients with a high level of spirituality compared to those who do not. nevertheless, applying spiritual care in the hospital setting or as adjunct therapy can be very challenging due to many reasons. first, the negative behaviour of the physicians towards spirituality in the healthcare setting because they may think there is no relation between healthcare and spirituality. on top of that, some of them also do not have a religious background so it is hard for them to consult and lead the patient to have spiritual counselling. besides, there is an ethical issue which addresses religion and spirituality as a sensitive issue to the patients, hence physicians should not query about them (mueller, plevak, & rummans, 2001). another challenge in applying spiritual care in the hospital setting or as an adjunct therapy is, not all countries offer religious and spiritual services. therefore, there are no proper guidelines developed to cater spiritual care (daher et al., 2015). without a guideline, the physicians had no clue in helping patients who need spiritual care (moghimian & irajpour, 2019). besides, the study reported by daher et al. (2015) showed difficulty faced by a pharmacist in attending to the spiritual needs of the patients. this is because, most of the patients with a religious background such as muslim and jewish are more concern about medicinal ingredients which contain pork, as it is prohibited in their religion. therefore, when the patients asked the pharmacist regarding this matter, the pharmacist had to refer to the pharmaceutical manufacturer as the manufacturer did not provide enough information regarding the origin of the ingredients as the pharmaceutical industries have a little to no knowledge regarding the suitable medication for religious people. recommendation to spiritual counselling future studies will need to employ a design which permits the essential necessities of spirituality in health care at the hospital (moghimian & irajpour, 2019). besides, according to daher et al. (2015), most of the participating pharmacists highly recommended a necessity for further resources to pre-existing databases from pharmaceutical manufacturers. this is because it helps both the patient and pharmacists in choosing a suitable therapy that is in the context of religious and spiritual belief of the patients. therefore, a database of medications that could recognize the origin of medication or excipients in the formulation, would make it easier for everyone. other studies also had suggested a change in the attitudes and behaviours of the healthcare members (moghimian & irajpour, 2019). reason for this is that most of them were not familiar with the prominence and methods of spiritual care services. furthermore, a comprehensive plan of the healthcare team such as designing a specific program of spiritual care is also recommended (moghimian & irajpour, 2019). the comprehensive plans must be adapted to religiousspiritual, pastoral, psychospiritual, and supportive spiritual needs of chronic patients. as a muslim, the application of quran verses and zikr that are being conducted concurrent with the patient's treatment showed a positive output (amin et al., 2017). chaplains are also a significant origin of spiritual care to facilitate speed recovery for christians (mueller, plevak, & rummans, 2001). they may give patients support, conduct spiritual counselling and achieve sacramental needs. discussion despite vast study on the relationship between religion, spirituality and health, the concern of religion and spirituality in patient counselling has been overlooked. this scoping review will build up education and perception more precisely on the importance of religious matters in the patient counselling. it is more focused on the associations of religions and spirituality in counselling to achieve better outcomes. therefore, it helps to identify the challenges and problems in applying spiritual care in medication counselling. the results from this review can guide health practitioners to improve the quality of medication counselling in any healthcare setting. thus, the study of the advantages and disadvantages of including religion and spirituality in the patient counselling is important to fully utilize the patient clinical outcomes. abu hanifah et al. (2021) journal of pharmacy, 1(2), 97-105 page 103 conventional medical counselling by healthcare professionals is conducted to deliver knowledge to the patient regarding their health condition and medication regardless their spiritual belief. it aims to educate patients in order to enhance their understanding about the progress of their health and guide them on the uses of the medication (sadruddin et al., 2017). it is a vital intervention to ensure correct usage of medication by patient and to monitor patient compliance towards their medication. from this, healthcare professionals are able to evaluate the effectiveness of pharmacotherapy plans. providing psychotherapy through psychological methods in the pharmacotherapy can result in better health outcomes. this is because psychotherapy helps to improve patient behavioral and lifestyle changes (oji, 2010). however, the problem of non-adherence towards the medication is still occurring and it will affect the health outcomes. therefore, it is significant to identify the cause of non-adherence among patients. our findings indicate that religiosity and faith have been found to help in reducing the levels of anxiety or depression among patients. this helps in controlling the body regulation and reduce the risk of having worsened health conditions. association of religious and spirituality variables in medical counselling will improve patient adherence on the medication. it is believed that religious and spirituality influence the psychology of the patient that affected their decision to pursue or seek care (daher et al., 2015). patient perception on medical treatment is a major determining factor in their willingness to receive the treatment. some religions emphasize the importance of seeking good health, thus this influences the patient's decision to improve their health. in the ethical and religious aspects in the patient counselling, physicians will genuinely prioritize the patients' well-being (d’souza, 2007). based on the previous studies, the component of compassionate love, humour, spirituality and guided imaginary has been included in the spiritual counselling to perform effective counselling on patients (kremer et al., 2014). it helps them to cope with the disease better. besides, this study also identifies the challenges to include religion and spirituality in medication counselling. although implementing religion and spirituality may be persuasive in community, there are certainly some difficulties for the healthcare professionals to conduct the medication counselling to the patient. there are some cases that people avoid and stop to receive medication when they discover the prohibited ingredients contained in the medication. this factor can lead to non-adherence to the medication regime. other than that, practising spiritual care as adjunct therapy can be very challenging. physicians show negative behaviour towards spirituality in the healthcare setting and claim such intervention is irrelevant in the medical setting. also, there is an ethical issue which addresses religion and spirituality as a sensitive issue to them (mueller, plevak, & rummans, 2001). other than that, not all countries offer religious and spiritual services and there are no proper guidelines developed to cater spiritual care (daher et al., 2015). thus, the physicians may lack skills and training regarding spiritual care. in order to overcome the challenge facing in practicing the religious and spirituality aspect in medication counselling, we have found several recommendations to improve patient health outcomes. pharmacists should have better knowledge on the ingredients contained in the medication. it is important for the pharmacist to be able to identify medications that contain prohibited materials and suggest an alternative of medication for the patient if available. this can help to provide suitable therapy that suits patients’ spiritual belief. besides, a comprehensive plan of practicing r/s in medication counselling by the healthcare team should be constructed (moghimian & irajpour, 2019). healthcare providers responsible to provide a proper spiritualitybased healthcare and educate the patient on the importance of spiritual care (moghimian & irajpour, 2019). other than that, it is recommended to fulfil religious demands by concerning patients’ spiritual background. conclusion patients’ counselling has evolved from the conventional approach to a recent wholesome approach with the inclusion of religion and spirituality aspects. the positive outcome from this new approach can be seen with patients and healthcare providers being the groups who gained the most benefits. good health outcomes are seen in most patients with the inclusion of religion and spirituality in counselling done in the healthcare setting. compassionate love, humour and guided imagery are found to be the components of religion and spirituality-based patient counselling. some challenges to implement wholesome patients' counselling have been identified. recommendations are given to overcome such challenges or improve the existing protocol for religion and spirituality-based patient counselling. as this approach of patients’ counselling is relatively new compared to the conventional method, more research regarding this area is highly encouraged. further research is important to refine the existing protocol of patient’s counselling with the inclusion of religion and spirituality components to maximize benefits from their implementation. acknowledgements we would like to express our sincere gratitude to associate professor dr. mohamad haniki bin nik mohamed and assistant professor dr. muhammad eid akkawi, lecturers from department of pharmacy practice international islamic university malaysia (iium) for their insights and recommendation to refine our scoping review. abu hanifah et al. 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(2007). spirituality and the care of patients at the end-of-life: an essential component of care. omega: journal of death and dying, 56(1), 33–46. https://doi.org/10.2190/om.56.1.d sadruddin, s., jan, r., jabbar, a. a., nanji, k., & tharani, a. (2017). patient education and mind diversion in supportive care. british journal of nursing, 26(10), s14–s19. https://doi.org/10.12968/bjon.2017.26.10.s14 introduction methodology results discussion conclusion acknowledgements conflict of interest references barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework page 70 zakaria et al. (2021) journal of pharmacy, 1(2), 70-78 perception towards insulin therapy and factors related to insulin refusal among insulin naive type 2 diabetes mellitus patients in primary care clinics pkd lipis noralina zakaria1*, narasimman baskaran2 and wan lin lim3 abstract introduction: insulin is one of the suggested treatments to prevent and reduce long-term diabetes complications. however, due to many factors such as socio-demographic factors, many type 2 diabetes mellitus patients refuse this treatment. this study aimed to determine perception towards insulin among naive type 2 diabetes mellitus patients, to calculate the prevalence of rejecting insulin therapy, and to find out factors related to the refusal. materials and methods: this cross-sectional study involved 188 insulin naive type 2 diabetes mellitus patients attending five primary health clinics in lipis district, pahang from october to november 2017. a five-point likert scale was used to determine perception towards insulin therapy and the chi-square test was used to assess the proportion of acceptance of the therapy. simple and multiple logistic regressions were utilised to study the associated factor(s). results: mean score of 60.5 ± 8.2 pointed towards a negative appraisal of insulin therapy. embarrassment to inject in public (69.1%) and concern of frequent hypoglycaemia (52.7%) were the most common perception. nearly half of the respondents (46.3%) refused insulin therapy upon suggestion. after adjusting the variables using multiple logistic regressions, only gender (adjusted or=0.20, 95% ci=0.10-0.40, p<0.001), educational level (adjusted or=0.17, 95% ci=0.06-0.50, p=0.001), age (adjusted or=1.04, 95% ci=1.01-1.08, p=0.012) and glycated haemoglobin level (adjusted or=1.33, 95% ci=1.07-1.67, p=0.013) remained significant. conclusion: in conclusion, insulin refusal among insulin naive patients was common in lipis with an overall negative perception towards therapy. by tailoring strategies according to the patient’s factors such as gender, educational level, age, and glycated haemoglobin level, the insulin refusal rate might decrease in the future. article history: received: 30 august 2020 accept: 3 may 2021 published: 31 july 2021 keywords: insulin refusal, insulin naive, type 2 diabetes mellitus how to cite this article: noralina zakaria, narasimman baskaran and wan lin lim (2021). perception towards insulin therapy and factors related to insulin refusal among insulin naive type 2 diabetes mellitus patients in primary care clinics pkd lipis. journal of pharmacy, 1(2), 70-78. doi: 10.31436/jop.v1i2.52 authors’ affiliation: 1 perkhidmatan klinik bergerak (bas) lipis, pejabat kesihatan daerah lipis, 27200 kuala lipis, pahang. 2 klinik kesihatan sungai koyan, 27200 lipis, pahang. 3 klinik kesihatan benta, 27100 jerantut, pahang *corresponding author: email address: noralinazakaria1389@gmail.com page 71 zakaria et al. (2021) journal of pharmacy, 1(2), 70-78 introduction diabetes situation in malaysia is worrying as the trend continues to increase by year. as reported by national health and morbidity survey (nhms) in 2011, the prevalence of diabetes in malaysia showed an increment from 11.6% in 2006 to 15.6% in 2011. by the year 2020, the prevalence of diabetes among malaysian adults will be expected to increase up to 21.6% (ministry of health, 2012). meanwhile, in pahang, the nhms survey 2015 concluded that 14.8% (147676) of adults 18 years and above are diagnosed with diabetes (ministry of health, 2015). throughout the year of therapy, ß cell function in patients with diabetes will diminish, causing treating diabetes with oral glucose lowering-drugs (oglds) alone will be insufficient. thus, insulin therapy is required for optimal control of glucose level. according to the 6th edition of malaysian cpg management of type 2 diabetes mellitus (t2dm), oglds remain the mainstay treatment of t2dm in combination with lifestyle intervention and appropriate dietary regimen. insulin therapy can be initiated for all ages and at any stage of t2dm. the variety of treatment options and regimens available make it possible for better personalisation of the treatment to the patient (ministry of health, 2018). insulin therapy should be considered when good glycaemic control is not achieved or maintained despite optimal dose and number of oglds. it is also suggested as initial therapy in newly diagnosed t2dm, particularly when the level of glycated haemoglobin (hba1c) > 10% or fasting plasma glucose (fpg) > 13.0 mmol/l (ministry of health, 2018). early use of insulin has proven to have greater improvement in hba1c and fpg with lower risk or no difference in the occurrence of hypoglycaemia (westphal et al., 2006; fonseca et al., 2011). besides, many complications can be prevented with the early introduction of insulin in the management of suboptimal controlled diabetes (gerstein et al., 2006; vinik, 2007; rubino et al., 2007). the insulin refusal rate among type 2 diabetes mellitus (t2dm) patients are high even though some eventually accept the treatment. to enhance patient quality of life and to prevent complications of uncontrolled diabetes, health care providers are responsible to help patients achieve the targeted blood glucose range. however, rejection of insulin therapy is one of the main challenges when treating diabetes patients, particularly those with poor control. studies done in several countries suggested that insulin naive patient refusal rate is varied. it ranged from 70.6% in singapore, 42.5% in bangladesh to nearly 28.2% in the united states of america region (wong et al., 2011; khan et al., 2008; polonsky et al., 2004). a high refusal rate was observed in a local study which discovered 51% of participants were reluctant to start insulin therapy (azmiah et al., 2011). patient’s acceptance of insulin therapy can be influenced by many factors such as socio-demographic factors, clinical factors or psychological factors. the combination of these factors makes the initiation of insulin therapy even more challenging. older age was one of the socio-demographic factors that contributed to insulin therapy refusal (tan et al., 2015; ghadiri-anari, et al., 2013; batais et al., 2016). gender, particularly female was more likely to refuse insulin therapy (polonsky et al., 2004; azmiah et al., 2011; hassan et al., 2013; kaur et al., 2018). other socio-demographic factors that were observed in previous studies included educational backgrounds (wong et al., 2011; tan et al., 2015; batais et al., 2016), employment status (khalili et al., 2016; tan et al., 2015), ethnicity (azmiah et al., 2011; tan et al., 2015) and marital status (khalili et al., 2016). meanwhile, the long duration of having diabetes and poor control of diabetes which was determined by a high level of hba1c was known clinical factors for insulin therapy refusal that were already addressed by previous literature (tan et al., 2015). despite the efficacy and benefits of insulin have been proven in many studies, these negative thoughts of patients towards insulin are a huge barrier for them to start insulin therapy. it is important to understand the patient perception of insulin and the reasons for refusal so that better service and counselling for the patient can be provided. therefore, this study was conducted to determine perception towards insulin among insulin naive t2dm patients. this study was also done to calculate the prevalence of rejecting insulin therapy in the lipis district. other than that, this study was done to find out the factors related to insulin therapy refusal. materials and methods a cross-sectional study was conducted in five primary health clinics in lipis district, pahang malaysia, namely klinik kesihatan (kk) benta, kk padang tengku, kk sungai koyan, kk merapoh and klinik bergerak komuniti lipis for two months from october to november 2017. a self-administered questionnaire (appendix 1) was developed by following a similar local study (azmiah et al., 2011). the first part of the questionnaire included socio-demographic details. the patient’s last hba1c reading was also recorded. the second part of the questionnaire consisted of 20 items (18 negative items and 2 positive items). scored on a five-point likert scale, where strongly disagree = 1, disagree = 2, unsure = 3, agree = 4, and strongly agree = 5. for calculation of the total score, the rating of the two positive items were in reverse, with strongly disagree = 5, disagree = 4, unsure = 3, agree page 72 zakaria et al. (2021) journal of pharmacy, 1(2), 70-78 = 2, and strongly agree = 1. the total possible score for the questionnaire was 100. the higher the score indicated a more negative appraisal of insulin therapy. data are presented as means ± standard deviation (sd) (chen et al., 2011). epi info version 7.2 stac calc was used to calculate the sample size for this study – sample size and power with 95% confidence level, 74.2% prevalence of rejecting insulin therapy, 5% precision and infinite population count (tan et al., 2015). the minimum sample size required was 353 after a 20% allowance for non-responders. respondent’s inclusion criteria were those diagnosed with type ii diabetes mellitus (t2dm) with the last hba1c reading ≥ 7.0 %, took two or more oglds for at least six months and never use insulin. patients diagnosed with type i diabetes mellitus, psychiatric disease or dementia and those with dexterity impairment were excluded from the study. respondents were conveniently recruited when having follow-up for their t2dm and the consent form, which was adapted from the national medical research register (nmrr), was disseminated to the selected study subjects (appendix 2). the prevalence of insulin refusal was calculated using the formula adapted from the centres for disease control and prevention website (centres for disease control and prevention. 2012). data were analysed using statistical package for social sciences (spss) version 21.0. the chisquare test was used to assess differences in proportion between those who accepted and those who rejected insulin therapy. simple and multiple logistic regressions were utilised to study the factors related to insulin therapy refusal. a p-value of less than 0.05 was considered to be statistically significant. the study was approved by the medical research ethics committee (mrec) malaysia and registered with the national medical research registry (nmrr id: nmrr-17-1843-37560) results a total of 201 patients were approached for the study but only 188 agreed to take part, yielding a response rate of 93.5%. since a self-administered questionnaire was used as the study instrument for this study, those with low literacy refused to participate. the majority of the respondents were malay (88.3%), single/widowed (70.2%), not working/retired (73.9%) with at least secondary education (50.5%) and an almost equal number of males and female participated in the study. their mean age was 57.8 years and the average earning was rm1469.15 ± 1270.76. the mean hba1c reading was 9.17% ± 1.60%. table 1: patient’s socio-demographic data and willingness to initiate insulin therapy (n=188) total willingness n (%) willing (%) unwilling (%) 188 (100.0) 101 (53.7) 87 (46.3) age 57.8* (11.5)ⱽ 55.3* (11.0)ⱽ 60.8* (11.3)ⱽ gender male female 85 (45.2) 103 (54.8) 27 (31.8) 74 (71.8) 58 (68.2) 29 (28.2) ethnic malay non-malay 166 (88.3) 22 (11.7) 86 (51.8) 15 (68.2) 80 (48.2) 7 (31.8) marital status single/wido wed married 132 (70.2) 56 (29.8) 65 (49.2) 36 (64.3) 67 (50.8) 20 (35.7) educational level not schooling/pri mary secondary tertiary 56 (29.8) 95 (50.5) 37 (19.7) 46 (82.1) 43 (45.3) 12 (32.4) 10 (17.9) 52 (54.7) 25 (67.6) working status not working working 139 (73.9) 49 (26.1) 84 (60.4) 17 (34.7) 55 (39.6) 32 (22.7) income rm0 – rm999 rm1000 – rm1999 rm2000 – rm2999 ≥ rm3000 65 (34.6) 82 (43.6) 25 (13.3) 16 (8.5) 33 (50.8) 44 (53.7) 15 (60.0) 9 (56.2) 32 (49.2) 38 (46.3) 10 (4.0) 7 (43.8) hba1c 9.2* (1.6)ⱽ 8.8* (1.2)ⱽ 9.6* (1.6)ⱽ *mean vstandard deviation hba1c: glycated haemoglobin page 73 zakaria et al. (2021) journal of pharmacy, 1(2), 70-78 the total possible score for the questionnaire is 100 and the higher the score indicates a more negative appraisal of insulin therapy. table 2 showed the total score for both respondents’ opinions regarding self-efficacy and insulin therapy (60.5 ± 8.2) indicating the respondents’ perceptions were negative towards insulin therapy. table 2: total score for self-efficacy and opinion regarding insulin mean ± sd opinion regarding self-efficacy 30.6 ± 5.7 opinion regarding insulin 29.9 ± 4.1 total score: 60.5 ± 8.2 the top three opinions regarding self-efficacy are feeling embarrassed to inject insulin in public (69.1%), the concern that hypoglycaemia would lead to permanent irreversible damage (60.1%) and fear of pain during insulin administration (57.4%). most respondents agreed that insulin might cause frequent hypoglycaemia (52.7%). meanwhile, almost half admitted they were unsure about most of the information regarding insulin such as insulin and needles are more expensive than oral therapy (42.6%), whether insulin can be stopped once initiated (41.0%). the prevalence of insulin refusal was presented in the figure below. four of every ten patients reported unwilling to start insulin therapy (46.3%). simple logistic regression was conducted to analyse the association between insulin therapy refusal and sociodemographic data, hence deciding the factor(s) that might contribute to insulin refusal. only factors with a p-value < 0.05 were deemed to be statistically significant. six factors that fulfilled the criteria, namely age, gender, educational level, employment status, and hba1c were further investigated with multiple logistic regressions. after analysing multiple logistic regressions, only gender, educational level, age, and hba1c remained significantly associated with the tendency to accept insulin therapy. while controlling for other factors, women were 20 % more likely to accept insulin therapy, compared to male respondents (adjusted or 0.20, 95 % ci 0.10-0.40, p = <0.001). respondents who received tertiary education were 83% more likely to resist insulin therapy compared to those who received only primary education or with no formal education (adjusted or 0.17, 95 % ci 0.06-0.50, p = 0.001). age and hba1c were inversely associated with insulin therapy acceptance. for every one-year increase in age, respondents were 1.04 times more likely to reject insulin therapy (adjusted or 1.04, 95 % ci 1.01-1.08, p = 0.012). similarly, for every 1.0% increment in hba1c level, subjects were 1.33 times more reluctant to initiate insulin therapy (adjusted or 1.33, 95 % ci 1.07-1.67, p = 0.013). discussion it is important to address a patient’s perception of insulin therapy as many patients were hesitant to use insulin due to their negative beliefs about insulin injection (hassan et al., 2013). the current study found out that the respondents have a prominent negative belief towards insulin therapy (total score 60.5 ± 8.2). this finding was comparable to a previous study where their insulin naive t2dm respondents scored 62.9 ± 7.1 indicating negative perceptions towards therapy (ghadiri-anari et al., 2013). this was understandable as patients tended to worry about the unfamiliar forms of treatment and become a barrier for the patient to start the treatment. by recognizing the patient’s hurdle, psychological insulin refusal (pir) can be overcome. providing the information and introducing the possible need for insulin early in treatment can improve patient’s perceptions towards insulin in general and overcome pir (polonsky et al., 2011). the majority of respondents admitted that they feel embarrassed to inject insulin in public (69.1%). it can be inconvenient and embarrassing as insulin needs to be injected at a certain time. this was supported by a previous finding where more than half of the subjects (55.9%) expressed that injecting insulin in public was embarrassing (azmiah et al., 2011). the absence of a private area may cause them to inject too early or might entirely omit the dose and eventually resulting in hypo/hyperglycaemia (chen et al., 2011; hunt et al., 1997). however, feeling embarrassed to inject insulin in public was the least worrying reason for patients in a previous study done in kubang pasu, kedah (17.5%) (tan et al., 2015). page 74 zakaria et al. (2021) journal of pharmacy, 1(2), 70-78 table 3: opinion regarding self-efficacy no. statements mean ± sd number (%) disagree unsure agree 1 feel embarrassed to inject insulin in public 3.8 ± 1.2 34 (18.1) 24 (12.8) 130 (69.1) 2 concern that hypoglycaemia would lead to permanent irreversible damage 3.6 ± 1.0 41 (21.8) 34 (18.1) 113 (60.1) 3 fear of pain during insulin administration 3.1 ± 1.1 70 (37.3) 10 (5.3) 108 (57.4) 4 fear of injection 2.9 ± 1.3 79 (42.0) 9 (4.8) 100 (53.2) 5 injecting insulin makes one feel as if he/ she is a drug addict. 3.1 ± 1.5 71 (37.8) 21 (11.2) 96 (51.0) 6 lack of confident in injecting insulin 3.1 ± 1.2 66 (35.1) 45 (23.9) 77 (41.0) 7 unable to arrange/ adjust daily activities as needed in insulin therapy 3.0 ± 1.3 81 (43.1) 36 (19.1) 71 (37.8) 8 worry about hypoglycaemia 2.6 ± 1.4 103 (54.8) 20 (10.6) 65 (34.5) 9 unable to arrange/ adjust diet as needed in insulin therapy 2.5 ± 1.1 109 (57.9) 16 (8.5) 63 (33.5) 10 lack of time to inject insulin systematically 2.4 ± 1.3 128 (68.1) 6 (3.2) 54 (28.7) table 4: opinion regarding insulin therapy no. statements mean ± sd number (%) disagree unsure agree 1 insulin causes hypoglycaemia frequently. 3.4 ± 1.0 37 (19.7) 52 (27.7) 99 (52.7) 2 insulin contains illegal substances. 2.9 ± 1.4 79 (42.0) 31 (16.5) 78 (41.5) 3 insulin causes a lot of adverse effects in the future. 2.9 ± 1.0 75 (39.9) 45 (23.9) 68 (36.2) 4 insulin and needles are more expensive than oral hypoglycaemic agents. 3.2 ± 0.9 40 (37.2) 80 (42.6) 68 (36.2) 5 insulin leads to renal failure. 2.9 ± 1.5 75 (39.9) 51 (27.1) 62 (33) 6 insulin therapy leads to dependency. 2.8 ± 1.2 72 (38.2) 54 (28.7) 62 (33) 7* insulin prevents long-term complications of uncontrolled diabetes. 3.1 ± 1.2 66 (35.1) 72 (38.3) 50 (26.6) 8 insulin therapy cannot be stopped once initiated. 2.7 ± 0.9 78 (41.5) 77 (41.0) 33 (17.5) 9* insulin is more effective than oral hypoglycaemic agents. 3.4 ± 0.9 95 (50.5) 62 (33.0) 31 (16.5) 10 insulin leads to blindness. 2.1 ± 0.9 113 (60.1) 65 (34.6) 10 (5.3) *questions 7 and 9 are the positive questions. page 75 zakaria et al. (2021) journal of pharmacy, 1(2), 70-78 figure 1: prevalence of insulin refusal among insulin naive type 2 diabetes mellitus in public patients in primary care clinics pkd lipis (n=188; 58.2% female; 93.1% malay) fear of problematic hypoglycaemia was found to be a secondary concern in the current study as most of the patients afraid that hypoglycaemia would lead to permanent irreversible damage (60.1%) and agreed that insulin can cause hypoglycaemia frequently (53.7%). one of the contributing factors could be because most of the respondents lived alone (70.2% of patients were single/widowed) and thus intensified the fear. the dawn study addressed the fear of hypoglycaemic events as one of the patients’ factors that contribute to a poor sense of well-being and thus affecting overall diabetes outcomes (korytkowski, 2002). the result was comparable to the findings in previous studies where 59.6% and 47.8% of patients respectively admitted that they refused insulin due to the fear of problematic hypoglycaemia (azmiah et al., 2011; tan et al., 2015). in the current study, 46.3% of patients with type 2 diabetes mellitus who visited public clinics in lipis denied insulin if the treatment was given to them. this percentage was comparable with previous studies done by khan et al., (2008) and azmiah et al., (2011) as 42.5% and 50.7% of their respondents respectively unwilling to initiate the treatment. meanwhile, higher refusal rates were recorded in previous studies done in various countries ranging from 60 to 80% of refusal rate (wong et al., 2011; tan et al., 2015; 1999; kaur et al., 2018). however, polonsky et al., (2004) reported the lowest rate with 28.2% of refusal. due to geographic factor as the current study was done in a rural setting, health promotion activities to patients are restricted, causing insufficient exposure to health information. urban populations have a better acceptance of insulin treatment and better access to health information related to disease and treatment (rubino et al., 2007). the current study found out that age was inversely associated with insulin therapy acceptance. for every one-year increase in age, respondents were 1.04 more likely to reject insulin therapy (p=0.012). however, a different finding was observed in previous literature as elderly respondents were more accepting of insulin therapy (r=-0.153, p=<0.05) (rubino et al., 2007). treating the elderly with diabetes is challenging as they are at an increased risk rate of concomitant illnesses such as hypertension, renal impairment, and heart diseases. polypharmacy is also one of the concerns as patients will be more concerned regarding possible drug-drug interaction and the idea of adding insulin into the treatment will cause more worries and cause them to refuse the therapy (ministry of health, 2015). however, several previous studies found no significant difference between age groups and willingness to start insulin therapy (tan et al., 2015; ghadiri-anari, et al., 2013; batais et al., 2016). very willing 12.8% (n=24) moderately willing 9% (n=17) slightly willing 31.9% (n=60) refused 46.3% (n=87) prevalence of insulin refusal among insulin naive type 2 diabetes mellitus (n=188) page 76 zakaria et al. (2021) journal of pharmacy, 1(2), 70-78 some previous studies discovered that male patients were more likely to accept the insulin initiation which male patients were said to be naturally less fearful of injection (polonsky et al., 2004; hassan et al., 2013; kaur et al., 2018). a local study was done by azmiah et al., (2011) also found out that female patients were 2.7 times more likely to refuse insulin initiation. however, in the current study female respondents were 20% more expected to accept insulin therapy, compared to males (p= <0.001). as found in previous studies, patients with higher educational backgrounds were less likely to refuse insulin initiation. wong et al., (2011) revealed that patients with tertiary level of education were more willing to start insulin therapy. same finding was observed in local study in which patient with at least a secondary education was 55.0% less likely to refuse insulin as patients with higher education are expected to be more receptive towards knowledge on disease and insulin therapy (tan et al., 2015). however, the current study found out that respondents who received tertiary education were 83.0% more likely to resist insulin therapy (p=0.001) and supported by a previous study done in saudi arabia as those with tertiary education were 48% unwilling to initiate insulin therapy (p=0.023) (batais et al., 2016). the recent study reported hba1c level was related to the refusal as for every 1.0% increment in hba1c level subjects were 1.33 times more reluctant to initiate insulin therapy (p=0.013). similar findings were recorded in previous studies where a significant relationship was found between patient’s willingness for insulin initiation with hba1c level (azmiah et al., 2011; tan et al., 2015). a different finding was observed by wong et al., (2011) in which those who were reluctant to start the treatment were among the lower mean hba1c group. compared to the other studies, patients with hba1c >7% were recruited in the recent study. according to previous literature patient with higher hba1c tends to refuse insulin therapy more than those with lower hba1c as a higher dose of insulin initiation is needed and thus higher concern for hypoglycaemia (hayward, 1997; abraira et al., 1995). however, some limitations should be noted. the current study has a limited number of respondents as those with the last hba1c reading < 7.0% were excluded from the study. the minimum sample size required was 353 after a 20% allowance for nonresponders. however, only 188 respondents agreed to participate in the study, causing difficulty to identify significant relationships from the data. the questionnaire given was self-administered. thus, those with low literacy tended to avoid participating in the study. conclusion overall, insulin naive t2dm patients in primary care clinics in the lipis district demonstrated a high level of negative perception towards insulin therapy (mean score = 60.5 ± 8.2). almost half of them (46.3%) were reluctant to start insulin therapy when being suggested by their doctors. most patients still have negative opinions regarding insulin and self-efficacy which resulted in a great influence on their willingness to start the therapy. in the future, factors that might be associated with insulin refusal should be addressed possibly in every patient so that the refusal rate can be reduced. acknowledgements conflict of interest the authors whose names are listed above certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in the manuscripts. references abraira, c., colwell, j. a., nuttall, f. q., sawin, c. t., nagel, n. j., comstock, j. p., ... & va csdm group. 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(2007). delayed initiation of subcutaneous insulin therapy after failure of oral glucose‐lowering agents in patients with type 2 diabetes: a population‐based analysis in the uk. diabetic medicine, 24(12), 1412-1418. https://doi.org/10.1111/j.1464-5491.2007.02279.x tan, w. l., asahar, s. f., & harun, n. l. (2015). insulin page 78 zakaria et al. (2021) journal of pharmacy, 1(2), 70-78 therapy refusal among type ii diabetes mellitus patients in kubang pasu district, kedah, malaysia. singapore medical journal, 56(4), 224227. doi: 10.11622/smedj.2014170 vinik, a. (2007). advancing therapy in type 2 diabetes mellitus with early, comprehensive progression from oral agents to insulin therapy. clinical therapeutics, 29(6), 1236-1253. https://doi.org/10.1016/j.clinthera.2007.07.005 westphal, s. a., & palumbo, p. j. (2006). a case for introducing insulin early in the treatment of type 2 diabetes mellitus. insulin, 1(2), 65-69. https://doi.org/10.1016/s1557-0843(06)80012-6 wong, s., lee, j., ko, y., chong, m. f., lam, c. k., & tang, w. e. (2011). perceptions of insulin therapy amongst asian patients with diabetes in singapore. diabetic medicine, 28(2), 206-211. https://doi.org/10.1111/j.1464-5491.2010.03195.x introduction materials and methods results discussion *questions 7 and 9 are the positive questions. conclusion acknowledgements conflict of interest references barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework page 63 taufek et al. (2021) journal of pharmacy, 1(2), 63-69. reflections of pharmacy students on experiential education in providing health services to people with history of substance use disorders in malaysia nor hidayah mohd taufek1,2*, syafiqah nadiah halimi1, norny syafinaz ab rahman1,2, che suraya zin1,2 and christopher j. turner3 abstract introduction: accreditation standards for pharmacy students’ education include the requirement for experiential education (ee) and acknowledge, in building life-long learning skills, the need to develop students’ skills in analysing their practice-based performance. the goal of this qualitative study was to assess students’ ability to reflect on their experience providing face-to-face care to patients with a history of substance use disorders (phsuds). materials and methods: interviews were conducted with undergraduate pharmacy students who provided care to phsuds in an ee programme. an interview guide was used to explore students’ experiences and their perceptions regarding the challenges they encountered, changes in their attitude towards phsuds and ways to improve their ability to care for phsuds. data relevant to the study was extracted from interview transcripts, manually sorted and coded using microsoft excel and subjected to thematic analysis. results: the themes identified were 1) increased self-confidence in face-to-face interactions with phsuds 2) increased empathy towards phsuds 3) reduction in stigmatised attitudes towards phsuds 4) positive nature of the interactions with phsuds 5) appreciation for the opportunity to apply knowledge in practice 6) recognition of the need for improved communication skill training 7) recognition of the need to improve training for dealing with unexpected questions and situations. conclusion: pharmacy students demonstrated reflective skills in identifying multiple positive learning outcomes stemming from a one-day ee programme involving phsuds. in addition, they provided insights useful to faculty in curriculum planning. article history: received: 4 august 2020 accept: 6 april 2021 published: 31 july 2021 keywords: experiential education, pharmacy students, people with a history of substance use disorders, reflections. how to cite this article: nor hidayah mohd taufek, syafiqah nadiah halimi, norny syafinaz ab rahman, che suraya zin and christopher j. turner (2021). reflections of pharmacy students on experiential education in providing health services to people with history of substance use disorders in malaysia. journal of pharmacy, 1(2), 63-69. 10.31436/jop.v1i2.31 authors’ affiliation: 1 department of pharmacy practice, kulliyyah of pharmacy, international islamic university malaysia, kuantan, malaysia. 2 substance use disorder research group, kulliyyah of pharmacy, international islamic university malaysia, kuantan, malaysia. 3 retired but formerly with skaggs school of pharmacy and pharmaceutical sciences, university of colorado anschutz medical campus, aurora, colorado. *corresponding author: email address: hidayahtaufek@iium.edu.my page 64 taufek et al. (2021) journal of pharmacy, 1(2), 63-69. introduction experiential learning was defined as "the process whereby knowledge is created through the transformation of experience” (kolb, 1984). it described a process of constructing knowledge that involves four learning modes of ‘experiencing’, ‘reflecting’, ‘thinking’, and ‘acting’ components in response to the learning situation and what is being learned (kolb, 1999). pharmacy students require experiential learning exposure to improve their problem solving skills beyond didactic lectures. the evaluation of actual scenario helps to boost students’ confidence to apply critical thinking and social skills (lisko & o’dell, 2010). students must be part of a practice change and improvement process to meet the needs of society (speedie, 2006). the importance of experiential learning in the education of pharmacy students is illustrated by its inclusion in accreditation standards for pharmacy education in multiple countries (accreditation council for pharmacy education, 2016; canadian council for accreditation of pharmacy programs, 2020; general pharmaceutical council, 2021). those standards require students to gain experience working in multiple pharmacy practice settings and encourage reflective practice. pharmacists must use reflection on their practice performance to improve future performance (speedie et al., 2012). it is important that experiential learning exposes pharmacy students to all segments of society. for example, the international pharmaceutical federation has encouraged professional pharmacy associations to address barriers in the provision of care for patients suffering from substance use disorders (international pharmaceutical federation, 2018). substance abuse is stigmatised and, accordingly, any stigmatised views held by pharmacy students against patients with a history of substance use disorders (phsuds) must be addressed. malaysian data on pharmacy students’ perceptions regarding phsuds is limited to one study that reported preconceived stigma towards phsuds lessened by experiential education interactions with phsuds (mohd taufek et al., 2021). that study focused on islamic principles rather than reflective learning. the goal of this qualitative study was to assess reflective practice by malaysian pharmacy student who provided care to phsuds. materials and methods this was a qualitative study employing a phenomenological approach to explore the experience of students who provided face-to-face health screening and counselling for phsuds. the study received ethical approval from the international islamic university malaysia (iium) research ethics committee (id no. irec 2019-026). eligibility criteria for participants were pharmacy students who successfully completed the iium drug abuse course phm 3282, volunteered to be trained in providing health screening services and consented to be interviewed. phsuds were patients/clients undergoing rehabilitation treatment by the malaysian government’s national antidrug agency (nada) and the non-governmental organization drug intervention community (dic) in kuantan district, pahang, malaysia. a one-day health screening programme was conducted for 120 phsuds at the iium faculty of pharmacy in collaboration with nada and dic. the screening services delivered by students are described in table 1. twenty out of 69 students who met the entry criteria volunteered to provide services and were trained regarding technical methods, knowledge and counselling for monitoring blood pressure, blood glucose, cholesterol and carbon monoxide testing. the research study was described to students who participated in the health screening programme after its completion and those who agreed to participate in the study were interviewed. one interviewer interviewed each student in a private room in the iium faculty of pharmacy between january and may 2019 at times mutually convenient to the interviewer and each student. all students were asked the same questions relating to student demographic information; students’ descriptions of their experiences; and their perceptions regarding the challenges they encountered, changes in their attitude towards phsuds, and ways to improve their ability to care for phsuds. the total duration of the interviews was 264 minutes and ranged from 21 minutes to 37 minutes per student. the variation in length was attributable to differences between the responses of individual students and the need for additional questions to clarify students’ responses. all participants preferred to communicate in malay and the interviews were audio-taped, transcribed verbatim and translated into english. the transcripts were compared several times across both languages and checked by two other researchers to ensure the meanings were interpreted accurately. the steps taken were guided by the need for translators to share the culture and the experiences of the students interviewed and be familiar with the context of the interviews to ensure accuracy in translating conceptual equivalence and interpretive insights (regmi, naidoo, & pilkington, 2010; clark et al., 2017). field notes were made during the interview with all the participants. the names of participants were kept anonymous for privacy protection. a thematic analysis of the transcripts was undertaken using inductive approach to identify, analyse and report recurring themes relevant to the study (braune & clarke, 2006; fereday & muir-cochrane, 2006). data (relevant phrases, sentences, etc.) were copied from each transcript page 65 taufek et al. (2021) journal of pharmacy, 1(2), 63-69. to a microsoft excel 2016 spreadsheet and manually sorted to facilitate the identification and coding of recurring themes. that process was conducted independently by three researchers and differences in the identification and coding of themes were resolved through discussion and referring to the field notes. summarizing the data was done by manually scrutinising and generalising the data sets as they were relatively small. there is no established ideal sample size when using thematic analysis. most qualitative studies use the concept of saturation, the point at which no new information or themes are observed in the data. (guest, bunce & johnson, 2006; willig, 2013; fugard & potts, 2015). table 1: activities during the health programme activity description a. blood pressure (bp) screening (omron automatic blood pressure monitor hem7121) students conducted screening of blood pressure using bp monitor to phsuds and counselled regarding healthy lifestyles. a. blood glucose test (accu-chek® performa blood glucose meter and lancing device no. 100608828, accuchek® performa test strips 06454003) students did blood glucose test using the test strip for glucometer and counsel phsuds regarding target blood glucose range. c. blood cholesterol test (cardiocheck® p.a., pts panels® cholesterol test strips c907) students measured the blood cholesterol using cholesterol measuring kit for phsuds and counselled regarding healthy lifestyles. d. carbon monoxide (co) test (picotm smokerlyzer® cp715879) using smokelyzer kit, students conducted measurements to detect the level of co, interpreted the level to phsuds and assess nicotine dependence using fagerstrom test. students provided counselling and information to phsuds regarding the facilities (public/private sectors) that are available to receive quit smoking services. results ten students consented to participate in the study and a thematic analysis using inductive approach was conducted on all ten transcripts. saturation was deemed to have been reached when no new themes were identified from the final three analyses. the demographic characteristics of the interview participants are presented in table 2. all participants were young adult females in the final year of their iium pharmacy programme. table 2: demographic characteristics of participants characteristics number (n =10) a. age (years) 23-25 10 b. gender female 10 c. marital status single 10 d. race malay 10 e. education undergraduate final year 10 table 3 lists the seven major themes identified by the thematic analysis and are illustrated by quotations from the transcripts. the themes identified were 1) increased selfconfidence in face-to-face interactions with phsuds 2) increased empathy towards phsuds 3) reduction in stigmatized attitudes towards phsuds 4) positive nature of the interactions with phsuds 5) appreciation for the opportunity to apply knowledge in practice 6) recognition of the need for improved communication skill training 7) recognition of the need to improve training for dealing with unexpected questions and situations. quotations from each participant are identified by the participant number (s1-s10). page 66 taufek et al. (2021) journal of pharmacy, 1(2), 63-69. table 3: themes identified from the transcripts positive nature of the interactions with phsuds: reported by all students. “we told them that their cholesterol level was high, they asked us what they should do to control it, they seemed like more motivated during health screening” (s2) “the positive part was when they were excited to know more about the information, i showed all the pamphlets, booklets, diagrams that we had. they also shared their experiences” (s3) “the acceptance was very good...they were not aggressive, i felt comfortable meeting different people with different behaviour… there was a client who told many stories...we didn’t even ask... but he told us from a to z....about his experiences...how many cigarettes he smoked before…now he has reduced the number of cigarettes” (s5) “their acceptance was very good because when we did the tests, they were all queuing up and even when we finished the session, they still wanted to do it...they were really happy with our services, they really wanted to know about their glucose and cholesterol level…their blood pressure” (s7) “it was interesting to interact with ex-drug users because i have never done it before, i did some counselling on healthy diet, they accepted our recommendation” (s10) appreciation for the opportunity to apply knowledge in practice: reported by all students. we told them that their cholesterol level was high, they asked us what they should do to control it, they seemed like more motivated during health screening” (s2) “the positive part was when they were excited to know more about the information, i showed all the pamphlets, booklets, diagrams that we had. they also shared their experiences” (s3) “the way how to assess the desire to quit smoking...the fagerstrom test...before this i didn’t know people who want to quit smoking need to take which medication…after the health screening when we assess their condition, then ok we can suggest...if the condition is like this, what do we want to give...for how long…when can we start...because we cannot immediately start…needs to see their condition as well” (s5) “we started to explain the physiology part because patient asked about how could smoking and blood pressure lead to stroke so we explained how the heart, blood and brain are connected, and patient understood and said that he never knew about this before, some patients were very curious to know about health information, so we had to spend 30 minutes for counselling” (s8) increased self-confidence in face-to-face interactions with phsuds: reported by all students. “in self-confidence, it improved, when the first person came, i was quite nervous to deal with them because i didn’t know how to talk to them but after i talked to them, they gave quite good response, and because they wanted to be helped” (s2) “it boosts my confidence as well… i feel like i want to do it again” (s4) “i felt confident providing counselling to them based on my knowledge…when we went to hospital, we only interviewed patients and were not able to counsel them, at the community pharmacy usually pharmacists helped so we did not manage to develop our confidence directly with patients, this time the confidence can be developed when we do it ourselves” (s9) increased empathy towards phsuds: reported by 8 out of 10 students. “because when we understand about how the drugs affect them, it changes our thought on them” (s2) “i respect them because they tried hard to improve their life” (s3) “they feel the stigma so that’s why they did not get the access to health services…we as a health care practitioner must not discriminate. this programme gives us more understanding and feel more empathy to this population” (s7) “after taking this course (drug abuse phm 3282) and involve with the patients, we understand the patients’ situation and the needs to be treated” (s8) reduction in stigmatised attitudes towards phsuds: reported by nine out of 10 students. “at first, i was quite scared because i thought maybe some of them could become aggressive...but throughout the programme everything was ok…i had stigma...but when i had direct contact with them, i can understand better about them” (s1) “i had this stigma on why we should spend a large amount of money in helping them...however i can see that it is a disease that affects their physiology. so, we must help them…. might be some of them had hiv... it was not like scared but to be more careful” (s2) “i was not sure about how to talk to them…but when i started to do it, i felt ok...and improved my stigma a bit…we need to involve ourselves more in this programme...there were some of my friends who didn’t want to participate because people who will attend are drug users” (s4) “previously i thought that it was quite scary if they attack us during the services but after learning from this programme we understand about the actual conditions and i did not have any bad thoughts anymore” (s8) page 67 taufek et al. (2021) journal of pharmacy, 1(2), 63-69. recognition of the need for improved communication skill training: reported by all students. “some of them were shy…sometimes i accidentally used medical terms” (s1) “when a patient came, i accidentally greeted them differently...not according to their culture or religion…i was not cautious enough about this...” (s3) “when i talked to them not all of them had eye contact with me…we don’t really know how to talk to them...some of them just wanted to chat about other things…may be when talking too fast with too much information it is difficult for them to understand” (s4) “because drug is a sensitive issue, so how to, the best way of communication with these people” (s6) “they were not really friendly, were quiet...so the communication was a bit challenging” (s7) “it helps us trying our best to communicate with the patient, we asked the lecturers if it was ok to ask certain questions, that we need to be careful on our wording and sentences, to check if there is any sensitive issues such as related to family members...for example can i ask about previous medication, why did they take it, but need to check their responses too whether it is appropriate to continue asking about certain questions or not” (s8) “i think students need to know what to ask...to avoid sensitive questions when patients told us their personal stories…my friend also asked me to exchange ideas about how to counsel the patients because we have never done it before, so we were not sure how, so we discussed with each other...how to make sure the patients understood the information” (s9) recognition of the need to improve training for dealing with unexpected questions and situations: reported by eight out of ten students. “there were questions that we did not know how to answer so we asked lecturers to help” (s1) “they asked about questions which were unfamiliar to me...there was also a patient showed us the mtac (medication therapy adherence clinic) booklet…but i have not yet learned about mtac, so i just browsed through it without a thorough understanding” (s3) “i did the smoking cessation part...the tool required correct technique otherwise we couldn’t get the results...previously i knew about fagerstrom test, but i didn’t read further...so i wanted to ask the patient, but i had not much ideas...and how we can encourage them to reduce smoking habit” (s5) discussion the study demonstrated the value of experiential education in revealing students’ reflective learning skills. for example, the students’ comments regarding increased empathy, reduced stigma, the positive nature of their interactions with phsuds and appreciation for the opportunity to apply knowledge in practice suggest they proved to themselves they were performing as healthcare professionals and that generated self-satisfaction and motivated further learning. in addition, the students’ comments regarding the application of knowledge in practice imply that their experience led them to quickly recognise that the application of didactic knowledge in practice calls for the integration of multiple skills. the study demonstrated that students could identify ways to improve future performance by reflecting on past performance (reflective practice) in ways useful for student learning but also for curriculum design. for example, the students identified the need for additional communication skill training but placed that responsibility on faculty rather than on themselves. pharmacy students, if they are to become independent practitioners, must be able to take charge of their own learning when a learning need is identified. evidencebased methods recommended for pharmacy student education include early and continuous experiential education which presents a programme-long opportunity for faculty to design experiential courses that foster students’ life-long learning skills (speedie et al, 2012). it is reasonable to expect pharmacy students to be uncomfortable when faced with unexpected patient questions but, reflecting the previous paragraph, it is important for faculty to consider curriculum design. small group discussion is an evidence-based teaching method that allows students to share their experiences with their peers and for the peers, based on their own experiences, to provide constructive comment (speedie et al, 2012). it is an important part of the learning process for students to recognise their experiences as opportunities for improvement rather than cause for embarrassment. it is reasonable to expect students who have presented examples of their practice-based performance and their ideas for improving that performance on multiple occasions over multiple years to their peers and faculty in small group discussion classes would graduate with ingrained and advanced continuous professional development skills. it is also reasonable to expect that a student who describes a positive learning experience to her/his peers would encourage the peers to seek out similar experiences for themselves. students’ ability to self-assess their learning is limited but the students’ comments on improved self-confidence are consistent with the literature including supervisorbased assessments of self-confidence in student pharmacists in early experiential education (dunning, heath, suls, 2004; hendry et al, 2016). the students’ comments about interest in repeating the experience help to validate their comments about increased selfpage 68 taufek et al. (2021) journal of pharmacy, 1(2), 63-69. confidence. the major limitation of this study was the small sample size and the lack of variability in the demographic characteristics of the volunteer students. larger studies with greater diversity in the study participants will be required before findings can be generalised. in addition, further studies will be required to assess the ability of malaysian pharmacy students to use reflective practice to improve future practice performance. there is a possibility of biased responses provided during the interview since the interviewer and participants knew each other but the interviewer did not involve in the health programme and there was only sharing of experience, without any assessment or incentives given to the participants that minimised the risk of biased findings. conclusion a one-day experience providing face-to-face care for phsuds resulted in multiple positive learning outcomes for malaysian pharmacy students. the students were able to recognise and appreciate improvements in their abilities to care for patients and provided information useful for curriculum design. acknowledgements the authors wish to thank the drug rehabilitation committee of national anti-drug agency kuantan district for sponsoring the health programme for phsuds, the pharmacists at the faculty of pharmacy iium (syed mohd syahmi syd mohmad faudzi, wan zuhaira amirah wan amran) who provided training to students and the pharmacy lecturers (muhammad eid akkawi, abdul rahman fata nahas, abdulkareem mohamed ahmed, siti hadijah shamsudin) who provided assistance and advice during the programme. conflict of interest the authors declare no conflict of interest. references accreditation council for pharmacy education (2016). accreditation standards and key elements for the professional program in pharmacy leading to the doctor of pharmacy degree. https://www.acpeaccredit.org/pdf/standards2016final.pdf (accessed 2021 march 23). braun, v., & clarke, v. 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(2021). experiential education as a strategy to preserve maqasid al-shariah by identifying and addressing stigmatic views held by pharmacy students of patients with substance use disorders. journal of pharmacy 1(1): 1-7. https://doi.org/10.31436/jop.v1i1.9 regmi, k., naidoo, j., & pilkington, p. (2010). understanding the processes of translation and transliteration in qualitative research. international journal of qualitative methods, 9(1). doi: 10.1177/160940691000900103. speedie m. (2006). introductory experiential education: a means for introducing concepts of healthcare improvement. american journal of pharmaceutical education, 70(6), 145. speedie, m. k., baldwin, j. n., carter, r. a., raehl c.l., yanchick v.a., & maine l.l. (2012). cultivating “habits of mind” in the scholarly pharmacy clinician: report of the 2011-12 argus commission. american journal of pharmaceutical education, 76 (6) s3; doi: 10.5688/ajpe766s3. willig, c. (2013). introducing qualitative research in psychology. 3rd ed. new york, ny: mcgraw-hill education. introduction materials and methods results ten students consented to participate in the study and a thematic analysis using inductive approach was conducted on all ten transcripts. saturation was deemed to have been reached when no new themes were identified from the final three analyses. the ... positive nature of the interactions with phsuds: reported by all students. increased self-confidence in face-to-face interactions with phsuds: reported by all students. “we told them that their cholesterol level was high, they asked us what they should do to control it, they seemed like more motivated during health screening” (s2) “in self-confidence, it improved, when the first person came, i was quite nervous to deal with them because i didn’t know how to talk to them but after i talked to them, they gave quite good response, and because they wanted to be helped” (s2) “the positive part was when they were excited to know more about the information, i showed all the pamphlets, booklets, diagrams that we had. they also shared their experiences” (s3) “it boosts my confidence as well… i feel like i want to do it again” (s4) “the acceptance was very good...they were not aggressive, i felt comfortable meeting different people with different behaviour… there was a client who told many stories...we didn’t even ask... but he told us from a to z....about his experiences...how many cigarettes he smoked before…now he has reduced the number of cigarettes” (s5) “i felt confident providing counselling to them based on my knowledge…when we went to hospital, we only interviewed patients and were not able to counsel them, at the community pharmacy usually pharmacists helped so we did not manage to develop our confidence directly with patients, this time the confidence can be developed when we do it ourselves” (s9) “their acceptance was very good because when we did the tests, they were all queuing up and even when we finished the session, they still wanted to do it...they were really happy with our services, they really wanted to know about their glucose and cholesterol level…their blood pressure” (s7) increased empathy towards phsuds: reported by 8 out of 10 students. “because when we understand about how the drugs affect them, it changes our thought on them” (s2) “i respect them because they tried hard to improve their life” (s3) “it was interesting to interact with ex-drug users because i have never done it before, i did some counselling on healthy diet, they accepted our recommendation” (s10) “they feel the stigma so that’s why they did not get the access to health services…we as a health care practitioner must not discriminate. this programme gives us more understanding and feel more empathy to this population” (s7) appreciation for the opportunity to apply knowledge in practice: reported by all students. “after taking this course (drug abuse phm 3282) and involve with the patients, we understand the patients’ situation and the needs to be treated” (s8) we told them that their cholesterol level was high, they asked us what they should do to control it, they seemed like more motivated during health screening” (s2) “the positive part was when they were excited to know more about the information, i showed all the pamphlets, booklets, diagrams that we had. they also shared their experiences” (s3) reduction in stigmatised attitudes towards phsuds: reported by nine out of 10 students. “at first, i was quite scared because i thought maybe some of them could become aggressive...but throughout the programme everything was ok…i had stigma...but when i had direct contact with them, i can understand better about them” (s1) “the way how to assess the desire to quit smoking...the fagerstrom test...before this i didn’t know people who want to quit smoking need to take which medication…after the health screening when we assess their condition, then ok we can suggest...if the condition is like this, what do we want to give...for how long…when can we start...because we cannot immediately start…needs to see their condition as well” (s5) “i had this stigma on why we should spend a large amount of money in helping them...however i can see that it is a disease that affects their physiology. so, we must help them…. might be some of them had hiv... it was not like scared but to be more careful” (s2) “i was not sure about how to talk to them…but when i started to do it, i felt ok...and improved my stigma a bit…we need to involve ourselves more in this programme...there were some of my friends who didn’t want to participate because people who will attend are drug users” (s4) “we started to explain the physiology part because patient asked about how could smoking and blood pressure lead to stroke so we explained how the heart, blood and brain are connected, and patient understood and said that he never knew about this before, some patients were very curious to know about health information, so we had to spend 30 minutes for counselling” (s8) “previously i thought that it was quite scary if they attack us during the services but after learning from this programme we understand about the actual conditions and i did not have any bad thoughts anymore” (s8) recognition of the need for improved communication skill training: reported by all students. “some of them were shy…sometimes i accidentally used medical terms” (s1) “when a patient came, i accidentally greeted them differently...not according to their culture or religion…i was not cautious enough about this...” (s3) “when i talked to them not all of them had eye contact with me…we don’t really know how to talk to them...some of them just wanted to chat about other things…may be when talking too fast with too much information it is difficult for them to understand” (s4) “because drug is a sensitive issue, so how to, the best way of communication with these people” (s6) “they were not really friendly, were quiet...so the communication was a bit challenging” (s7) “it helps us trying our best to communicate with the patient, we asked the lecturers if it was ok to ask certain questions, that we need to be careful on our wording and sentences, to check if there is any sensitive issues such as related to family members...for example can i ask about previous medication, why did they take it, but need to check their responses too whether it is appropriate to continue asking about certain questions or not” (s8) “i think students need to know what to ask...to avoid sensitive questions when patients told us their personal stories…my friend also asked me to exchange ideas about how to counsel the patients because we have never done it before, so we were not sure how, so we discussed with each other...how to make sure the patients understood the information” (s9) recognition of the need to improve training for dealing with unexpected questions and situations: reported by eight out of ten students. “there were questions that we did not know how to answer so we asked lecturers to help” (s1) “they asked about questions which were unfamiliar to me...there was also a patient showed us the mtac (medication therapy adherence clinic) booklet…but i have not yet learned about mtac, so i just browsed through it without a thorough understanding” (s3) “i did the smoking cessation part...the tool required correct technique otherwise we couldn’t get the results...previously i knew about fagerstrom test, but i didn’t read further...so i wanted to ask the patient, but i had not much ideas...and how we can encourage them to reduce smoking habit” (s5) discussion conclusion a one-day experience providing face-to-face care for phsuds resulted in multiple positive learning outcomes for malaysian pharmacy students. the students were able to recognise and appreciate improvements in their abilities to care for patients and pr... acknowledgements the authors wish to thank the drug rehabilitation committee of national anti-drug agency kuantan district for sponsoring the health programme for phsuds, the pharmacists at the faculty of pharmacy iium (syed mohd syahmi syd mohmad faudzi, wan zuhaira ... conflict of interest the authors declare no conflict of interest. references barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework md salim et al. (2021) journal of pharmacy, 1(1), 54-62. page 54 stability of extemporaneous rifampicin prepared with x-temp® oral suspension system. salma nadirah md salim1, mohd danial mohd murshid1 and amirah mohd gazzali1,* abstract introduction: rifampicin is a first line antituberculosis drug that is commonly used in the treatment of tuberculosis, both in adults and paediatric patients. however, there is a lack of liquid formulation for rifampicin in the market due to the small market size and the physicochemical properties of the drug itself. an innovative new mix called x-temp® oral suspension system (oss) has been available in the market as a choice of vehicle for extemporaneous suspension. aim: the aim of this study was to prepare rifampicin suspension in the x-temp® oss and evaluate its stability following storage at two temperatures – refrigerated (5 °c ± 3 °c) and in a stability chamber (30 °c ± 2 °c/rh 75% ± 5%). materials and method: this study investigates the physicochemical and microbiological stability of rifampicin formulated in x-temp® oss. the rifampicin suspension was prepared at 25mg/ml and kept in two types of amber-coloured storage bottles. the bottles were stored in an open and close storage system at 5 oc (refrigeration) and 30 °c/75% rh (non-refrigerated) and the stability of the product was evaluated at specified time intervals. results: it was found that the content of rifampicin remained above 90% of the original concentration throughout the study as required by the standard references. visual appearance, colour, odour and ph remained unchanged throughout the study period and the extemporaneous preparation was not susceptible to microbial contamination. conclusion: results from this stability study confirmed that the x-temp® oss is a suitable vehicle for the preparation of extemporaneous rifampicin liquid formulation. article history: received: 16 august 2020 accepted: 26 october 2020 published: 8 january 2021 keywords: extemporaneous, rifampicin, xtemp®, suspension, tuberculosis. how to cite this article: md salim, s. n., murshid, m. d. m. & gazzali, a. m. (2021). stability of extemporaneous rifampicin prepared with x-temp® oral suspension system. journal of pharmacy, 1(1), 54-62. authors’ affiliation: 1 department of pharmaceutical technology, school of pharmaceutical sciences, universiti sains malaysia, 11800 minden, penang, malaysia. *corresponding author: email address: amirahmg@usm.my tel:+ 60194883800 md salim et al. (2021) journal of pharmacy, 1(1), 54-62. page 55 introduction extemporaneous preparations or compounding is defined as the mixing of ingredients listed in a prescription or drug formula. this term generally refers to a manual process, performed for individual orders and specific patients (quick et al., 1997). although not being conducted often in the current pharmacy practice, it remains an important area that needs to be taken care of by pharmacists. extemporaneous products usually need to be prepared for certain patient populations who may have special clinical needs, which could not be met by licensed medicinal products available in the markets. in addition, the fact that certain medications are not available in paediatric-friendly dosage forms also necessitate reformulation by pharmacists as extemporaneous preparations. stability of extemporaneous preparation is an important factor that needs to be considered by formulation pharmacists. since the preparations are unlicensed, the safety risks are higher (jackson & lowey, 2010) and hence it is very important to ensure that the formulation is stable and safe for consumption. common extemporaneous preparations are usually evaluated to ensure their stability as a way to support the practice. rifampicin or also known as rifampin is a first line antituberculosis (tb) drug and is important in the treatment of tb during both the active and continuous phases. it has a very low water solubility (1400 mg/l at 25 °c) with high susceptibility to photodegradation in aqueous form (yalkowsky & he, 2003). it is also unstable in the presence of heat, air and moisture (osol, hoover, & et al., 1975). in clinical settings, rifampicin is commonly available in capsule form whilst oral liquid products for paediatric are rarely produced, which might be related to its instability problems (glass & haywood, 2006). hence, it is a common practice to prepare rifampicin extemporaneously in hospitals, clinics and pharmacies for this specific population. this practice has its challenges to ensure good acceptance and compliance from patients due to several issues such as poor palatability and dispersibility in aqueous solution. to ensure good dispersibility, rifampicin is commonly formulated as suspension with suitable suspending agents and thickeners. one particular study by nahata et al. (1994) described the difficulty to withdraw the prescribed amount of rifampicin from extemporaneous preparations due to problems of powder wetting and dispersion faced by syrup-based formulations. they reported that the presence of a suspending agent is important to ensure accurate dosing each time. stability of extemporaneous rifampicin is also being described in the literature. rifampicin is light-, heat-, air and moisture-sensitive, hence this may lead to challenges in the storage of its liquid preparations. readers are directed to other publications that discuss the stability of extemporaneously prepared rifampicin suspensions (glass & haywood, 2006; krukenberg, mischler, massad, moore, & chandler, 1986; nahata, morosco, & hipple, 1994). currently in the clinical settings, rifampicin oral liquid products are commonly prepared by using syrups such as simple syrup and cherry syrup. the mixture of orasweet® and ora-plus® syrups are also being suggested. in recent years, an innovative and new mix called xtemp® oral suspension system (oss) has been available in the market as a choice of vehicle for extemporaneous syrup and suspension. it is a complete oss with suspending agent, stabilizer and is mildly flavoured (orange flavour), with minimal preservation. the availability of this oss could help to accelerate the preparation process of extemporaneous products and potentially offer good stability and palatability of the prepared product. the presence of suspending agents in xtemp® has made it a suitable carrier to be used in the extemporaneous preparation of low water-soluble drug powder such as rifampicin. however, with the multiple problems associated with rifampicin stability, a study on rifampicin in x-temp® oss is needed. hence, this study was designed to ensure that the oss has the ability to preserve rifampicin efficiently. we decided to conduct the stability study independently to investigate the suitability of x-temp® as a carrier for rifampicin. the aim of this study was to prepare rifampicin suspension in the x-temp® oss and evaluate its stability following storage at two temperatures – refrigerated (5 °c ± 3 °c) and in a stability chamber (30 °c ± 2 °c/rh 75% ± 5%). the product was kept in either a plastic or a glass bottle and parameters including physical characteristics (colour, clarity and odour), ph, microbial presence and available concentration were evaluated at specific time intervals in two systems; open and closed systems. detailed methods and results are presented in the following sections. materials and methods materials and instruments rifampicin capsules 300 mg (rifasynt®), x-temp® oss and hdpe plastic bottles were obtained from bioscenergy international pv. ltd. methanol (qrëc®) was of ar grade and was used as received. nutrient agar (merck, darmstadt, germany) was used for microbial tests. uv/vis spectrophotometer (kinesis hitachi model u-2800, leicestershire, uk) was used to evaluate the concentration of rifampicin in the oss, ph meter (hanna precision ph meter model ph 211, merck, darmstadt, germany) was used to assess the ph value of the preparation. a designated stability chamber was used to md salim et al. (2021) journal of pharmacy, 1(1), 54-62. page 56 store the preparation. preparation of rifampicin oos rifampicin capsules were opened in order to obtain the powder. the powder then was mixed with oss in a mortar according to the amount and concentration needed. rifampicin oss was prepared at a concentration of 25 mg/ml by using 300 mg rifampicin capsules. the method used for mixing these two ingredients is the geometric dilution whereby the rifampicin capsule content was triturated in a portion of syrup, syrup was added bit by bit until it becomes a slurry paste, the slurry paste was retriturated and the remaining syrup was added gradually while mixing. both open and close systems experiments were conducted in two types of container; glass and plastic bottle. open system rifampicin oss were prepared and stored in glass and plastic bottles, each containing 300 ml of the product at a concentration of 25 mg/ml. the bottles were kept at two different temperatures, 5 °c ± 3 °c (refrigerated) and 30 °c ± 2 °c/rh 75% ± 5% (non-refrigerated) (asean guidelines on stability study of drug product). each day, 4 ml (equal to 100 mg of rifampicin) was withdrawn and transferred into an empty bottle, to simulate the patient's behaviour of opening, withdrawing and closing the bottle every day. at a specific time interval (0, 7, 14, 30, 60 days), the samples withdrawn were evaluated for the physical characteristics (colour, clarity and odour), ph value, rifampicin concentration and microbial presence, as explained in the following subsections. all experiments were conducted in triplicates (n=3). close system rifampicin oss were prepared and stored in glass and plastic bottles, each containing 50 ml of the product. the bottles were kept at two different temperatures, 5 °c ± 3 °c (refrigerated) and 30 °c ± 2 °c/rh 75% ± 5% (nonrefrigerated). at a specific time interval (0, 14, 30, 60, 90 days), three bottles from each group were evaluated for the physical characteristics (colour, clarity and odour), ph value, rifampicin concentration and microbial presence, as explained in the following subsections. all experiments were conducted in triplicates (n=3). evaluation of physical characteristics and ph value the physical appearance of the withdrawn suspension was inspected at each time point and was compared with the characteristics recorded at day 0. the suspensions were then shaken well to ensure homogenous dispersion and the required volume was withdrawn from the bottles. no bubble was retained after shaking. the ph value of the product was measured by using a ph meter at each time point. any changes were recorded accordingly. evaluation of rifampicin concentration the concentration of rifampicin at each time point was determined through spectrophotometry approach. briefly, 0.1 ml of rifampicin oss was diluted in 10 ml of methanol (primary dilution) and subsequently 0.1 ml from the primary dilution was further diluted with 10 ml of methanol (secondary dilution). the absorbance of the secondary dilution was measured at 480 nm and the amount of rifampicin presence in the product was calculated in reference to a standard calibration curve of rifampicin prepared at 5, 10, 15, 20 and 25 µg/ml (r2= 0.9983). evaluation of microbial presence this test was conducted to determine the presence of escherichia coli, aerobic bacteria, yeast and mould in the product. a culture media was prepared by dissolving 3g of nutrient agar dehydrated powder in 150 ml of distilled water. the suspension was boiled and kept under vigorous stirring. the solubilized agar was then sterilized by autoclaving at 121 °c for 15 minutes. subsequently, 0.1 ml of the tested product was pipetted into a sterile petri dish and the agar was then poured into the same dish, before being placed in an incubator at 37 °c for 2 days (europe, 2011). results the experiments were conducted to determine the potential of x-temp® oss to produce a stable extemporaneous oral liquid product of rifampicin. the u.s pharmacopeia and european pharmacopeia standard was used as a reference in determining the level of rifampicin concentration needed to be present in the product throughout storage and the microbial limit that is allowed to be available in such a product. the calibration curve of rifampicin was prepared between 5 to 25 µg/ml and this range is within the linear range of rifampicin as reported in the literature (tilinca et al., 2017). open system as presented in table 1 and 2, the physical characteristics (of the suspension remained unchanged throughout the study period (60 days) for both glass and plastic bottles. the ph of the suspension was maintained between 4.17 to 4.35 for plastic bottles and 4.17 to 4.33 for glass bottles, showing no obvious ph changes in 60 days. the concentration of rifampicin in the preparation was also successfully maintained between the accepted ranges of 90% to 110%. microbial growth was also absent. this finding was observed for both storage conditions, showing that the x-temp® oss has the ability to preserve and stabilize the rifampicin suspension in 60 days, in an open system. the overall stability data are summarized in figure 1, figure 2, table 1 and table 2. md salim et al. (2021) journal of pharmacy, 1(1), 54-62. page 57 figure 1: the comparison of rifampicin concentration (%) against time (days) in open system at temperature 5 ºc (n ≥ 3) figure 2: the comparison of rifampicin concentration (%) against time (days) in open system at temperature 30 ºc (n ≥ 3) close system the physical characteristics of rifampicin suspension stored under close system were also maintained throughout the 90 days study period. fluctuations in terms of concentration could be seen as presented in figure 3 and figure 4. the overall characteristics of the rifampicin oss is presented in table 3 and 4, for plastic and glass bottles respectively. figure 3: the comparison of rifampicin concentration (%) against time (days) in close system at temperature 5 ºc (n ≥ 3) figure 4: the comparison of rifampicin concentration (%) against time (days) in close system at temperature 30 ºc (n ≥ 3) 0 20 40 60 80 100 120 0 7 14 30 60 p e rc e n ta g e o f ri fa m p ic in (% ) time (days) 5℃ (glass bottles) 5℃ (plastic bottles) 0 20 40 60 80 100 120 0 7 14 30 60 p e rc e n ta g e o f ri fa m p ic in ( % ) time (days) 30℃ (glass bottles) 30℃ (plastic bottles) 0 20 40 60 80 100 120 0 14 30 60 90 p e rc e n ta g e o f ri fa m p ic in ( % ) time (days) 5℃ (glass bottles) 5℃ (plastic bottles) 0 20 40 60 80 100 120 0 14 30 60 90p e rc e n ta g e o f ri fa m p ic in ( % ) time (days) 30℃ (glass bottles) 30℃ (plastic bottles) md salim et al. (2021) journal of pharmacy, 1(1), 54-62. page 58 table 1: open system/plastic bottle test specification temperature time (days) 0 7 14 30 60 visual appearance colour: red clarity: opaque odour: orange 2-8 ºc red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange 30 ºc red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange ph 4 to 5 2-8 ºc 4.29 4.29 4.23 4.23 4.37 30 ºc 4.17 4.25 4.31 4.33 4.35 assay 90.0% to 110.0% (usp 2013) ± sem 2-8 ºc 100.0% ± 0.0 101.8% ± 5.6 101.8% ± 3.1 94.7% ± 4.1 94.7% ± 4.1 30 ºc 100.0% ± 0.0 97.7% ± 5.9 97.7% ± 4.7 100.0% ± 2.3 97.7% ± 0.7 microbial limit aerobic bacteria <1000 cfu/g yeast & mould <100 cfu/g 2-8 ºc conforms conforms conforms conforms conforms 30 ºc conforms conforms conforms conforms conforms table 2: open system/glass bottle test specification temperature time (days) 0 7 14 30 60 visual appearance colour: red clarity: opaque odour: orange 2-8 ºc red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange 30 ºc red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange ph 4 to 5 2-8 ºc 4.31 4.23 4.17 4.17 4.33 30 ºc 4.19 4.25 4.38 4.30 4.38 assay 90.0% to 110.0% (usp 2013) ± sem 2-8 ºc 100.0% ± 0.0 102.2% ± 6.3 100.0% ± 12.6 108.7% ± 4.7 108.7% ± 4.7 30 ºc 100.0% ± 0.0 100.0% ± 5.8 109.6% ± 3.5 103.9% ± 0.6 101.9% ± 5.7 microbial limit aerobic bacteria <1000 cfu/g yeast & mould <100 cfu/g 2-8 ºc conforms conforms conforms conforms conforms 30 ºc conforms conforms conforms conforms conforms md salim et al. (2021) journal of pharmacy, 1(1), 54-62. page 59 table 3: close system/plastic bottle test specification temperature time (days) 0 14 30 60 90 visual appearance colour: red clarity: opaque odour: orange 2-8 ºc red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange 30 ºc red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange ph 4 to 5 2-8 ºc 4.17 4.17 4.16 4.16 4.36 30 ºc 4.17 4.65 4.70 4.78 4.79 assay 90.0% to 110.0% (usp 2013) ± sem 2-8 ºc 100.0% ± 0.0 104.5% ± 8.2 104.5% ± 8.2 104.5% ± 8.2 110.0% ± 8.1 30 ºc 100.0% ± 0.0 110.0% ± 30.8 109.0% ± 0.9 101.4% ± 3.9 101.4% ± 4.6 microbial limit aerobic bacteria <1000 cfu/g yeast & mould <100 cfu/g 2-8 ºc conforms conforms conforms conforms conforms 30 ºc conforms conforms conforms conforms conforms table 4: close system/glass bottle test specification temperature time (days) 0 14 30 60 90 visual appearance colour: red clarity: opaque odour: orange 2-8 ºc red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange 30 ºc red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange red, opaque, orange ph 4 to 5 2-8 ºc 4.19 4.23 4.18 4.16 4.36 30 ºc 4.19 4.60 4.71 4.81 4.78 assay 90.0% to 110.0% (usp 2013) ± sem 2-8 ºc 100.0% ± 0.0 109.6% ± 7.1 105.6% ± 8.0 110.0% ± 7.2 99.4% ± 2.5 30 ºc 100.0% ± 0.0 110.0% ± 1.1 110.0% ± 1.5 92.8% ± 3.1 96.6% ± 2.1 microbial limit aerobic bacteria <1000 cfu/g yeast & mould <100 cfu/g 2-8 ºc conforms conforms conforms conforms conforms 30 ºc conforms conforms conforms conforms conforms md salim et al. (2021) journal of pharmacy, 1(1), 54-62. page 60 discussion the suspension was prepared from capsules of rifampicin 300 mg. nahata et al. (1994) described four preparation methods that can be used to prepare rifampicin suspension. in this study, we used one of the methods proposed which is triturating rifampicin capsule content in a portion of the carrier, adding more carriers, re-triturating the slurry and adding the remaining carrier while mixing. the authors reported that suspension prepared from capsules could lead to lower-than-expected rifampicin concentration as compared to those prepared from intravenous (iv) preparations (nahata et al., 1994). in this study, we did not compare the stability of the preparations made from iv rifampicin, as the finding obtained from this study showed that the concentration of rifampicin remained above 90% of the original concentration throughout the course of study. the fluctuations in concentration are expected as presented in other previous studies (baniasadi, shahsavari, namdar, & kobarfard, 2015; nahata et al., 1994). however, the amount of the drug present in the formulation must be ensured to be within the accepted limit (90% – 110%) as outlined by pharmacopoeias. based on visual inspection, the rifampicin powder was properly wetted by the oss and the suspension formed was thick and slurry-like. the presence of three suspending agents in x-temp® oss, which are microcrystalline cellulose, carboxymethylcellulose sodium (cmc-na) and xanthan gum, explains its effectiveness in ensuring the stability of the suspension and prevent caking of rifampicin powder at the bottom of the container. in compounded medications, microcrystalline cellulose is used as an adsorbent, a suspending agent and a capsule diluent (marques-marinho, f. d., & viannasoares, c. d. 2013). in addition, microcrystalline cellulose is also considered as a component of the vehicle used for oral suspension (united states pharmacopeia 2011). marques-marinho f. d., & vianna-soares also reported in their publication that cmc-na acts as a capsule disintegrant, a stabilizer, a suspending agent, an emulsifying agent (0.25-1%), a gelling agent (3-6%) and a viscosity-increasing agent (0.1-1%) in compounded medicines. its application in compounding pharmacies is primarily due to its viscosity-increasing properties. viscous aqueous solutions are used to suspend powders intended for either topical or oral use (marques-marinho, f. d., & vianna-soares, c. d. 2013). xanthan gum is described as anionic microbial polysaccharides, which is believed to have excellent performance in stabilizing suspensions as it will form gel-like network structure by intermolecular interaction (xue, d., & sethi, r. 2012). the x-temp® oss could also wet the rifampicin powder well. this ability is important as it relates to the dispersion of the powder and prevents fluctuations in the rifampicin concentrations. previously reported work on rifampicin suspension by nahata et al., (1994) showed the use of simple syrups was believed to have poor powder wettability, which contributed to a big fluctuation in rifampicin concentration available in the suspension over time, which is risky in the administration of drugs such as rifampicin (nahata et al., 1994). dhanapal et al. (2012) reported on the formulation and evaluation of rifampicin suspension with single suspending agent. the authors used carboxymethylcellulose sodium, sodium starch glycolate, xanthan gum and carbomer 934, with the presence of flavouring agents and preservatives. it was shown that xanthan gum as a single suspending agent would give the best dispersion characteristics as compared to the others (dhanapal, manavalan, chandar, & chenthilnathan, 2012). the combination of agents would undoubtedly improve the dispersion criteria, and this seems to be the case with x-temp® oss. the rifampicin suspension prepared by using xtemp® oss did not show any retained bubbles after shaking. there is no tendency of floating ingredients upon standing as compared to what mentioned by haslam and his colleagues when they used ora-sweet®, ora-plus® and simple cherry syrup as their suspension vehicle (haslam, egodage, chen, rajewski, & stella, 1999). bubbles will affect the volume of preparation withdrawn; hence the presence of less bubbles is an advantage in liquid pharmaceuticals. the ph of the rifampicin oss was aimed at between 4 and 5. this is in accordance with the stability of rifampicin, which has been reported to be at the maximum between the two ph values (haslam et al., 1999). it was also reported elsewhere that acetate buffers influence the degradation of rifampicin at over ten times degradation rate while chloroacetate and phosphate buffers degrade the drugs three times faster (k.c jindal et al., 1995). x-temp® oss contains citric acid and sodium acid phosphate as buffers. the presence of both ingredients will produce a citrate-phosphate buffer which is also known as mcilvaine buffer. the buffer system used in x-temp® oss covers a ph range from 2.2 to 8.0. the presence of citric acid also serves as an antioxidant which will prevent the oxidative side reaction of rifampicin. the findings of this study as shown in table 1, 2, 3 and 4 proved that the ph of rifampicin in x-temp® oss remained at between ph 4 and 5 throughout the study, which makes the x-temp® oss a suitable carrier for this drug. the rifampicin oss also prepared void of any microbial contamination, despite being prepared at nonsterile room condition. this is an important characteristic of an oral extemporaneous preparation, as the shelf life of such product is usually limited by the growth of microorganisms besides the stability of the active md salim et al. (2021) journal of pharmacy, 1(1), 54-62. page 61 ingredient itself. the incorporation of preservative(s) is usually needed for extemporaneous products to be safe and stable for a longer period. the preservatives used in this oss covers a wide range of microorganisms, which is the reason for the absence of microbial contamination throughout the study period. moreover, the ph of the oss that maintained between 4 and 5 is also a non-favourable condition for the growth of common microorganisms, which might be an added value in the preservation of the rifampicin oss (jin & kirk, 2018). several publications reported on the stability of rifampicin extemporaneous preparation by using different syrups (allen & erickson, 1998; baniasadi et al., 2015; krukenberg et al., 1986). the products were kept at either room temperature or under refrigeration. krukenberg and coworkers showed a four-week stability period for 1% rifampicin suspensions formulated using syrup nf, two commercially available simple syrup, wild cherry syrup and fruit-flavoured syrup. allen jr. l.v. and his team reported a stability period of four weeks for 1% rifampicin suspensions prepared using simple syrup, wild cherry syrup or fruit-flavoured syrup and stored under refrigeration. baniasadi. s. and his colleagues also reported a stability period of 28 days (4 weeks) for a preparation of 120 ml of 10 mg/ml rifampicin (1%) prepared by using simple syrup and stored under refrigeration. nahata et al. (1994) found that rifampicin was stable in extemporaneous preparation for 56 days (8 weeks) in room temperature while a recent study by cober et al. (2010) said that at least there are 99% of the initial rifampicin throughout the 60 days study period. the formulation was prepared by using rifampicin 20mg/ml (2%) in a mixture of ora-plus® with either ora-sweet® or ora-sweet sf and stored at room temperature (cober, johnson, lee, & currie, 2010). concerning the x-temp® oss, the combination of different carefully chosen materials has managed to preserve the rifampicin and stabilize the suspension. the stability was shown to be preserved for 60 days in the open system. the type of bottles was previously described as an important factor in determining the stability of rifampicin suspension (dhanapal et al., 2012). there were also reports on the increasing concentration of rifampicin during storage time, in which the authors believe that this is due to the binding of rifampicin to the plastic bottles and incomplete wetting and dispersion of the rifampicin powder during preparation (baniasadi et al., 2015). however, allen and coworkers did not observe this. they reported that the stability of 25mg/ml (2.5%) preparations of rifampicin, extemporaneously prepared in 3 vehicles; 1:1 ora-sweet:ora-plus, 1:1 ora-sweet sf:ora-plus, and cherry syrup, stored in polyethylene terephthalate (pet) prescription bottles in the dark was 28 days in both 5 °c and 25 °c (allen & erickson, 1998). this could mean that the binding of rifampicin to plastic bottles may happen with certain types of plastics. hence, care must be taken to choose good plastic bottles such as pet and hdpe for storage to ensure that the concentration of rifampicin remains stable throughout the declared stability period. another possible explanation would be that the suspension system itself has managed to prevent the adsorption of rifampicin on the bottles. this, however, would necessitate a more detailed study on the adsorption characteristics of the extemporaneous rifampicin suspension on different packaging materials. conclusion the price of syrups used in extemporaneous preparations is an important factor being considered by healthcare institutions. the ability of a carrier system to ensure the stability of the product is also important. xtemp® oss can help to maintain the stability of rifampicin extemporaneous preparations for up to 60 days in an open system and up to 90 days in a close system. in such practice, the patients do not need to visit the hospital too often to obtain their drug supply. the pharmacy department can also prepare stocks of rifampicin suspension and keep in a close system for up to 90 days. this is highly convenient and can increase the working efficiency in a busy pharmacy. acknowledgements the authors acknowledged the contribution of mr. amiruddin mat jalil in providing the training to the students involved in conducting the microbiological assays, mr. mohd asro ramli for assisting the students in using the stability chamber and puan suhana hairani salleh and puan juwita johari for assisting the students in the lab. conflict of interest the authors report no conflicts of interest. the authors alone are responsible for the content and writing of this article. the authors acknowledge the contribution of bioscenergy international pv. ltd. for supplying the xtemp® oss, rifampicin capsules and plastic hdpe bottles used in this study. md salim et al. 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(2003). handbook of aquoues solubility data. boca raton, florida: journal of american chemical society. https://www.npra.gov.my/images/guidelines_central/asean_common_technical_requirements_actr/asean-guideline-on-stability-study-of-drug-produc-2013-2820th-accsq-ppwg29.pdf https://www.npra.gov.my/images/guidelines_central/asean_common_technical_requirements_actr/asean-guideline-on-stability-study-of-drug-produc-2013-2820th-accsq-ppwg29.pdf https://www.npra.gov.my/images/guidelines_central/asean_common_technical_requirements_actr/asean-guideline-on-stability-study-of-drug-produc-2013-2820th-accsq-ppwg29.pdf https://www.npra.gov.my/images/guidelines_central/asean_common_technical_requirements_actr/asean-guideline-on-stability-study-of-drug-produc-2013-2820th-accsq-ppwg29.pdf http://www.ijpsr.info/docs/ijpsr15-06-04-010.pdf http://www.ijpsr.info/docs/ijpsr15-06-04-010.pdf barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework page 75 mohd kamaruzihan & soe (2023) journal of pharmacy, 3(1), 75-85 a comparative study: impact of screen time on sleep quality among university students and school children nurul qistina mohd kamaruzihan1 and may khin soe2,* abstract introduction: screen time has been found to affect sleep quality negatively. despite numerous studies proving that poor sleep quality and excessive screen time is prevalent among school children and university students, a comparative study on both age groups is yet to be explored extensively so far. this study focused more on school-aged children and university students, as they are often associated with sleep deprivation. the main objectives are to assess the association between screen time and sleep quality among schoolchildren and university students and to compare the effects of screen time on sleep quality between both age groups. method: this study was conducted in kulliyyah of pharmacy, iium kuantan, and six primary schools around kuantan involving 100 undergraduate pharmacy students and 100 primary schoolchildren aged 10 to 12 years old. the participants were assessed using a self-administered online questionnaire consisting of demographic background, electronic device use, and pittsburgh sleep quality index (psqi). the result was analysed using spss 23.0 software— descriptive analysis and chi-square test to determine the association between duration of screen time and sleep quality. results: the mean duration of screen time among the participants is 5.5 hours (± 0.102). 56.5% participants have poor sleep quality. the psqi score for ug students is significantly higher (mean score 6.7 ± 2.741) compared to children (mean score 5.54 ± 2.812) respectively (p value=0.001). the duration of screen time is weakly related to sleep quality. however, respondents with excessive screen time of more than 12 hours have a higher mean psqi score. conclusion: the findings revealed that majority respondents have poor sleep quality, independent of screen time. further research with larger sample size is suggested for clearer comprehensive results. article history: received: 9 june 2022 accepted: 31 january 2023 published: 31 january 2023 keywords: screen time, quality of sleep, psqi, pharmacy students, school children how to cite this article: mohd kamaruzihan, n. q. & soe, m. k. (2023). a comparative study: impact of screen time on sleep quality among university students and school children. journal of pharmacy, 3(1). 75-85 doi: 10.31436/jop.v3i1.176 authors’ affiliation: 1 kulliyyah of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, 25200 kuantan, malaysia. 2 department of basic medical sciences, kulliyyah of pharmacy, jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia. *corresponding author: email address: may_soe@iium.edu.my page 76 mohd kamaruzihan & soe (2023) journal of pharmacy, 3(1), 75-85 introduction it is a common phenomenon to see people carrying their smartphones everywhere, including going to the bed. the term “screen time” is referred to the activities done in front of a screen, such as watching television, working on a computer and laptop, or playing video games, which also includes spending time on social media (kaneshiro neil, 2019). excessive screen time can be defined as the time spent in front of the screen for more than recommended. the recommended screen time is ideally 2 hours per day but it may vary according to the demographic background such as age group and occupation (leblanc et al., 2017). excessive screen time is often associated with short sleep duration and poor sleep quality. for example, university students often use laptops to do their assignments until late at night. adolescents often use smartphones to access social media and to play games. according to a study done by bhat et al. (2018), younger participants use electronic social media before bedtime more than older participants (bhat et al., 2018). this finding is associated with insomnia and sleep disturbance on the particular group of population. according to ohayon et al. (2017) on the national sleep foundation’s sleep quality recommendation, several key indicators for good sleep quality include sleep latency of up to 15 minutes, fewer awakenings, and reduced wake after the sleep onset (ohayon et al., 2017). the recommended sleep duration for schoolchildren aged 6-13, teenagers aged 14-17, and young adults aged 18-25 according to the national sleep foundation is 9-11 hours, 8-10 hours, and 79 hours respectively (national sleep foundation recommends new sleep times | sleep foundation, 2015). hence, the recommended sleep duration is longer for younger ages. however, not all are aware of the importance of maintaining a good sleep quality that is essential for growing children and truly practicing it. this study mainly focuses on school-aged children and university students that are categorised as young adults as they are often associated with sleep deprivation and poor sleep quality. a study done among college students at king abdulaziz university on the relationship between sleep quality and the level of internet addiction resulted in 54.4% of the participants having poor sleep quality (abdulrahman khayat et al., 2018). students were well-aware of the importance of getting optimal sleep duration and quality, but very few were actually practising it or making it a priority habit (dowdell & clayton, 2019). this might lead to several consequences that may affect their health and academic performance. one factor that may influence sleep quality is excessive screen time, which is supported by many studies. night-time phone usage is commonly associated with sleep quality where first-year university students reported having a very short duration of sleep compared to the recommendations (whipps et al., 2018). the presence of smartphones nearby before sleep might disturb their sleep pattern. moreover, a previous study found that sleep deprivation was caused by media device use, whereby 38.4% students agreed that the major cause of their sleep deprivation was internet addiction (ranasinghe et al., 2018). as there are many studies proving that poor sleep quality and excessive screen time are prevalent among schoolchildren and university students, it is important to determine the association between them and whether excessive screen time influences their sleep quality in a negative way. this study aims to assess the association between the duration of screen time and sleep quality among schoolchildren and university students, to determine the effects of excessive screen time on sleep quality, and to compare the effects of a longer duration of screen time on sleep quality between schoolchildren and university students. the hypotheses suggested in this study is excessive screen time causes poor sleep quality in schoolchildren and university students. methodology a cross-sectional study was conducted quantitatively among schoolchildren aged ten to twelve years old from primary schools in kuantan and undergraduate pharmacy students in iium kuantan campus. the participants were assessed using a close-ended questionnaire, which consists of the demographic background of the participants, electronic device use, and the pittsburgh sleep quality index (psqi). there are seven components in the psqi which are subjective sleep quality, sleep latency, sleep duration, sleep efficiency, sleep disturbance, use of sleep medication, and daytime dysfunction. the questionnaire was provided in english and malay. after receiving approval from iium research ethics committee, this study was subsequently approved by the ministry of education and pahang state education department. the survey was distributed by their respective teacher to the primary school students with the permission of the school headmasters. parental consent was needed for schoolchildren to participate in this study. all participants were required to submit their consent form before answering the survey and their participation was voluntary. the result was analysed using spss 23.0 software— descriptive analysis and chi-square test to determine the association between screen times and sleep quality and to compare the result between schoolchildren and university students. the sample size was calculated with 95% confidence interval, 80% power and 0.29 odds ratio, the minimum recommended sample size is 98. therefore, 200 (100 primary schoolchildren and 100 university students) were selected randomly as study subjects. page 77 mohd kamaruzihan & soe (2023) journal of pharmacy, 3(1), 75-85 1. inclusion criteria 1. primary school children aged 10 to 12 years old, studying in a school in kuantan, pahang, able to write and understand english or malay language, consented by their parents or guardian. 2. undergraduate pharmacy students currently studying in year 1, 2, 3, or 4 in kulliyyah of pharmacy, able to read and understand english. 3. participation in both populations is voluntary. 2. exclusion criteria 1. children with significant physical and mental disabilities or chronic illness, age below 9 and above 12, or did not receive consent from their parents. 2. students from other kulliyyah in iium kuantan, postgraduate students, students who cannot read nor understand english, students with chronic illness and physical or mental disabilities. results 1. demographic background table 1 shows the descriptive statistics of the sociodemographic factors for both students and children. the mean age for the undergraduate (ug) students and children is 20.60 (±1.303) and 11.13 (±0.825) years old, respectively. 100% participants from the university are malay while 91% from the school and minority of them are from other races. the calculated mean bmi for both groups are 21.33 (ug students) and 19.23 (children). the basal metabolic rate (bmr) is calculated from the formula shown in figure 1. the mean bmr for ug students is 1411.33 (±168.07) kcal per day while for children is 1233.71 (±161.99) kcal per day. majority of the ug students are lightly active (47%) while majority of the children are moderately active (40%). total calorie requirement is calculated from the formula shown in figure 2 with the calculated mean of 1859.97 (±281.08) kcal for ug students and 1738.18 (±311.03) kcal for children. table 1: socio-demographic factors of participants socio-demographic factors ug students children n (%) n (%) age* (years) 20.60 (±1.30) 11.13 (±0.83) gender male 26 (26.0) 46 (46.0) female 74 (74.0) 54 (54.0) race malay 100 (100.0) 91 (91.0) chinese 0 2 (2.0) india 0 6 (6.0) others (melanau) 0 1 (1.0) bmi* 21.33 (±3.90) 19.23 (±4.68) height* (cm) 159.23 (±8.12) 142.44 (±13.03) weight* (kg) 54.10 (±3.90) 39.10 (±11.21) bmr* 1411.33 (±168.07) 1233.71 (±161.99) activity factor level sedentary 43 (43%) 26 (26%) lightly active 47 (47%) 32 (32%) moderately active 10 (10%) 40 (40%) very active 0 2 (2%) extremely active 0 0 tcr* (kcal) 1859.97 (±281.08) 1738.18 (±311.03) notes: *expressed in means ± sd abbreviations: bmi, body mass index; bmr, basal metabolic rate; tcr, total calorie requirement; psqi, pittsburgh sleep quality index. page 78 mohd kamaruzihan & soe (2023) journal of pharmacy, 3(1), 75-85 as for the activity factor level, 47% of ug students are lightly active followed by 43% sedentary and 10% moderately active. meanwhile, 40% of schoolchildren are moderately active followed by 32% lightly active, 26% sedentary, and 2% very active. bmrmen = 66.5 + (13.75 x weight in kg) + (5.003 x height in cm) − (6.775 x age in years) bmrwomen = 655.1 + (9.563 x weight in kg) + (1.850 x height in cm) − (4.676 x age in years) figure 1: harris-benedict equation for calculation of bmr total caloric requirements (tcr) = bmr x activity factor • little/ no exercise: bmr x 1.2 • light exercise: bmr x 1.375 • moderate exercise (3-5 days/week): bmr x 1.55 • very active (6-7 days/week): bmr x 1.725 • extra active (very active and physical job): bmr x 1.9 figure 2: calculation for tcr 2. pattern of smartphone usage the most used electronic devices among the participants are smartphones (81%), followed by computer/laptop (10%), tablet/ipad (4.5%), television (4%), and video games (0.5%) (table 2). all ug students own a smartphone while 81% of schoolchildren own it. the mean age of first using smartphone for ug students is 13 years old, while for children is 8 years old. ug students spend average time using smartphone more than children do, which is 5-6 hours per day and 3-4 hours per day, respectively. most children spend more than 2 hours on electronic devices for academic purposes, but most ug students spend more than 2 hours for social media and entertainment. other purposes of using electronic devices mentioned by the participants from the survey are online shopping, work-related purpose, reading e-books and online news, searching information, and communication. majority of ug students (98%) and children (57%) used smartphone in the last hour before bedtime. most of ug students take smartphones to bed with them (88%) while majority of children (60%) are not. in addition, majority use smartphone as alarm clock, to text message and browse social media before sleep. table 2: pattern of smartphone usage among participants questions ug students n (%) children n (%) total % what type of electronic devices you use the most? smartphone 84 (42%) 78 (39%) 81.0 television 1 (0.5%) 7 (3.5%) 4.0 computer/ laptop 10 (5%) 10 (5%) 10.0 tablet/ ipad 5 (2.5%) 4 (2%) 4.5 video games 0 (0%) 1 (0.5%) 0.5 do you own a smartphone? yes 100 (50%) 81 (40.5%) 90.5 no 0 (0%) 19 (9.5%) 9.5 age of first using a smartphone* 13.81 (±2.39) 8.8 (±2.53) page 79 mohd kamaruzihan & soe (2023) journal of pharmacy, 3(1), 75-85 average time spent per day using smartphone (hours) 5.5 3.5 purpose of using electronic devices and daily usage academic no use 0 (0%) 0 (0%) 0.0 <30 minutes 12 (6%) 4 (2%) 8.0 30 min-1 hours 20 (10%) 13 (6.5%) 16.5 1-2 hours 20 (10%) 21 (10.5%) 20.5 >2 hours 48 (24%) 62 (31%) 55.0 social media no use 0 (%) 20(10%) 10.0 <30 minutes 1 (0.5%) 17 (8.5%) 9.0 30 min-1 hours 13 (6.5%) 19 (9.5%) 16.0 1-2 hours 33 (16.5%) 29 (14.5%) 31.0 >2 hours 53 (26.5%) 15 (7.5%) 34.0 video games no use 47 (23.5%) 19 (9.5%) 33.0 <30 minutes 21 (10.5%) 23 (11.5%) 22.0 30 min-1 hours 11 (5.5%) 24 (12%) 17.5 1-2 hours 13 (6.5%) 20 (10%) 16.5 >2 hours 8 (4%) 14 (7%) 11.0 entertainment no use 1 (0.5%) 17 (8.5%) 9.0 <30 minutes 2 (1%) 22 (11%) 12.0 30 min-1 hours 10 (5%) 27 (13.5%) 18.5 1-2 hours 33 (16.5%) 21 (10.5%) 27.0 >2 hours 54 (27%) 13 (6.5%) 33.5 kamaruzihan & soe (2023) journal of pharmacy, 3(1), 75-85 page 80 others no use 41 (20.5%) 38 (19%) 39.5 <30 minutes 23 (11.5%) 30 (15%) 26.5 30 min-1 hours 15 (7.5%) 19 (9.5%) 17.0 1-2 hours 13 (6.5%) 4 (2%) 8.5 >2 hours 8 (4%) 9 (4.5%) 8.5 electronic devices used in the last hour before bedtime smartphone 98 (49%) 57 (28.5%) 77.5 television 1 (0.5%) 44 (22%) 22.5 computer/ laptop 25 (12.5%) 2 (1%) 13.5 tablet/ ipad 12 (6%) 5 (2.5%) 8.5 video games 3 (1.5%) 3 (1.5%) 3.0 do you take your smartphone and/or tablet to bed with you? yes 88 (44%) 40 (20%) 74.0 no 12 (6%) 60 (30%) 26.0 do you use your smartphone and/or tablet as your alarm clock? yes 92 (46%) 60 (30%) 74.0 no 8 (4%) 40 (20%) 24.0 do you text or use a messaging app before sleep? yes 57 (28.5%) 79 (39.5%) 68.0 no 43 (21.5%) 21 (10.5%) 32.0 do you play games on your smartphone and/or tablet before sleep? yes 21 (10.5%) 61 (30.5%) 41.0 no 79 (39.5%) 39 (19.5%) 59.0 do you use social media on your smartphone before sleep? yes 92 (46%) 61 (30.5%) 76.5 no 8 (4%) 39 (19.5%) 23.5 notes: *expressed in means ± sd kamaruzihan & soe (2023) journal of pharmacy, 3(1), 75-85 page 81 3. sleep quality a psqi score of more than five indicates poor sleep quality. 56.5% participants have poor sleep quality. the mean global score for ug students is higher compared to children where their mean score is 6.73 and 5.54, respectively (table 3). other components that are significantly higher among ug students are component 1 (subjective sleep quality) and component 7 (daytime dysfunction). the association of gender, age group (ug students and children), and screen time with sleep quality were determined using chi-square independence test (table 4). the result shows an association between age group and sleep quality, (p=0.004). meanwhile, there is no significant association found between gender and screen time with sleep quality. figure 3: graph association between screen time and sleep quality. table 3: scores of each component of the pittsburgh sleep quality index (psqi) and the global psqi score psqi ug students children p-value mean±sd mean±sd component 1: subjective sleep quality 1.44 (0.857) 0.81 (0.775) .000a component 2: sleep latency 1.07 (1.008) 0.82 (0.821) .108 component 3: sleep duration 1.57 (0.924) 1.56 (0.701) .765 component 4: sleep efficiency 0.32 (0.634) 0.46 (0.881) .538 component 5: sleep disturbance 1.05 (0.500) 1.06 (0.565) .975 component 6: sleep medication 0.09 (0.473) 0.09 (0.429) .749 component 7: daytime dysfunction 1.19 (0.720) 0.74 (0.705) .000a global score 6.73 (2.741) 5.54 (2.812) .001a a significant at p<0.05 table 4: association of gender, age group, and screen time with psqi sleep quality using chi-square test. variables pearson chi-square value df asymptomatic significance (2-sided) exact sig. (2-sided) exact sig. (1-sided) gender 0.634a 1 .426 .460 .258 age group 8.972a 1 .004 .004 .002 screen time 7.825b 6 .251 a. 0 cells (0.0%) have expected count less than 5. b. 2 cells (14.3%) have expected count less than 5. the minimum expected count is 0.87. page 82 mohd kamaruzihan & soe (2023) journal of pharmacy, 3(1), 75-85 4. other parameters of 29 respondents who are overweight, 15 of them are having poor sleep quality, while 7 out of 13 respondents who are obese have poor sleep quality. an eta coefficient test was used to determine whether there is an association between bmi and sleep quality. thus, the following null hypothesis was tested: h0 = there will be no association between bmi and respondents’ sleep quality. there was no significant association between bmi and sleep quality, η = 0.011. thus, we cannot reject the null hypothesis and no significant association was found between bmi and respondents’ sleep quality. discussion 1. pattern of electronic device use we found out that most respondents used smartphones the most, followed by computer and laptops. previous studies showed that smartphones and laptops were the most used electronic devices among ug students and adolescents (dowdell & clayton, 2019; ramesh et al., 2020). while all ug students owned a smartphone, 40.5% children in this study were using their parent’s smartphone. in this study, it is observed that ug students spent more screen time compared to the children. a previous study was conducted on the average time spent on electronic devices among schoolchildren. it was found that they spent 5 hours per day on electronic devices, indicating that children are exposed to excessive screen time (unplagan et al., 2018). the prevalence of excessive screen time was also undeniably high among university students (demirci et al., 2015). most of the respondents in our study spent more than 2 hours on electronic devices for academic purposes, by which the children spent more for academic compared to ug students. this is reasonable in this era of covid-19 pandemic where all of the teaching and learning method has been switched to online learning. social media becomes the second lead purpose of using electronic devices which the ug students preceded the children. similarly, a study found that social media was used as a reference material to obtain current information in order to stay updated (nazir et al., 2020). they tend to check their smartphone before sleep, or using it as an alarm (dowdell & clayton, 2019; lebourgeois et al., 2017). our study observed majority of them take their smartphone to bed, whether as their alarm or to check messaging app and social media before bed, or to play video games. pharmacists could play an important role in educating the children on the effect of excessive screen time on sleep quality by campaigning at school as a community project. educating the parents is also important to provide strict control on the children’s screen time. 2. evaluation of sleep quality the mean psqi global score for ug students and children suggested poor sleep quality for both groups. however, the results were just slightly over the threshold value (psqi global score >5). this study found that majority of the respondents have poor sleep quality. this result is similar with a study by ramesh et al. (2020) which found that 43.5% of the participants had good sleep quality while 56.43% of the participants had poor sleep quality. our study observed a significant difference of the psqi global score between ug students and children with a higher score among students, indicating ug students have worse sleep quality than children do. in support with these findings, a study done by nurismadiana & lee (2018) on the prevalence of sleep quality among university students in a public university found that the prevalence of the students having poor sleep quality is higher among undergraduate students. students are sleep deprived due to the tight academic schedule, academic pressure, and lack of supervision from parents (dowdell & clayton, 2019). 3. sleep quality and screen time, gender, bmi there is weak evidence of association between duration of screen time and sleep quality (p=0.251). in support for this finding, previous studies found no significant relation between duration of screen time before bed and sleep quality among medical students (ramesh et al., 2020; yeluri et al., 2020). they also found no association between genders, passive screen time, and sleep quality. however, another study by hrafnkelsdottir et al. (2020) found that greater screen time was associated with irregular sleep pattern in terms of bedtime, wake time, rest duration, and sleep duration among adolescents. there is no significant relation between daytime dysfunction and screen time in this study. however, respondents with excessive screen time of more than 12 hours have a higher mean psqi score, specifically for the component of daytime dysfunction. similar to a previous study, majority of their respondents experienced poor sleep quality and daytime lethargy because of excessive smartphone use at late-night (syed nasser et al., 2020). daytime sleepiness might affect the student’s academic performance because they are unable to focus during the class. sleep disturbance among adolescents are closely related to excessive screen time (greever et al., 2017; tao et al., 2017). excessive screen time can disturb sleep quality although the exact mechanism is unknown. it is proposed that excessive screen time can disrupt circadian rhythm (blume et al., 2019; lely et al., 2014), delay bedtime onset (dowdell & clayton, 2019; ghekiere et al., 2019), reduce sleep duration (boonluksiri, 2018; whipps et al., 2018), and increase daytime sleepiness page 83 mohd kamaruzihan & soe (2023) journal of pharmacy, 3(1), 75-85 (hershner & chervin, 2014). this study found no significant difference of sleep quality between genders, which is similar to previous study that found lack of consistency in gender differences concerning sleep quality (farah et al., 2019). we found weak association in terms of bmi, activity level, and sleep quality, which is not in agreement with previous studies where they found association between sleep quality and bmi specifically in overweight and obese respondents (krističević et al., 2018; j. wang et al., 2019). sleep quality might affect body weight because sleep deprivation leads to hormonal imbalance, which promotes weight gain through the production of leptin and ghrelin, hormones that control appetite (beccuti & pannain, 2011). this study used a self-administered questionnaire to assess the screen time and sleep quality, thus recall bias could not be avoided. in addition, it limits the in-depth interpretation of the data, as the study method is a quantitative study. further study in assessing qualitative aspects should be conducted to study more on the impacts of excessive screen time on sleep quality and other healthrelated parameters. next, since the children need to use recall techniques to answer the questionnaire, bias could not be excluded. covid-19 pandemic has also put a strain on this research. data collection is limited to online survey for the participants in kulliyyah of pharmacy and limited contact with schoolchildren with strict standard of procedure to prevent the virus transmission. further similar research with larger sample size focusing on children and adolescents is suggested for clearer comprehensive results. conclusion overall, this study found a promising significant finding for the association between duration of screen time and sleep quality among ug students and children. the majority of respondents developed poor sleep quality with a higher prevalence among ug students, specifically in the aspects of subjective sleep quality and daytime dysfunction, but both components are independent of screen time. this study highlights the effects of excessive screen time on sleep quality and the importance of having good sleep quality for the children and university students. conflict of interest the authors declare no conflict of interest in the journey of publishing this research article. references abdallah, a. a., mahfouz, m., mohammed, s., emam, s. a., ahmed, t., & rahman, a.-e. 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(2020). electronic gadget screen-time, sleep quality & quantity and academic performance in medical students. the journal of the association of physicians of india, 68(1), 102. barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework rozi & sabere (2021) journal of pharmacy, 1(1), 19-26. page 19 a review on conventional and novel topical ocular drug delivery system. mohamad faeznudin rozi1, awis sukarni mohmad sabere1* abstract ocular drug delivery is a very challenging area for ophthalmologists and drug delivery scientists due to the structural and barrier complexity of the eye. barriers such as different layers of cornea, sclera, conjunctival blood flow, and tear dilution limit the efficacy of drug delivery to the anterior part of the eye in addition to more barriers present to the posterior part. due to these, scientists have designed and studied various delivery systems to increase drug delivery and treatment efficacy to the eye. among conventional ocular drug delivery systems, ophthalmic solution or eye drop is widely used and preferred by consumers. conventional dosage forms available in the market are emulsion, suspension, ointment and polymeric gels. several ocular formulations such as nanoformulations, liposomes, ocular inserts, and ocular mini-tablets are also being widely studied as future treatments to improve ocular drug delivery and as an alternative to conventional drug delivery. this review intends to summarise several conventional and novel topical formulations for ocular drug delivery. article history: received: 4 august 2020 accepted: 28 october 2020 published: 8 january 2021 keywords: drug delivery system, ocular drug delivery, ophthalmic solutions. how to cite this article: rozi, m. f. & sabere, a.s. m. (2021). a review on conventional and novel topical ocular drug delivery system. journal of pharmacy, 1(1), 19-26. authors’ affiliation: 1 department of pharmaceutical chemistry, kulliyyah of pharmacy, international islamic university malaysia (iium), jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia *corresponding author: email address: awissabere@iium.edu.my tel:+609 5714931, fax: +09 5716775 tel:+609 rozi & sabere (2021) journal of pharmacy, 1(1), 19-26. page 20 introduction eyes are one of the important organs in a human body. as an organ of sense, it allows humans to observe and interact with their surroundings. generally, the eyeball is divided into two parts, namely the anterior and the posterior segments. the anterior segment of the eyeball contains the cornea, iris, lens, conjunctiva, ciliary body and aqueous humour while the posterior segment consists of sclera, choroid, retinal pigment epithelium, neural retina, optic nerves and vitreous humour (monkhouse, 2007), as illustrated in figure 1. even though the eyeball has many structures, only the anterior part is exposed. the remaining structure is covered and protected by the orbit in which the eyeball is situated. the small exposed portion of the eye is prone to various infections even though it has several protective mechanisms such as the eyelashes, the eyelids and tears (tortora & derrickson, 2015). however, eye diseases are not only limited to infections. other diseases such as glaucoma, cataract, and allergic conjunctivitis can also inflict the eyes. thus, a treatment is needed whenever infections or diseases are present in the eyes. figure 1 human eye anatomy. for the treatment, topical instillation of the agents is the preferable approach as it is easy, convenient and noninvasive. eye drops are the most commonly used conventional topical ophthalmic dosage form due to the ease of administration and patient compliance (patel, cholkar, agrahari, & mitra, 2013). however, it is less effective in certain situations or treatments due to the very low ocular bioavailability and low drug permeation into the ocular tissue as a result of tear turnover, nasolacrimal drainage, and blinking. these barriers have been taken into consideration in increasing the effectiveness of the topical ophthalmic dosage form. (souza, dias, pereira, bernardi, & lopez, 2014). as a result, various conventional and novel drug delivery systems have been developed and studied such as emulsion, suspension, ointment, and one that uses the technological development of lipid-based system and polymeric system. conventional topical ocular drug delivery system nowadays, there are several types of ocular drug delivery system in the market including ophthalmic eye drops which are highly used by patients. others are emulsion, suspension, ointment and polymeric gel preparation. eye drops topical eye drops are the most convenient, noninvasive and patient compliant among topical eye preparations. however, according to pahuja, arora, & pawar (2012), there are a few barriers encountered by eye drops in treatments. based on the study, a large number of patients faced difficulties in instilling the drops. besides, the tear drainage that increases with the volume of eye drops can lead to the loss and dilution of the solution. other than that, the amount of the drug absorbed into the ocular tissue cannot be estimated due to the limited holding capacity of the eye pocket. benzalkonium chloride, which is commonly used as preservative, may also cause several problems such as the peeling of the corneal epithelium cells at their borders which inhibits the growth of the cells and enlarges the intercellular spaces in the superficial cells of the cornea (pahuja et al., 2012). even though ghate & edelhauser (2006) stated that benzalkonium chloride could improve the corneal permeability of various drugs, the negative side effects caused by it should not be ignored. due to these limitations, patel et al. (2013) suggested the use of viscosity enhancer to increase the contact time, permeation enhancer to increase the uptake of active ingredient, and cyclodextrin as a carrier for hydrophobic molecules to increase the bioavailability of the topical eye drop. emulsion the interest in using emulsion in the past has been resuscitated by submicron emulsion (ranged between 0.1 μm and 0.3 μm) with non-ionic surfactant to increase its stability (ghate & edelhauser, 2006). patel et al. (2013) stated that emulsion-based formulation could enhance both the solubility and the bioavailability of ocular drugs. generally, there are two types of emulsion that are already available in the market as a vehicle for active pharmaceutical ingredients, namely oil in water (o/w) and water in oil (w/o) emulsions (vandamme, 2002). among these two emulsions, (o/w) type is preferable as it is less irritating to the eye and has better ocular tolerance. according to liang et al. (2008), emulsion-based formulation can offer several benefits in ocular formulation such that it improves the precorneal residence time, enhances the drug’s corneal permeation, increases the bioavailability, and it has sustained-release properties. precorneal residence time can also be improved by using emulsion with chitosan as its surface coating. this is based on the pharmacokinetic study done by yamaguchi et al. (2009) using chitosan-coated emulsion in comparison to non-coated emulsion on the eyes of male albino rabbits. the results showed improvement on the emulsion mean residence time (1.5 times) and half-life (1.8 times) of the rozi & sabere (2021) journal of pharmacy, 1(1), 19-26. page 21 drug in comparison to non-coated emulsion. however, ophthalmic emulsions come with their own limitations. they have low stability and are prone to various types of instability phenomena such as flocculation, coalescence and creaming (aldrich et al., 2013). flocculation occurs when the dispersed phase comes out from the suspension and forms flakes. coalescence is another instability process by which the dispersed droplets in the suspension are continuously combined to form larger droplets. other than that, one phase in the emulsion can migrate either to the top or the bottom depending on their relative densities, forming a separated layer between the two phases known as creaming. thus, the study suggested the use of surfactants to improve the kinetic stability of the emulsion products. suspension suspension can be defined as a dispersion of finely insoluble active pharmaceutical ingredients in a solvent (patel et al., 2013). in other words, it is a concentrated solution of active pharmaceutical ingredients. this type of ocular drug delivery system has several benefits over ophthalmic drops. the main benefit is that it can improve the drug’s contact time and duration of action due to the insoluble suspension that retains in the precorneal pocket instead of being washed away or diluted by the tear. the improvement of the duration of the drug action is also due to the different particle sizes of the suspended particles. the small particles will replenish the absorbed drug while the large particles will be retained in the precorneal pocket and undergo slow dissolution (remington, 2011). according to ghate & edelhauser (2006), prednisolone acetate suspension is the most effective to go across the cornea and suppress corneal inflammation compared to prednisolone phosphate solution. there were also four weeks randomised, double-blinded, multicentre phase ii clinical trial done on 1% and 2% repabimide suspension over placebo. this trial revealed that both suspensions are well tolerated and effective in treating dry eye compared to the placebo (kinoshita et al., 2012). in addition, higher concentration of suspension was found to be more effective than one with a lower concentration. despite all the benefits, suspension also has several drawbacks. for example, due to the high viscosity of tobradex®, scoper et al. (2008) experimented by reducing the viscosity and improving its pharmacokinetics along with bactericidal activity. this resulted in a new suspension formulation, tobradex st®, which showed better formulation characteristics, pharmacokinetics, bactericidal characteristic and patient compliance. another drawback of suspension formulation is that it needs to be shaken to reach the required dosage level. this will decrease patient compliance and vary the dosage of the drug delivered to the eye. ghate & edelhauser (2006) stated patient compliance as a limiting factor in ocular drug efficacy as the efficacy will increase with dosing frequency. with low patient compliance, the efficacy of the suspension might also be affected. ointment according to rathore & nema (2009), ointment is a mixture of semisolid and solid hydrocarbon, such as paraffin, which is non-irritating to the eye and melts at body’s physiological temperature. commonly, there are two types of ointment, namely simple-based ointment which is made up of one continuous phase of ointment and compound-based ointment which consists of two-phase system like emulsion. when applied to the eye, the ointment will break into small drops that will remain in the conjunctival sac for a longer period of time (baranowski, karolewicz, gajda, & pluta, 2014). this action leads to the major advantage of ointment, such that it serves as a drug depot in conjunctival sac which enhances and prolongs its absorption (ghate & edelhauser, 2006). according to ali & lehmussaari (2006), the desirable attributes of ointment development should include several factors such that it needs to be non-irritating to the eye, uniform, easily manufactured, and it does not cause excessive blurred vision. even though it can enhance and prolong drug absorption, ophthalmic ointment faces a major drawback that can reduce its efficacy. according to sasaki et al. (1999), the application of ointment can lead to the blurring of vision and occasional irritation, resulting in low patient compliance. due to this, it is usually being applied at night before sleep (rathore & nema, 2009). polymeric gel ocular gel is another dosage form of delivering drugs to the eye topically. gels are made up of various materials such as mucoadhesive polymers which are important for the localised delivery of active ingredients. mucoadhesive polymers have been used in ophthalmic gels to increase their efficacy (shaikh, raj singh, garland, woolfson, & donnelly, 2011). this polymer provides an attachment for the drug carrier to a biological tissue resulting in an extended contact time and an improved ocular bioavailability (ali & lehmussaari, 2006). there are two types of ophthalmic gels, namely preformed gel and in-situ forming gel. according to ranch et al. (2017), ophthalmic preformed gel is less preferable as a dosage form because it is present as a gel substance at room temperature. this property has a limited use in ophthalmic drug delivery because of low accuracy and reproducibility administration of drugs, often producing blurry vision, crusty eyelids, and lachrymation. due to this, in situ gels become a focus in gelling system as it provides both advantages of solution and gel. in situ forming gel is a viscous liquid preparation that will change to a gel phase using either one of these three mechanisms which are ph triggered, temperature triggered, rozi & sabere (2021) journal of pharmacy, 1(1), 19-26. page 22 or ion activated. it is preferred over the preformed gel as it is more comfortable, easily administered as a drop, and causing less to no problem to the vision (rathore, 2010). kaur, singh, & kanwar (2000) stated that good in situ forming gel criteria should include low viscosity, freeflowing property to be administered as a drop, and strong gel formation to withstand the sheer force of the conjunctiva. according to gurtler & gurny (1995), it is difficult to administer accurate dose with preformed gel due to the variation of the amount of drug delivered during administration. however, with in situ gel-forming formulation, it is possible to administer accurate and reproducible quantities of dose. moreover, relatively prolonged action duration of in situ forming gel reduces the administration frequency and thus increases patient compliance. novel topical ocular drug delivery system even though conventional topical ocular preparations are being widely used nowadays, some drawbacks are still present in terms of their usage, efficacy and safety. due to these, various approaches have been made and studied. one of the approaches is by utilising nanotechnology in the ocular drug delivery system through nanoparticles and nanomicelles. there are also several other approaches to improve ocular delivery system such as liposomes and ocular inserts. nanoparticles sahoo, dilnawaz, & krishnakumar (2008) defined nanoparticles as any particles with a diameter not bigger than one micrometer, comprising of various natural or synthetic polymers, lipids, phospholipids, or metals. there are two types of nanoparticles: nanocapsules and nanospheres. in nanocapsules, the drug is encapsulated inside the polymeric capsule while in nanospheres, the drug is uniformly dispersed throughout the polymeric matrix (patel et al., 2013). one of the advantages of nanoparticles is that it can extend the drugs delivery to the tissues as the uptake and the distribution of nanoparticles depend on their size (gaudana, jwala, boddu, & mitra, 2009). this is proven in the study done by sakurai, ozeki, kunou, & ogura (2001) on the significance of particle size in tissue distribution. the study concluded that the smaller particle size could be distributed further to the tissue area where no large particle is present. many other approaches have been developed using the nanoparticle technology. one of them is solid lipid nanoparticles. solid lipid nanoparticles have several advantages such as they can improve corneal absorption, enhance corneal ocular bioavailability for both hydrophilic and lipophilic drug, allow autoclave sterilisation, and they do not display any biotoxicity since physiological lipids are used during the preparation process (seyfoddin, shaw, & alkassas, 2010). other than that, solid lipid nanoparticles also show sustained drug release properties based on an in vivo study done by cavalli, gasco, chetoni, burgalassi, & saettone (2002). solid lipid nanoparticles of tobramycin showed sustained drug release for up to six hours in comparison to short duration of tobramycin eye drops with an equal dose. de campos, diebold, carvalho, sánchez, & alonso (2004) performed a study on chitosan fluorescent nanoparticles and found that the nanoparticles were stable upon incubation with lysozyme and did not affect the viscosity of mucin dispersion. the study found that the amount of chitosan fluorescent in cornea and conjunctiva were higher for the nanoparticles compared to the controlled chitosan fluorescent solution, and the amount was constant up to 24 hours. after 24 hours of incubation with chitosan nanoparticles, the cell survival was remarkable and the viability of the recovered cell was nearly 100 percent. aside from that, there is also a study done by motwani et al. (2008) on submicroscopic reservoir using nanoparticles. in this study, mucoadhesive chitosan-sodium alginate nanoparticles were used to deliver gatifloxacin to the eye. as a result, it was found that this system underwent a fast release for the first hour and continued on slow release for the rest of 24 hours study. the outcome reduces the frequency of dosing which then increases its patient compliance. nanomicelles according to patel et al. (2013), nanomicelles are the most frequently used carrier system to deliver therapeutic agents into clear aqueous solution. nanomicelles are made up of amphiphilic molecules which are surfactants or polymers in nature that will self-assemble into micelles. there are three different types of micelles, namely regular micelles, reverse micelles, and unimolecular micelles (trivedi & kompella, 2010). regular micelles are amphiphilic copolymers which self-assemble in aqueous medium while reverse micelles are amphiphilic copolymers which self-assemble in non-aqueous medium. unimolecular micelles on the other hand are made up of the block of copolymer which has several hydrophobic and hydrophilic regions in one molecule. this enables it to self-assemble into a micelle from one molecule. between these three types, the reverse micelle is a good candidate to encapsulate and deliver hydrophilic drug as it forms micelles with the polar part facing towards the interior covering of the hydrophilic substances. in addition, qiu, zhang, yan, jin, & zhu (2007) mentioned that reverse micelles could also be used to encapsulate polymeric particles. nanomicelles pose several advantages as a drug delivery system. nishiyama & kataoka (2006) pointed out that it requires a simple preparation and it has the ability to improve drug solubility, lower the toxicity, raise circulation time, and increase tissue penetration with well targeted delivery properties. in vivo studies on rabbits done by civiale, licciardi, cavallaro, giammona, & mazzone (2009) suggested that nanomicellar formulations are a better option for topical delivery of small molecules compared to rozi & sabere (2021) journal of pharmacy, 1(1), 19-26. page 23 suspension. in another study, cholkar, patel, dutt vadlapudi, & k. mitra (2012) concluded that nanomicellar formulations could efficiently transverse ocular tissues and deliver drug to the back of the eye tissues. however, the conventional micelles face a little drawback. it is not stable over a long period of time, having short period of sustained release, inadequate suitability for hydrophilic drugs, and system optimisation is needed for each drug (torchilin, 2006). thus, these need to be considered for improvement. liposomes liposomes have an aqueous core containing drug which is enclosed by one or more phospholipid bilayers. according to patel et al. (2013), liposomes with the ability to encapsulate both hydrophobic and hydrophilic drugs can be classified into three types, namely small unilamellar vesicles (10–100 nm), large unilamellar vesicles (100–300 nm), and multilamellar vesicles (contain more than one bilayer). these liposomes are promising means in delivering ophthalmic drug due to the presence of natural phospholipids, cell-like membranes and excellent biocompatibility (gan et al., 2013). other than that, liposomes can attach to the hydrophobic corneal epithelium on which they constantly release the bound drug content, enhancing pharmacokinetics, and reducing the toxic side effect (chetoni, burgalassi, monti, najarro, & boldrini, 2007). additionally, budai et al. (2007) stated that sustained release of the drug could be produced by using multilamellar vesicles depending on the nature of the selected lipid composition. a study using a rabbit model was performed by shen & tu (2007), where they measured the concentration-time profile of ganciclovir in aqueous humour after been instilled with liposomes containing ganciclovir and ganciclovir solution. the results showed that the area under the curve for liposomes containing ganciclovir was 1.7 times bigger than the ganciclovir solution. the drug distribution of liposomal formulation was higher in sclera, cornea and vitreous humour. another study was performed by habib, fouad, & fathalla (2008) comparing fluconazole solution and fluconazole loaded liposomes on the rabbit keratitis model. after 21 days, liposomal formulation was found successful at eliminating the infection and superior to the solution. these two studies clearly show that liposomal formulation is a better delivery system than solution. however, liposomes also suffer several drawbacks. this formulation tends to be unstable, degraded, and aggregated while its fuses cause leakage of entrapped drugs during storage and after administration (zhang & wang, 2009). thus, mehanna, elmaradny, & samaha (2010) suggested surface modification and polymerisation to be carried out to enhance the performance of liposomes. ocular inserts ocular inserts are sterile preparation with a thin, multilayered and drug-impregnated solid or semisolid devices placed into conjunctival sac whose size and shape are especially designed for ophthalmic application (kumari, sharma, garg, & garg, 2010). the main purpose of the ocular inserts is to improve the contact duration between the delivery system and the conjunctival tissue to ensure a prolonged release that suits topical or systemic treatment. according to kumar, bhowmik, harish, duraivel, & kumar (2013), there are two types of ocular inserts, namely soluble and insoluble ocular inserts. soluble ocular inserts are generally defined as erodible, monolithic polymer that undergoes slow dissolution while releasing the drug and does not require removal from the eye. the insoluble type of ocular inserts is made up of insoluble polymer that can deliver drug by a variety of methods and at a predetermined rate, but it needs to be removed from the eye when empty. sultana, jain, aqil, & ali (2006) viewed the delivery of ocular inserts as more controlled, sustained and continuous. in doing so, it maintains an effective drug concentration in the target tissue and minimises the number of applications. however, based on the review, they found that the usage of this delivery system is less popular among users due to physiological factors such as patients’ unwillingness to abandon the traditional liquid and semisolid medication, and occasional therapeutic failures such as unintentional expulsion from the eye and membrane rupture. ocular inserts give several advantages such that they increase contact time, exhibit prolonged release, reduce systemic side effect, reduce dosing frequency, produce accurate dosing, increase shelf-life compared to aqueous solutions and elimination of preservative, thus leave less sensitivity reaction (kumari et al., 2010). however, ocular inserts also come with their own disadvantages. the foreign-body sensation in the eye can lead to discomfort, causing low patient compliance, excessive lachrymation which accompanies with irritation, drug dilution, and concentration reduction (friedrich, saville, cheng, & rootman, 1996). kumari et al. (2010) also mentioned some other disadvantages of ocular inserts such as unwanted migration in the conjunctival sac, unintended loss, and difficulties to place or remove as well as interference with the vision. conclusion there are various types of ocular drug delivery systems found in the literature and the market. despite that, drug delivery remains a conundrum and major challenge for ocular and formulation scientists due to the complexity of the eye structure. until now, topical eye drops remain the most preferred approach for eye treatment especially for the anterior application due to the ease of administration. however, eye drops formulation faced several major drawbacks that can reduce its efficacy such as loss of active agents by tear drainage, low corneal permeability, and reduced patient compliance following frequent administration. due to these, several conventional ocular drug delivery systems have been developed as options to improve treatment efficacy. the examples are ocular rozi & sabere (2021) journal of pharmacy, 1(1), 19-26. page 24 emulsion, suspension, ointment and polymeric gels. aside from these conventional delivery systems, scientists are developing more ocular delivery systems such as nanomicelles, nanoparticles, liposomes and ocular inserts. these novel systems are developed to further increase the efficacy and safety in the application of ocular drug delivery. despite that, there still possessed several drawbacks. it is hoped that the future novel systems would be able to overcome all the drawbacks while retains its efficacy, safety and improve patient compliance. acknowledgements this work was supported by the international islamic university malaysia research initiative grant scheme (rigs 16-288-0452). conflict of interest none. references aldrich, d. s., bach, c. m., brown, w., chambers, w., fleitman, j., hunt, d., & tin, g. w. 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(2002). microemulsions as ocular drug delivery systems: recent developments and future challenges. progress in retinal and eye research, 21(1), 15-34. yamaguchi, m., ueda, k., isowaki, a., ohtori, a., takeuchi, h., ohguro, n., & tojo, k. (2009). mucoadhesive properties of chitosan-coated ophthalmic lipid emulsion containing indomethacin in tear fluid. biological and pharmaceutical bulletin, 32(7), 12661271. zhang, j., & wang, s. (2009). topical use of coenzyme q10-loaded liposomes coated with trimethyl chitosan: tolerance, precorneal retention and anti-cataract effect. international journal of pharmaceutics, 372(1-2), 6675. barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework azhar et al., (2023) journal of pharmacy, 3(1), 38-52 page 38 stability study of royal jelly in alginate-pectin beads muhammad fitri azhar1, nurul ain mohammad hamdi1, muhammad salahuddin haris1,2,* abstract introduction: the stability of royal jelly (rj) beads is a critical aspect to ensure the product is safe, efficacious, and possesses an acceptable quality for consumers. this study aims to establish storage duration and condition to ensure the stability of rj in alginate-pectin beads. methods: in this study, two types of packaging material have been chosen, namely polyethylene (pet) opaque bottles and glass containers. samples of rj beads were stored in four different storage conditions that include freezer, laboratory environment, real-time (30 °c, 75% rh) and accelerated (40 °c, 75% rh) stability chambers at different sampling points (0, 14 days, 1 month, 3 months). the rj beads were characterised for physicochemical properties and 10-hydroxy-2decenoic acid (10-hda) content in the rj-encapsulated beads. results and discussion: the colour of rj beads in the refrigerator remained whitish grey throughout the study but colour change in room temperature (laboratory) is observable starting from 1-month time point. the particle size of rj beads stored in accelerated stability chamber had a decreasing pattern with significance (p < 0.05) for both different types of storage container. no significant difference (p > 0.05) between sphericity coefficient values of rj beads stored in glass and pet container in refrigerator, room temperature and real-time stability chamber at 0 month and 14-day time point. constant peaks of 10-hda appeared for rj samples stored in all storage conditions at 14-day time point. nonetheless, at 1-month and 3-months, peak area starts to show decreasing trend for beads stored in room temperature, real time and accelerated stability chambers. conclusion: the study showed that the rj beads exhibited convincing stability for 3 months. article history: received: 12 september 2022 accepted: 6 january 2022 published: 31 january 2023 keywords: stability study, alginate-pectin beads, royal jelly how to cite this article: azhar, m. f., mohammad hamdi, n. a. & haris, m. s. stability study of royal jelly in alginate-pectin beads. journal of pharmacy, 3(1)., 38-52 doi: 10.31436/jop.v3i1.191 authors’ affiliation: 1 department of pharmaceutical technology, kulliyyah of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, 25200 kuantan, malaysia. 2 ikop pharma sdn. bhd., jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia. *corresponding author: email address: solah@iium.edu.my azhar et al., (2023) journal of pharmacy, 3(1), xx-xx page 39 introduction stability can be defined as the ability of a pharmaceutical or nutraceutical product to withstand physical, chemical, or microbiological changes or decomposition when exposed to various environmental conditions (association of southeast asian nations, 2013). in general, the purpose of stability testing is to provide the evidence on the effect of time on the product under the influence of a variety of environmental factors, such as temperature, humidity, and light to establish a shelf life or expiry date for the pharmaceutical product hence recommending appropriate storage conditions (association of southeast asian nations, 2005). the stability of a pharmaceutical product is complex, often being dependent on multiple physical, chemical, and microbiological factors that may or may not interact with each other (aulton, 2018). in this stability study, two types of packaging material have been chosen, namely polyethylene (pet) opaque bottles and glass containers. plastic and glass are the most used primary packaging materials. glass is widely used for packaging pharmaceuticals because of its excellent barrier properties, relative inertness, and compatibility with pharmaceuticals (polshettiwar, 2021). on the other hand, the growing use of plastics as a pharmaceutical packaging material is because of the significant advantages and consumer preference. plastics are lightweight and shatterproof. plastics are also easily shaped and sealed, which gives great versatility in the design of the pack (andjelković et al., 2021). stability study was executed to investigate the characteristics of royal jelly (rj) encapsulated in alginatepectin beads in different storage conditions with varying temperatures and relative humidity (rh). in general, a drug product should be evaluated under distinctive storage conditions that test its thermal stability and, if applicable, its sensitivity to moisture or potential for solvent loss (association of south east asian nations, 2005). alginate-pectin beads filled in pet opaque plastic bottles and glass containers were randomly sampled and later were kept in refrigerator (2-8 °c), room temperature (25 °c), a real time (30 °c and 75% rh) and an accelerated (40 °c and 75% rh) stability chambers. at different stability time points (0-day, 14-days, 1-month and 3-months), samples were taken out from each different storage condition for later characterisation. in this stability study, all the sample were characterised for physical appearance, particle size, sphericity coefficient, microscopic morphology, 10-hda content and compression testing. a suitable storage condition is crucial to ensure the quality of rj as its bioactivities are primarily influenced by its storage condition. the physical and chemical constituent of rj can change when it is stored improperly resulting in the loss of its functional properties. therefore, it is important to evaluate the effect of storage conditions on the quality of rj. materials the rj beads filled in pet opaque bottle and clear glass bottle were randomly sampled and stored (n=30) into four different storage conditions: in refrigerator, laboratory, accelerated and real time stability chambers. the kbf 240 accelerated and max 1400 real time stability chambers were provided from capromax (selangor, malaysia). methodology the stability study of rj beads was executed based on the pharmaceutical product guideline written in the asean guideline on stability study of drug product (2013). as mentioned earlier, rj bead samples were stored in refrigerator (2-8 °c), laboratory (25 °c), real time (30 °c, 75% rh) and accelerated (40 °c, 75% rh) stability chambers. at predetermined stability time point: 0-day, 14-days, 1-month and 3-months, samples were taken out (n=30) from each different storage condition. the stability samples were observed and characterised for physical appearances, particle size, sphericity coefficient, microscopic morphology, 10hda content and compression testing. the physical appearance of rj beads, namely under its organoleptic properties at different time points was compared to the appearance of the beads at 0-month stability time point. 1. determination of the diameter and sphericity coefficient of rj beads to achieve statistical result, thirty rj beads were randomly chosen and rinsed with distilled water after thirty minutes of gelation time. image analysis software (image j, national institute of health, usa) was used to measure the diameter of each of the rj beads taken. statistical data such as mean, median, and mode were generated automatically. the sphericity coefficient (sc) of rj beads was calculated using the following equation by houghton & amidon (1992) (equation 1): sc=dmin/dmax (eq. 1) where, dmin and dmax are minimum and maximum ferret’s diameters of the rj beads, respectively (shaiqah et al., 2020). beads with a sc value approaching 1 are considered ideal and spherical (azhar et al., 2021). azhar et al., (2023) journal of pharmacy, 3(1), xx-xx page 40 2. 10-hda analysis rj beads were immersed in the phosphate-buffer solution (pbs) with concentration 0.1 m and ph 6.8. the samples were subjected under vigorous stirring for 30 minutes until the alginate-pectin coating disintegrated. then, 25 ml of water and methanol (1:1, v/v) were added and the suspension formed was centrifuged at 4000 rpm for 10 minutes using rotofix 32 from andreas hettich gmbh & co. (tuttlingen, baden-württemberg). the 10-hda content in the rj beads was obtained by analysing the supernatant solution spectrophotometrically at 215 nm using hplc (waters e2695, waters corporation, milford, usa). 3. surface morphology prior to sem imaging, 10 to 15 rj beads were rinsed with an increasing gradient of ethanol concentration of 10%, 50%, 70%, 90%, and 100% and left air-dried for 30 minutes at room temperature (25-30 °c) for proper sample dehydration. the surface morphology of the beads was evaluated by using a scanning electron microscope at 100 and 500 times of magnification (sem, fei, quanta 450, thermofisher scientific, oregon, usa). 4. compression testing brookfield ct3 texture analyser (middleboro, usa) was used for uniaxial compression of a single rj bead. for statistically significant results, 30 rj beads were randomly selected from the samples that had been dipped previously in simulated gastric fluid or simulated intestinal fluid for 30 minutes. cylindrical aluminium probe with 6 mm diameter was attached to compress the bead at 1.0 mm/s. the trigger load of 0.05 n and peak deformation of up to 50% of the initial bead diameter were set. equation 2 was utilised to calculate young’s modulus, e (pa). 𝑬 = 𝟑 × (𝟏 − 𝝊𝟐) × 𝑭 √𝒅 × 𝑯𝟑 (eq. 2) where, d: diameter of the bead (m) f: trigger load (n) h: deformation of the bead (m) v: poisson’s ratio results and discussion 1. physical appearance rj is whitish grey colour. it is a complex compound that consist of water (60%-70%), proteins (27%-41%), carbohydrates (30%), lipids (8%-19%), free amino acids, trace mineral, and water-soluble vitamin (maghsoudlou, sadeghi mahoonak, mohebodini, & toldra, 2019). the colour of rj is a critical parameter as it acts as indicator of its freshness and suitability (zheng, wei, wu, hu, & dietemann, 2012). figures 1, 2, 3 and 4 display the appearance of rj beads at 0-day, 14-days, 1-month and 3-months stored in refrigerator, room temperature, real time, and accelerated stability chambers. the colour of rj beads in both type of packaging stored in the refrigerator remained whitish grey throughout this study (figure 1). in comparison, at 1-month and 3-months stability time points, the colour of the beads stored at room temperature turned purplish grey (figure 2). changing of rj beads colour also occurred in both accelerated and real time stability chambers at 1-month and 3-months stability time point for both packaging, where the beads changed from whitish grey to dark brownish yellow (figure 3 and figure 4). this result is in agreement with previous studies that reported browning reaction of rj during storage at room temperature as early 1-month storage (chen & chen, 1995; qiao, wang, liu, & zhang, 2018). this effect is attributed to the millard reaction or also known as non-enzymatic reaction where a chemical reaction occurs between amino acids present in rj and reducing sugar that produce brown colour. chen & chen, (1995) proposed that browning reaction was stimulated by the higher temperature in which this reaction was sensitive to the ambient temperature and higher temperature stimulate the reaction rates. during maillard reaction, a wide range of reaction products is formed, leading to significant alteration that affect nutritional value of nutraceuticals (starowicz & zieliński, 2019). in contrast, the colour change of rj beads at room temperature (laboratory) is observable starting at 3-months. at room temperature, pet bottle is believed to reduce and delay the change of colour of the rj beads. the opaqueness of the bottle aids in protecting the beads from direct sunlight and its subsequent heat changes. hence, at room temperature, pet bottle can serve as the best potential candidate for the storage of rj beads and can be considered for future recommendation in the market. azhar et al., (2023) journal of pharmacy, 3(1), xx-xx page 41 figure 1. the physical appearance of rj beads stored in refrigerator at four stability time points (0-day, 14-day, 1month and 3-month) stored in glass container stored in pet container s ta b il it y t im e p o in t 0 th d a y 1 4 th d a y 1 st m o n th 3 rd m o n th 1 mm 1 mm 1 mm 1 mm 1 mm 1 mm 1 mm 1 mm azhar et al., (2023) journal of pharmacy, 3(1), xx-xx page 42 figure 2. the physical appearance of rj beads stored in room temperature at four stability time points (0-day, 14-day, 1-month and 3-month) stored in glass container stored in pet container s ta b il it y t im e p o in t 0 th d a y 1 4 th d a y 1 st m o n th 3 rd m o n th 1 mm 1 mm 1 mm 1 mm 1 mm 1 mm 1 mm 1 mm azhar et al., (2023) journal of pharmacy, 3(1), xx-xx page 43 figure 3. the physical appearance of rj beads stored in real-time stability chamber at four stability time points (0-day, 14-day, 1-month and 3-month) stored in glass container stored in pet container s ta b il it y t im e p o in t 0 th d a y 1 4 th d a y 1 st m o n th 3 rd m o n th azhar et al., (2023) journal of pharmacy, 3(1), xx-xx page 44 figure 4. the physical appearance of rj beads stored in accelerated stability chamber at four stability time points (0day, 14-day, 1-month and 3-month) stored in glass container stored in pet container s ta b il it y t im e p o in t 0 th d a y 1 4 th d a y s 1 st m o n th 3 rd m o n th azhar et al., (2023) journal of pharmacy, 3(1), xx-xx page 45 2. particle size the particle size of the rj beads was measured and analysed in four different storage conditions that include refrigerator, laboratory, real time, and accelerated stability chambers. for better overview, figure 5 descriptively illustrates particle size of the randomly selected rj beads (n=30) at different stability study points. the mean bead diameter ranges between 1777 ± 121 µm to 1823 ± 199 µm. reduction of particle size was observed in all storage conditions for both types of beads container. however, only the particle size of rj beads stored in accelerated stability chamber had a decreasing pattern with significance (p<0.05) for both types of storage container. the reduction in particle size of rj beads stored in accelerated stability chambers across all stability time points might be an evident sign that 10-hda undergoes chemical and physical degradation when exposed to higher temperature (40 °c). on top of that, occurrence of total water loss in the beads via evaporation and disruption of polymeric cross-linking of the biopolymer by high temperature explains the further reduction of the bead diameter over time (bannikova, rasumova, evteev, evdokimov, & kasapis, 2017; vargas, pereira, guimarães, waldman, & pereira, 2018). nevertheless, rj beads stored in refrigerator, room temperature and real-time stability chamber manifests insignificant size reduction (p>0.1) from 0-month till 3-months for both types of storage container. the statistical analysis using two-factorial anova is also insignificant between all groups. figure 5: (a) particle size (µm) of the rj beads at different stability time point stored in pet container (n=30) (b) particle size (µm) of the rj beads at different stability time point stored in glass container (n=30) (a) (b) azhar et al., (2023) journal of pharmacy, 3(1), xx-xx page 46 3. sphericity coefficient the sphericity coefficient of rj beads should be near to 1.0 as indication of perfect circular shape is attained during electrospraying process (azhar et al., 2021). figure 6 illustrates sphericity coefficient values of the randomly selected rj beads (n = 30) in four storage conditions. all the sphericity coefficient values did not reach below 0.75. it shows that the spherical shape of the beads is maintained regardless of the storage conditions and time points. on top of that, there was no significant difference (p > 0.05) between sphericity coefficient values of rj beads stored in glass and pet bottle in refrigerator, room temperature and real-time stability chamber at 0 month and 14-day time point. however, the sphericity coefficient values of rj beads decreased in significant manner (p<0.05) for both containers inside accelerated stability chambers. it ranged from 0.81 at 1 month to 0.77 at 3 months in glass container and from 0.81 at 1 month to 0.76 at 3 months in pet container. these results are in agreement with the findings that increasing storage temperature of calcium-alginate beads yields more irregular internal structure, hence decreasing the rupture strength of the beads (jeong, kim, lee, cho, & kim, 2020). nevertheless, the sphericity coefficient of rj beads in both type of storage container stored in refrigerator manifests insignificant changes for 3-months stability time point indicating good physical stability of rj beads. figure 6: (a) sphericity coefficient of the rj beads at different stability time point stored in glass container (n=30) (b) sphericity coefficient of the rj beads at different stability time point stored in pet container (n=30) (a) (b) azhar et al., (2023) journal of pharmacy, 3(1), xx-xx page 47 4. microscopic morphology figures 7 and 8 manifest the microscopic morphology of random rj beads sample under different magnification of sem at 0 and 3-month storage in refrigerator, room temperature, real time, and accelerated stability chambers. it is observed that microscopic morphology of the beads remained relatively indifferent after 3 months of storing in both glass and pet container. figure 7: sem photographs of rj beads of selected samples at 0-month time-point (a: surface at magnification 100 µm; b: surface at magnification 500 µm) figure 8: sem photographs of rj beads of selected samples at 3-month time point (a: surface at magnification 100 µm; b: surface at magnification 1 mm) 5. 10-hda assay content 10-hda is one of the main bioactive compounds of rj that is only found in rj in nature (khazaei, ansarian, & ghanbari, 2018). thus, the present of 10-hda can be used as a marker to validate the freshness and quality of rj (antinelli et al., 2003; kim & lee, 2010). table 1 summarises the variation of 10-hda content in four different conditions. analysis using one-way anova inferred that the difference in 10-hda content (%, w/v) in rj beads stored in accelerated stability chamber was the only one that is statistically significant (p<0.05) for 1-month and 3-months compared to 0-month time point (table 1). both types of storage container, pet and glass type manifest similar findings with no significance (p> 0.05). nonetheless, the 10-hda content of rj in alginatepectin beads store in refrigerator remain the same throughout the study for both type of storage container. this result is in accordance with the colour changes reported in this study. changing of rj beads colour is correlated with degradation of 10-hda content. maintaining required amount of 10-hda is encouraged as a low 10-hda content implies a low rj activity (muñoz, decap, ruiz, arbildua, & monasterio, 2011). the variation of 10-hda content (mg/ml) between stability time points and type of container can be optimised by proper standardisation of the procedure in preparing the samples. in addition to the abovementioned measure, the declining pattern of 10-hda content occurred in the beads stored in room temperature and both real-time and accelerated stability chambers signals rj deterioration. nutraceutical products, which include the formulation of rj beads contain numerous phytoconstituents of different chemical classes (maghsoudlou et al., 2019). these constituents may undergo various interas well as intra-molecular reactions under the influence of varied environmental conditions, such as heat, humidity, air and/or light during processing, formulation and storage of the material (maghsoudlou et al., 2019; ramadan & alghamdi, 2012). 10-hda is a major fatty acid in rj can degrade under the influence of heat and light which may, in turn, alter the actual content, shorten shelf-life and reduce therapeutic efficacy of the final products. recent studies propose that 10-hda is the leading indicator in the determination of the freshness, however, inconsistent stability of this fatty acid will cause as long-term challenge for the standardisation of rj formulation (shen et al., 2015). in this study, 10-hda represents an adulteration indicator and should be above 1.4% for fresh rj (abdulqader yaslam bazeyad, ahmad t y p e o f st o ra g e c o n d it io n r e fr ig e ra to r (2 -8 °c ) r o o m t e m p e ra tu re ( 2 5 °c ) r e a lti m e (3 0 °c  2 , r h 7 5 %  5 ) a c c e le ra te d (4 0 °c  2 , r h 7 5 %  5 ) a b b a a b a b azhar et al., (2023) journal of pharmacy, 3(1), xx-xx page 48 abdullah al-ghamdi, & yehya zaki alattal, 2022). the values for the analysed rj samples were within the limits proposed by the iso rj international standard (international organization for standardization, 2016), which sets the minimum concentration of 10-hda is 1.4% for pure rj. in brief, the best storage conditions to preserve critical quality attributes (cqa) of rj beads especially the 10-hda content itself is the refrigerator (2-8 °c) with the most practical and versatile packaging of pet. the low temperature of storage condition reduces the phenomenon of oxidation and hydrolysis reaction subsequently minimising physical and chemical degradation of rj beads and later extending its shelf-life (muresan et al., 2016). 6. compression testing determination of degree of deformation in compression testing is crucial to assess the physical stability of the rj beads during storage and transportation as well as its bioavailability when exposed to human gastric and intestinal environment (rayment et al., 2009). the higher the young’s modulus being expressed in the compression testing, the higher the force needed for the beads to resist deformation phenomenon (lee, zhang, & ryu, 2018). figures 9, 10 and 11 summarise all the young’s modulus values of rj beads being stored in two types of containers (glass and pet) with four different storage conditions; refrigerator, room temperature, real-time and accelerated stability chambers. all the young’s modulus values were above 7 x 1000 pa at all stability time points when the beads were tested air-dried as well as when being dipped into simulated gastric solution for 2 hours regardless the type of container used (figure 9 and figure 10). this proves that the encapsulation of rj beads using alginate and pectin confers marked improvement of mechanical protection to the formulation in these both conditions. however, when the beads were subjected under simulated intestinal fluid for 2 hours, the values of young’s modulus exhibited substantial decreasing trends from 7.7 x 1000 pa at 0 month to 2.32 x 1000 pa at 3 month with statistical significance (p<0.05) (figure 11). it was inferred that the decreasing pattern of young’s modulus is due to the reduction rj beads rigidity in alkaline environment as compared to in acidic condition (abu, rasel, & hasan, 2012). more open and porous polymeric network formed during rigorous swelling in alkaline environment. by increasing the dipping time of the beads in the simulated intestinal fluid, the activity contributes to the gradual reduction of the deformation and its corresponding young’s modulus values. the reason for this is that mannuronic and guluronic acids residue of alginate have pka values of 3.38 and 3.65 respectively. the beads are stabilised by intermolecular hydrogen bonding network in a gastric environment where the ph value (1.5 – 3.5) is lower than the pka of the uronic acid (pawar & edgar, 2012). meanwhile in simulated intestinal condition as the ph rises above the pka of the polysaccharides, the beads are expected to disintegrate due to the deprotonation of the polysaccharides of alginate beads leading to electrostatic repulsion and eventually disintegration of the beads (chuang et al., 2017; marciani et al., 2019). table 4: results of intraday and interday precision for the simultaneous quantification of inh and pyr in pure form storage conditions stability time point 10-hda content (%, w/v) in glass container 10-hda content (%, w/v) in pet container refrigerator (2-8 °c) 0 month 1.83 ± 0.1 1.83 ± 0.1 14 days 1.83 ± 0.7 1.83 ± 0.7 1 months 1.83 ± 0.3 1.83 ± 0.9 3 months 1.83 ± 0.1 1.83 ± 0.2 laboratory (25 °c) 0 month 1.83 ± 0.1 1.83 ± 0.1 14 days 1.83 ± 0.6 1.83 ± 0.5 1 months 1.79 ± 0.4 1.79 ± 0.7 3 months 1.77 ± 0.6 1.77 ± 0.1 real time (30 °c  2, rh 75 %  5) 0 month 1.83 ± 0.1 1.83 ± 0.1 14 days 1.83 ± 0.9 1.83 ± 0.2 1 months 1.78 ± 0.6 1.78 ± 0.3 3 months 1.75 ± 0.2 1.75 ± 0.7 accelerated (40°c + 2, rh 75 %  5) 0 month 1.83 ± 0.1 1.83 ± 0.1 14 days 1.83 ± 0.2 1.83 ± 0.6 1 months 1.74 ± 0.8 1.74 ± 0.1 3 months 1.71 ± 0.1 1.71 ± 0.1 azhar et al., (2023) journal of pharmacy, 3(1), xx-xx page 49 figure 9: (a) young’s modulus of the rj beads (airdried) at different time point stored in glass container (b) young’s modulus of the rj beads (air-dried) at different time point stored in pet container. figure 10: (a) young’s modulus of the rj beads (dipped in simulated gastric fluid) at different time point stored in glass container (b) young’s modulus of the rj beads (dipped in simulated gastric fluid) at different time point stored in pet container. (a) (b) (a) (b) azhar et al., (2023) journal of pharmacy, 3(1), xx-xx page 50 figure 11: (a) young’s modulus of the rj beads (dipped in simulated intestinal fluid) at different time point stored in glass container (b) young’s modulus of the rj beads (dipped in simulated intestinal fluid) at different time point stored in pet container. conclusion in conclusion, the study showed that the rj beads exhibited convincing stability for 3 months when it is stored at low temperature. analysis of 10-hda content in the rj-encapsulated beads as well as observation of its physicochemical properties that includes physical appearances, particle size, sphericity coefficient, microscopic morphology, and compression testing showed that the results are highly consistent across all stability time points. environmental conditions especially temperature must be considered during beads preparation and storage since room temperature will accelerate physicochemical changes of rj beads. therefore, rj beads must be stored at low temperature (refrigerator) to maintain the stability of formulation and provide a longer shelf life. besides, this study also exhibits that colour changes of rj beads is well correlated with the degradation of the 10-hda compound. the investigation using different types of container substance proves similar results regardless of the storage conditions. in practicality, pet container offers higher superiority to the consumers compared to glass container as packaging for rj beads due to its convenience in terms of low cost and lightweight. on top of that, opaqueness and inertness of pet container serve as additional advantages to protect the rj beads from gradual deterioration to sunlight, heat and moisture. conflict of interest the authors declare that there is no conflict of interest. references abdulqader yaslam bazeyad, ahmad abdullah alghamdi, & yehya zaki alattal. 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(2022) journal of pharmacy, 2(2), 149-158 page 149 knowledge, attitude, and practice towards antibiotic use among the public in the city of kuantan, pahang state, malaysia. muhammad eid akkawi1, nashaat abdulkareem al-shami2, yaser mohammed al-worafi3, abdullah abdulmajid abdo ahmed1, abdulkareem mohammed al-shami4* abstract background: public knowledge and attitudes towards antibiotic use play a vital role in the success of controlling antibiotic resistance. this study aimed to assess the public’s knowledge, attitudes, and practice (kap) towards antibiotic use in kuantan, malaysia. methods: this cross-sectional study was conducted among the general public above 18 years old. the data was collected in public places using a pre-validated questionnaire. software statistical package for social sciences (spss) was used for statistical analyses. results: out of 410 participants who answered the questionnaire, 229 (55.9%) of them were female and 288 (70.3%) had a college/university degree. it was found that 258 participants (62.9%) have good knowledge and 306 of them (74.6%) have positive attitude towards antibiotic use. however, 160 respondents (39%) believe that antibiotics can kill viruses and 183 respondents (44.6%) stated that they would ask the doctor to give them antibiotics in case they have common cold symptoms. having a good knowledge was significantly related to the age (p=0.005), employment status (p<0.001), education level (p<0.001), and the field of work or study (p<0.001). furthermore, 313 of the sample (76.3%) had an overall good practice. however, 162 participants (39.5%) do not complete their antibiotic course. additionally, 57 participants (13.9%) share and 69 of them (16.8%) use the leftover antibiotics. showing a good practice correlated to the field of work/study (p =0.04), level of knowledge (spearman's rho = 0.373; p<0.001) and attitude (spearman's rho = 0.55; p<0.001). conclusion: although the majority of the public showed good kap, there are still important gaps that need to be filled. therefore, a public educational campaign is needed to improve the knowledge, attitude and practice towards antibiotic use. article history: received: 12 may 2022 accepted: 17 july 2022 published: 31 july 2022 keywords: antibiotics use; public; knowledge; attitude; practice; malaysia how to cite this article: akkawi, m. e., al-shami, n. a., alworafi, y. m., abdo ahmed, a. a. & al-shami, a. m. knowledge, attitude, and practice towards antibiotic use among the public in the city of kuantan, pahang state, malaysia. journal of pharmacy, 2 (2)., 149-158. doi: 10.31436/jop.v2i2.157 authors’ affiliation: 1department of pharmacy practice, faculty of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, indera mahkota, kuantan, pahang, malaysia. 2the islamic hospital, alabdali, amman, jordan. 3college of pharmacy, university of science and technology of fujairah, fujairah, uae. 4faculty of pharmacy, picoms international university college, taman batu muda, 68100 batu caves, kuala lumpur, malaysia. *corresponding author: email address: dhamarali2002@yahoo.com; drkareem@picoms.edu.my tel: 0060124241740 shirazi et al. (2022) journal of pharmacy, 2(1), 16-30 page 150 introduction antibiotics are the mainstay treatment of bacterial infections. however, as antibiotic resistance has escalated rapidly, healthcare professionals are facing a problem of prescribing an effective antibiotic even for common bacterial infections. at the same time, the development of new antibiotics constantly declined (lópez romo & quirós, 2019). antibiotic resistance happens naturally as part of microbial evolution, but it can be spread up by human activities (sartelli et al., 2017). such activities include overuse of antibiotics, improper prescribing of antibiotics and self-medication with antibiotics. furthermore, the antibiotic agents that have been utilized unnecessarily and extensively can increase the antibiotic resistance (michael, dominey-howes, & labbate, 2014). in many developing countries, antibiotics can be obtained as over the counter drugs. moreover, antibiotics can also be purchased through online marketing platforms (like, aliexpress and lazada). all these situations increase the accessibility towards antibiotics hence promote their overuse (ventola, 2015). apart from that, another contributing factor in the arising of antibiotic resistance is inappropriate prescribing of antibiotics and this practice can affect the therapeutic outcome as well as can cause undesirable side effects to the patient. if antibiotics are prescribed at subtherapeutic amounts, then this practice can lead towards the advancement of the microbial resistance mechanism by genetic alteration (ventola, 2015). this suggests that antibiotics are widely misused and health-care professionals especially doctors, can play a vital role regarding this issue. this situation is confirmed by the data from the centers for disease control and prevention (cdc). it was reported that prescribing antibiotics to the outpatient is declined by 5% in the united states of america (usa) from 2011 to 2014 but according to cdc about 30% of these prescriptions were not needed to be done (center for disease control and prevention, 2017). on the other hand, a study that was carried out in primary care clinics within malaysia showed that the rate of antibiotic prescribing was 21.1% with evidence of improper and excessive use of antibiotics for conditions that were self-limiting (ab rahman et al., 2016). inadequate knowledge of the proper use of antibiotics was reported in various developing countries. for example, a moderate knowledge and behavior score and a low belief score of the were observed in oman (jose et al., 2013). likewise, chen et al reported incorrect knowledge among residents of changhua county in taiwan(chen et al., 2005). the inadequate knowledge has contributed to the high increase in self-medications with these agents (c o omolase et al., 2007; ekambi et al., 2019). for instance, antibiotic users believed that antibiotics are always necessary for the treatment of flu or flu-like symptoms. also, people usually stop taking antibiotics when symptoms improve (chen et al., 2005; jifar & ayele, 2018; vanden eng et al., 2003). in a study conducted by the world health organization (who) among publics in 12 different countries it showed that 64% of them believed that cold and flu can be resolved when antibiotics are given. moreover, the survey also showed that 34% of the participants thought that antibiotic should be stopped when they feel better (who media centre, 2016). malaysia is not an exception. a couple of studies showed poor to moderate levels of knowledge the public in some malaysian states (choo et al., 2018; chow & nor liana, 2020; ka keat & chew charn, 2012). however, there was no study focused on pahang state, the largest state in the malaysian peninsula. the current study aims to examine the knowledge, attitude, and practice regarding antibiotics use focusing among the public in kuantan, pahang state, malaysia. methodology design and setting of a study a cross-sectional study was conducted over two months from october to december 2019 among the general public in kuantan, pahang, malaysia. the data was collected through a validated structured questionnaire using the convenience sampling technique. the inclusion criteria were: (1) adults aged 18 years and over; (2) able to read and understand malay language and (3) aware of the term ‘antibiotics’ (individuals were asked if they know the meaning of antibiotic agent). individuals filled out the questionnaire in the presence of a researcher to explain the questions and to make sure that individuals’ answers were based on their current knowledge. study population and sample size the participants included in this study were the general public adults. the sample size was calculated by the raosoft® online sample size calculator. based on the census malaysia, there were approximately 529600 people in kuantan city in 2019 and population size was also confirmed from the office of kuantan municipal council (mpk). the estimated sample size was 384 individuals with a margin error of 5% and confidence 95% and 50% distribution. however, the collected sample size was 410 participants. development and validation of the study instrument the questionnaire used in this study was developed from previous studies (ab rahman et al., 2016; ka keat & chew charn, 2012; oh et al., 2011; zahreddine et al., 2018) performed among different nations. the questionnaire was initially developed in english language and translated into bahasa melayu language. the questionnaire has been translated into bahasa melayu and subjected to a process of forward and backward translation. the accuracy and meaning of the translated versions both forward and backward were checked, and recommended adjustments were discussed shirazi et al. (2022) journal of pharmacy, 2(1), 16-30 page 151 before being finalized. then, the questionnaire was validated by five academic staff from the faculty of pharmacy at international islamic university malaysia (iium). the feedback received from them was collected to refine the questionnaire conformity and amendments are made to the questionnaire. furthermore, the questionnaire was tested with a pilot study which was conducted by distributing the modified questionnaire to 30 individuals from the public in kuantan. the internal consistency for the questionnaire was assessed using cronbach’s α test. the cronbach’s α values for knowledge, attitude and practice sections were 0.791, 0.762 and 0.784, respectively. the final questionnaire comprised of four different sections, section a consists of eight demographic features such as (age, gender, ethnicity, marital status, education...etc.). meanwhile, section b recorded participants’ knowledge on antibiotic use and it consists of nine statements concerning fundamental knowledge of antibiotics including functions of antibiotics, indications of antibiotics, identification of antibiotics, course of antibiotics, and antibiotic resistance. participants were required to respond with either “yes”, “no”, or “not sure”. section c constituted seven attitude statements and participants were asked to answer using a 5-point likert scale (1=strongly agree; 2=agree; 3=not sure; 4=disagree and 5=strongly disagree). section d documented participants’ practice of antibiotic use, which comprised of 8 questions. regarding the scoring system, incorrect answers or “not sure” answers were given 0 points meanwhile correct answers” were given 1 point in part b (knowledge section) with a maximum score of 9 points. in part c and part d, negative attitude and bad practice were given 0 points meanwhile positive attitudes and good practice were given 1 point. scores for each section were summed up and good knowledge, positive attitude and good practice were considered when a participant scored more than half of the total points. statistical analysis software statistical package for social sciences (spss) was used. descriptive statistics were used in data analysis and results were presented in forms of frequency and percentage. frequency tables were drawn based on the data analysis. the relationship between demographic characteristics and knowledge, attitude, and practice were analyzed using pearson’s chi-squared or fisher’s tests. spearman’s correlation coefficient was used to test for correlation between the scores of the different sections. results participants’ characteristics out of 410 participants, about half of the participants (55.9%) were female. about half of the participants (52.9%) aged between 18-30 years old, followed by 3145 years old (22%). approximately half of the participants were married and most of them 240 (55.4%) were malay. also, three-quarters of participants (70.3%) had a college or university degree. the majority of the respondents (82.6%) were working or studying in a nonhealthcare related field. all details regarding demographic characteristics are shown in table 1. knowledge regarding antibiotic use knowledge regarding antibiotic utilization was evaluated by using nine statements. about two-thirds of the participants (62.9%) showed good knowledge with median and mean±sd scores of 5 and 5.23±2.22, respectively. the majority of respondents (81%) knew that antibiotic can kill bacteria. however, 39% of them answered “yes” or “not sure” when asked if antibiotic can kill viruses. on the other hand, (85.4%) were aware that not completing the antibiotic course can reduce its effectiveness. table 1 shows the comparison of knowledge score between different groups whereby the total knowledge score of the participants towards antibiotic use was found to be significantly affected by the age (p=0.005), ethnicity (p=0.014), employment status (p<0.001), education level (p=7.2857) and the field of work or study (p=<0.001). a high knowledge level was noted among 18-30 years old participants (69%) followed by other age groups. malay participants (69%) showed a significantly higher knowledge level as compared to other ethnicities. students (74%) portrayed significantly higher knowledge scores compared to other people. also, high education was associated with higher level of knowledge. on the other hand, no statistical difference was found based on the gender or the area of residency. attitude towards antibiotics use most of respondents (74.6%) showed a positive attitude towards a rational use of antibiotics with a median of 5. more than 75% of the participants disagreed to selfmedicate with antibiotics, to keep stocks of antibiotics at home, or to consume unfinished antibiotics from previous use. also, 55.4 % of the respondents disagreed with asking the healthcare professional to give them antibiotics for common cold. on the other hand, about half of the participants (51.7%) tend to stop the antibiotics when their condition gets better. table 3. shirazi et al. (2022) journal of pharmacy, 2(1), 16-30 page 152 table 1: patients’ characteristics and their correlation with the knowledge, attitude and practice. (n: 410) patients’ characteristics n (%) good knowledge (%) p value positive attitude (%) p value good practice (%) p value gender 0.422 0.001 0.102 female 229 (55.9) 61 66.9 72.4 male 181 (44.1) 65 80.8 79.5 age 0.005 0.460 0.060 18-30 years old 217 (52.9) 69 71.4 72.8 31-45 years old 90 (22) 63 78.9 86.7 46-60 years old 69 (16.8) 54 76.8 79.7 ˃60 years old 34 (8.3) 41 79.4 64.7 highest education <0.001 (fisher’s exact test) 0.024 (fisher’s exact test) 0.519 (fisher’s exact test) college or university 287 (70) 0 50 50 secondary education 102 (24.9) 32 68.4 73.7 primary education 19 (4.6) 40 73.5 73.5 no formal education 2 (0.5) 74 75.6 77.7 employment status <0.001 (fisher’s exact test) 0.170 (fisher’s exact test) 0.153 (fisher’s exact test) employed 181 (44.1) 69 79.6 80.7 student 94 (22.9) 46 68.5 68.5 self-employed 92 (22.4) 45 64.5 74.2 unemployed 31 (7.6) 74 73.4 74.5 retiree 12 (2.9) `67 83.3 91.7 field of work or study (occupation) <0.001 0.004 0.004 non-healthcare related 336 (82.6) 85 87.3 88.7 healthcare related 71 (17.4) 58 72 73.7 shirazi et al. (2022) journal of pharmacy, 2(1), 16-30 page 153 table 2: knowledge about antibiotic use (n:410) questions correct answer n (%) incorrect answer n (%) do you think antibiotics can be used to kill bacteria? 332 (81) 78 (19) do you think antibiotics can be used to kill viruses? 250 (61) 160 (39) do you think antibiotics are used to relieve cough and colds? 254 (62) 156 (38) do you think antibiotics are consumed to cure inflammation? 283 (69) 127 (31) do you think penicillin is one of the antibiotics? 206 (50.2) 204 (49.8) do you think paracetamol is an antibiotic? 311 (75.9) 99 (24.1) the incomplete course of antibiotics can reduce treatment effectiveness. 350 (85.4) 60 (14.6) the consumption of antibiotics needlessly can cause bacteria to be resistant against antibiotics. 270 (65.9) 140 (34.1) antibiotic resistance is considered as a global problem. 234 (57.1) 176 (42.9) table 3. participants’ response towards attitude questions (n: 410) positive attitude negative attitude statement strongly disagree n(%) disagree n(%) not sure n(%) agree n(%) strongly agree n(%) i prefer getting antibiotics from a private clinic or a pharmacy without having to see a doctor. 267 (65.1) 62 (15.1) 42 (10.2) 8 (2.0) 31 (7.6) i will ask doctors or pharmacists to give me antibiotics if i suffer from common cold symptoms 170 (41.5) 57 (13.9) 69 (16.8) 44 (10.7) 70 (17.1) shirazi et al. (2022) journal of pharmacy, 2(1), 16-30 page 154 i tend to stop taking antibiotics when my health condition improves. 154 (37.6) 48 (11.7) 53 (12.9) 51 (12.4) 104 (25.4) i will consume my unfinished antibiotics from previous use. 270 (65.9) 49 (12) 47 (11.5) 19 (4.6) 25 (6.1) i will keep stocks of antibiotics at home for future use. 277 (67.6) 54 (13.2) 33 (8.0) 23 (5.6) 23 (5.6) negative attitude positive attitude i will consume the antibiotics as instructed on the label. 11(2 .7) 4 (1.0) 20 (4.9) 71 (17.3) 304 (74.1) table 4. practice of the study participants regarding antibiotic use (n:410) yes n(%) no/sometimes n(%) i check for the expiry date of the antibiotics before consuming them. 78 (19) 332 (81) do you usually complete the course of antibiotics prescribed? 248 (60.5) 162 (39.5) have you ever shared antibiotics with other sick patients? 57 (13.9) 353 (86.1) do you usually consume the leftover antibiotics from previous course of illness? 69 (16.8) 341 (83.2) shirazi et al. (2022) journal of pharmacy, 2(1), 16-30 page 155 table 5: correlation between the different domains of the survey (n:410). section score spearman's rho knowledge attitude practice knowledge median (iqr): 5 (3) mean (sd): 5.3 (2.2) correlation coefficient 1.00 0.385* 0.373* p value < 0.001 < 0.001 attitude median (iqr): 5 (2) mean (sd): 4.2 (1.5) correlation coefficient 0.385* 1.00 0.550* p value < 0.001 < 0.001 practice median (iqr): 3 (1) mean (sd): 3.1 (0/9) correlation coefficient 0.373* 0.550* 1.00 p value < 0.001 < 0.001 *correlation is significant at the 0.01 level (2-tailed). the data analysis demonstrated that gender (p=0.001), level of education (p = 0.024) and the field of work or study (p=0.004) of the participants significantly affect their attitude toward the antibiotic use. on the other hand, no association was found between the attitude towards antibiotic use and age, ethnicity, employment status, area of residency and the education level. table 1. practice regarding antibiotics use most of the respondents (62.5%) reported that they get their antibiotics from hospitals or governmental clinics. a respiratory tract infection was the most common reason of getting antibiotic prescription (69.5%). four out of the eight questions in the practice section were used to evaluate the participant’s practice (table 4). the majority of the participants (76.3%) had good practice with a median of 3. only 13.9% of the respondents reported sharing their antibiotics with other people before. also, a comparable percentage of participants (16.8%) use their leftover antibiotics. remarkably, about two-fifth of the respondents do not complete the course of a prescribed antibiotic. table 4. table 1 shows the comparison of practice scores between different groups. respondents who worked in a health-related field had higher practice score compared to their counterparts (p=0.04). nevertheless, no statistical significance was found between the practice of antibiotic use and gender, age, ethnicity, employment status or education level. correlation between the three domains it was found that the scores of knowledge, attitude and practice sections are significantly correlated. table 5. the same applied to the status of each domain. good knowledge, positive attitude and good practice were correlated to each other (p < 0.001). discussion the current study evaluated the knowledge, attitude and practice of the general public lives in kuantan city the capital of pahang state, the largest state in the malaysian peninsula. overall, the general public had a good knowledge about antibiotic use as the majority of the participants answered more than half of the questions correctly. also, most of the participants (74.6%) showed positive attitude towards antibiotic use. these findings contradict the results reported from studies conducted in malaysia (chow & nor liana, 2020; oh et al., 2011) and other countries (jifar & ayele, 2018; mouhieddine et al., 2015), where the participants showed low to moderate knowledge as well as negative attitude related to antibiotic use. this discrepancy could be attributed to the differences in the participants’ characteristics and the scoring systems used. for instance, most of the participants in the above-mentioned studies (chow & nor liana, 2020; jifar & ayele, 2018; oh et al., 2011) had primary or secondary education compared to 74% having high education in our study. also, the above studies categorized the knowledge into poor, moderate and good levels. about one-third of the participants thought that antibiotic can kill viruses and be relieve common cold symptoms. however, this percentage was higher in previous malaysian studies (choo et al., 2018; ka keat & chew charn, 2012; oh et al., 2011). related to this issue, about one-quarter of the respondents stated that they would ask doctors or pharmacists to give them antibiotics if they suffer from common cold symptoms. likewise, this percentage was much higher in other studies from malaysia (chow & nor liana, 2020; ka keat & chew charn, 2012; oh et al., 2011) and the usa (vanden eng et al., 2003). patients’ expectation of having an antibiotic prescription when visiting a doctor was reported as one of the contributing factors for inappropriate antibiotic prescribing (teixeira rodrigues et al., 2013). shirazi et al. (2022) journal of pharmacy, 2(1), 16-30 page 156 the most common negative attitude was the tendency to stop antibiotic when the condition improves which was reported by more than one-third of the participants. similar finding was previously reported from malaysia (ka keat & chew charn, 2012; oh et al., 2011), lebanon (mouhieddine et al., 2015) and ethiopia (jifar & ayele, 2018). previous studies reported an association between the participants’ knowledge and their demographics (ka keat & chew charn, 2012; mouhieddine et al., 2015; oh et al., 2011). likewise, our study found that age, ethnicity, employment, education level and occupation significantly affect the knowledge level. however, only gender, level of education and occupation affected the participants’ attitude. regarding the practice of antibiotic use, the majority of respondents had good overall practice. however, in alignment to the attitude of stopping antibiotic when the condition improves, 39.5% of them do not complete the prescribed antibiotic course. this is a common practice that frequently reported in the literature (chen et al., 2005; chow & nor liana, 2020; islahudin et al., 2014). although, not finishing antibiotic course is widely thought to increase bacterial resistance, modern evidence does not support this belief (langford & morris, 2017). however, completing the antibiotic course is essential to ensure eradication of the pathogens (michael, dominey-howes, labbate, et al., 2014). a fewer participants showed kinds of selfmedication practice with antibiotics. they share their antibiotic with sick patients (13.9%) and use the leftover antibiotic from previous course (16.8%). these findings are similar to what have been reported from malaysia before (ka keat & chew charn, 2012; oh et al., 2011) and other countries in the far east (chen et al., 2005; you et al., 2008). nevertheless, they are much lower than those reported from some other countries in the middle east (jose et al., 2013; langford & morris, 2017). this could be attributed to less strict legislations of selling antibiotics in community pharmacies in the middle east countries. good practice regarding antibiotic use was significantly associated with the occupation and employment status. other demographic characteristics like gender and education level were reported as influencing factors on practice in another malaysian study conducted in perak state (choo et al., 2018). level of knowledge, attitude and practice regarding antibiotic use were correlated to each other’s. participants who had good knowledge were more likely to have positive attitude and consequently good practice. it is logical that people who have higher knowledge regarding antibiotic to be wiser while using such kind of medications. conclusion this study identified the knowledge, attitude and practice towards antibiotics use among public in kuantan. the study found that most of the participants showed an overall good knowledge coupled with positive attitude and good practice. however, significant percent of the participants showed inadequate knowledge about the role of antibiotics in viral infections which resulted in requesting antibiotic when they have common cold symptoms. although only a small portion of respondents use and share the leftover antibiotics, this would translate into the practice of about 75000 people live in kuantan. therefore, a public educational campaign is needed to improve the knowledge, attitude and practice towards antibiotic use. strengths and limitations the current study evaluated the three aspects (knowledge, attitude and practice) related to antibiotic use using an updated questionnaire based on a comprehensive literature review. the sample involved in the study was big enough to represent the city of kuantan. however, this study has some limitations. first, the majority of the participants had college/university degree which could underestimate the problem of irrational use of antibiotics among laymen population. second, the study was conducted in several public places in the downtown of kuantan city which may not reflect the actual kap of people living in rural areas. acknowledgements the authors would like to thank financial assistance received from sultan ahmad shah medical centre (sasmec) research grant 2021 and research cluster research initiative grant scheme (rc-rigs21). both research grants were provided by the international islamic university malaysia. the funders had no role in the study design and data interpretation. conflict of interest the authors declare no conflict of interest related to this study. funding none. ethical considerations ethical approval was gotten from the international islamic university malaysia research ethics committee (irec) (ref: iium/504/14/11/2/ irec-2019 231). written informed consent was obtained from respondents who agreed to participate in the study. authorship statement ama, naa conceptualized and designed the study and revised the manuscript, aaaa collected the data and wrote the draft manuscript, mea analyzed the data and revised the manuscript, yma designed the study and revised the manuscript. shirazi et al. 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(2018). knowledge of pharmacists and parents towards antibiotic use in pediatrics: a cross-sectional study in lebanon. pharmacy practice, 16(3). https://doi.org/10.18549/pharmpract.2018.03.1194 introduction antibiotics are the mainstay treatment of bacterial infections. however, as antibiotic resistance has escalated rapidly, healthcare professionals are facing a problem of prescribing an effective antibiotic even for common bacterial infections. at the ... apart from that, another contributing factor in the arising of antibiotic resistance is inappropriate prescribing of antibiotics and this practice can affect the therapeutic outcome as well as can cause undesirable side effects to the patient. if anti... inadequate knowledge of the proper use of antibiotics was reported in various developing countries. for example, a moderate knowledge and behavior score and a low belief score of the were observed in oman (jose et al., 2013). likewise, chen et al repo... methodology design and setting of a study a cross-sectional study was conducted over two months from october to december 2019 among the general public in kuantan, pahang, malaysia. the data was collected through a validated structured questionnaire using the convenience sampling technique. t... study population and sample size the participants included in this study were the general public adults. the sample size was calculated by the raosoft® online sample size calculator. based on the census malaysia, there were approximately 529600 people in kuantan city in 2019 and pop... development and validation of the study instrument the questionnaire used in this study was developed from previous studies (ab rahman et al., 2016; ka keat & chew charn, 2012; oh et al., 2011; zahreddine et al., 2018) performed among different nations. the questionnaire was initially developed in eng... the final questionnaire comprised of four different sections, section a consists of eight demographic features such as (age, gender, ethnicity, marital status, education...etc.). meanwhile, section b recorded participants’ knowledge on antibiotic use... regarding the scoring system, incorrect answers or “not sure” answers were given 0 points meanwhile correct answers” were given 1 point in part b (knowledge section) with a maximum score of 9 points. in part c and part d, negative attitude and bad pra... statistical analysis software statistical package for social sciences (spss) was used. descriptive statistics were used in data analysis and results were presented in forms of frequency and percentage. frequency tables were drawn based on the data analysis. the relationsh... results participants’ characteristics out of 410 participants, about half of the participants (55.9%) were female. about half of the participants (52.9%) aged between 18-30 years old, followed by 31-45 years old (22%). approximately half of the participants were married and most of them... knowledge regarding antibiotic use knowledge regarding antibiotic utilization was evaluated by using nine statements. about two-thirds of the participants (62.9%) showed good knowledge with median and mean±sd scores of 5 and 5.23±2.22, respectively. the majority of respondents (81%) k... table 1 shows the comparison of knowledge score between different groups whereby the total knowledge score of the participants towards antibiotic use was found to be significantly affected by the age (p=0.005), ethnicity (p=0.014), employment status (... attitude towards antibiotics use most of respondents (74.6%) showed a positive attitude towards a rational use of antibiotics with a median of 5. more than 75% of the participants disagreed to self-medicate with antibiotics, to keep stocks of antibiotics at home, or to consume unfini... the data analysis demonstrated that gender (p=0.001), level of education (p = 0.024) and the field of work or study (p=0.004) of the participants significantly affect their attitude toward the antibiotic use. on the other hand, no association was foun... practice regarding antibiotics use most of the respondents (62.5%) reported that they get their antibiotics from hospitals or governmental clinics. a respiratory tract infection was the most common reason of getting antibiotic prescription (69.5%). four out of the eight questions in t... table 1 shows the comparison of practice scores between different groups. respondents who worked in a health-related field had higher practice score compared to their counterparts (p=0.04). nevertheless, no statistical significance was found between... correlation between the three domains it was found that the scores of knowledge, attitude and practice sections are significantly correlated. table 5. the same applied to the status of each domain. good knowledge, positive attitude and good practice were correlated to each other (p < 0.001). discussion the current study evaluated the knowledge, attitude and practice of the general public lives in kuantan city the capital of pahang state, the largest state in the malaysian peninsula. overall, the general public had a good knowledge about antibiotic u... the most common negative attitude was the tendency to stop antibiotic when the condition improves which was reported by more than one-third of the participants. similar finding was previously reported from malaysia (ka keat & chew charn, 2012; oh et a... regarding the practice of antibiotic use, the majority of respondents had good overall practice. however, in alignment to the attitude of stopping antibiotic when the condition improves, 39.5% of them do not complete the prescribed antibiotic course. ... conclusion this study identified the knowledge, attitude and practice towards antibiotics use among public in kuantan. the study found that most of the participants showed an overall good knowledge coupled with positive attitude and good practice. however, signi... strengths and limitations the current study evaluated the three aspects (knowledge, attitude and practice) related to antibiotic use using an updated questionnaire based on a comprehensive literature review. the sample involved in the study was big enough to represent the city... acknowledgements the authors would like to thank financial assistance received from sultan ahmad shah medical centre (sasmec) research grant 2021 and research clusterresearch initiative grant scheme (rc-rigs21). both research grants were provided by the internation... conflict of interest the authors declare no conflict of interest related to this study. funding none. ethical considerations ethical approval was gotten from the international islamic university malaysia research ethics committee (irec) (ref: iium/504/14/11/2/ irec-2019 231). written informed consent was obtained from respondents who agreed to participate in the study. authorship statement ama, naa conceptualized and designed the study and revised the manuscript, aaaa collected the data and wrote the draft manuscript, mea analyzed the data and revised the manuscript, yma designed the study and revised the manuscript. references barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework mohd taufek et al. (2022) journal of pharmacy, 2(2), 36-44 page 36 reflective practice of experiential learning visit to drug rehabilitation centres improved pharmacy curriculum prospects and services in promoting community engagement nor hidayah mohd taufek1,2,*, nor ilyani mohamed nazar1, norny syafinaz ab rahman1,2, syahrir zaini2, christopher john turner3 abstract introduction: reflective practice has been proven effective in improving students' learning outcomes from various health disciplines, including pharmacy. as healthcare providers, pharmacists have frequent contact with vulnerable patients with risky behaviours thus require appropriate attitudes and positive perceptions to ensure equal services. these intangible components require self-reflection of individual learning processes. this study aimed to investigate the view of pharmacy students towards an experiential learning opportunity at drug rehabilitation centres via a reflective practice approach. method: pharmacy students from international islamic university malaysia participated in a reflective practice exercise following a visit to drug rehabilitation centres in pahang, malaysia, in 2018. a total of 43 of the 69 students consented for their reflective writings to be analysed for this research. thematic content analysis was conducted, and emerging themes were identified. results: students reflected on several themes, which were 1) sense of responsibility and volunteerism, 2) perspective on pharmacy knowledge application, 3) professional and societal engagement, 4) understanding people's behaviour 5) personal inadequacy. conclusion: pharmacy students have effectively utilised a reflective practice approach to address specific issues and needs of the local community related to substance use disorders which were beneficial to improve the curriculum and encourage community engagement activities. article history: received: 16 october 2021 accepted: 23 march 2022 published: 31 july 2022 keywords: reflective practice, experiential learning, pharmacy students, community engagement, substance use disorders how to cite this article: mohd taufek, n. h., mohamed nazar, n. i., ab rahman, n. s., zaini, s. & turner, c. j. reflective practice of experiential learning visit to drug rehabilitation centres improved pharmacy curriculum prospects and services in promoting community engagement. journal of pharmacy, 2(2)., 36-44 doi: 10.31436/jop.v2i2.112 authors’ affiliation: 1 substance use disorder research group, kulliyyah of pharmacy, international islamic university malaysia, 25200 kuantan, malaysia. 2 qualitative research-methodological application in health sciences research group, kulliyyah of pharmacy, international islamic university malaysia, 25200 kuantan, malaysia. 3 retired but formerly with skaggs school of pharmacy and pharmaceutical sciences, university of colorado anschutz medical campus, aurora, colorado, usa. *corresponding author: email address: hidayahtaufek@iium.edu.my mohd taufek et al. (2022) journal of pharmacy, 2(2), 36-44 page 37 introduction reflective practice is an essential component that integrates the theory and practice for students in health profession disciplines. students are exposed to various tools on reflective practice through writing, reflecting on experiences, observation, and developing metacognition to enhance their learning and practice (mann, gordon, & macleod, 2009). it is a powerful method for students to express their emotions based on good and bad experiences during practice (ganesh & ganesh, 2010) then share their learning process and discovery through self-reflection (lie et al., 2010). there are various self-reflection techniques and tools that allow students to improve written communication skills, think critically, and link the learned information to practical applications. examples include writing reflective diaries that require students to identify their limitations, reflect on learning incidents and create change among themselves (zimmerman et al., 2007); e-portfolios reported to improve students’ self-assessment skills (hj. ebil et al., 2020; marinho et al., 2021); and role playing in simulated experiences (kilgour et al., 2015). a study on physiotherapy students in malaysia has shown that students reflected on applying theory into practice, self-development, improving communication skills and adaptation to clinical environment through reflective diary (ramli, joseph & lee, 2013). in the united states, a pie-recap method was an eight-domain format used in reflective journal writing to encourage students to prioritise, identify, explore, recollect, evaluate, and challenge/solidify their knowledge. the method as mentioned above needs to be conducted incongruent with an action plan for professional development experiences and growth in curricular, cocurricular and affective domains (hughes et al., 2019). qualitative researchers have used diaries for data collection when there is limited access to individual subjects' experiences with a certain situation (elliot, 1997; zimmerman & wieder, 1997). the main advantages of using the 'diary-interview' method in healthcare research include accommodating different response modes and participants' priorities and elucidating the contexts within which help-seeking occurred. in the context of students visiting drug rehabilitation centres, the first-hand experience of students learning addressing marginalized population is best documented on reflective diaries where students can express their feelings, perspective and thoughts. it has been reported that diaries play significant roles as a record of reflection on the experience of an illness and means of understanding what is 'taken for granted' in accounts of health and illness (elliot, 1997). boud et al. (1985) have categorised six stages of increasing depth of reflection: attention to feelings, association, integration, relationshipseeking, validation, appropriation, and outcome. in pharmacy education, the training methods provided to address substance use disorders (suds) at the undergraduate level vary in course structures, experiential visits, and average hours of didactic instructions (thomas & muzyk, 2018). however, only a few studies have investigated these components. a study by keating et al. (2019) has highlighted essential findings on pharmacy education addressing mental health discipline. people with suds are closely linked to mental and certain brain-related illnesses due to physical and psychological trauma related to suds. yet, few opportunities for experiential education or contact-based learning with former drug users have been available in malaysia to explore various aspects such as drug-related health issues including chronic diseases which require tailored intervention. contact-based learning and sharing personal recovery stories have improved student attitudes towards patients with mental illnesses as reported in a previous study where a brief experiential visit to a mental health hospital has integrated students’ learning and values related to their expertise in supporting patients with mental disorders (keating et al., 2019). this study aimed to explore the reflections of pharmacy students from a visit to drug rehabilitation centres by using a reflective diary method. methodology ethics approval was received from the international islamic university malaysia (iium) ethics committee (id no. irec 2020-116). a retrospective qualitative study using thematic content analysis of data from reflective diaries was conducted to describe reflections of experiences of pharmacy students following a visit to drug rehabilitation centres in pahang, malaysia. all (69) reflective diaries in the 2018 archives were retrieved and 43 of the 69 students consented to the study. this was the first cohort that used reflective diary as an assessment for an elective course, drug abuse phm 3282. this study was conducted between october until december 2020 at the kulliyyah of pharmacy, iium. the visit involved six settings which were private and government drug rehabilitation centres which included shelters for children and recovering drug users for adults. students were divided into groups for each setting, shelter homes for children, male adults, female adults, drop-in centres for active drug users for males and females and a community integration centre. during the visit, students listened to the briefing by the staff and interviewed the residents. then individual reflective diaries written in english language was submitted for assessment after the visit. the diary consisted of six parts which were 1) description of activities, 2) significant experience/observation, 3) learning from experience/observation, 4) additional learning needs and plan to achieve learning, 5) summary of learning and 6) meaningful interaction with the team (peers, participants, residents, preceptors). mohd taufek et al. (2022) journal of pharmacy, 2(2), 36-44 page 38 the reflective diaries were de-identified from the participant information and analysed anonymously. two researchers conducted the thematic content analysis to compare and cross-check the themes identified from the diaries. one researcher independently constructed, compared, and cross checked to produce a final list of these codes. the coding decisions were agreed upon by two researchers. the summary of the data was done by manually scrutinising and generalising the data sets. in this research, the six stages of the braun and clarke thematic analysis method (2006) were used: (1) data familiarisation: the reflective journals were repeatedly read, and the thoughts on meaningful data were jotted down; (2) generating initial codes: after reading the content of the reflective journals, meaningful contents were coded; (3) searching themes: related and similar codes were analysed and grouped to form potential themes; (4) reviewing themes: the themes were repeatedly read, and the relevance of their context was confirmed; (5) defining and naming themes: the nature of each theme was examined, and the data described by each theme were confirmed; and (6) writing the report. a protocol was used to guide the analysis to four categories from the reflective diaries: habitual action/non‐reflection, understanding, reflection, and critical reflection (kember et al., 2008). the questions used to guide the participants in completing diaries were not used in the data analysis. results table 1. describes the relevant excerpts from five themes emerged from reflective diaries of 43 students. 1. sense of responsibility and volunteerism students reported their responsibility to volunteer in the society and the drug rehabilitation centres to educate the residents, community and reduce stigma to ensure sustainable recovery of former drug users. they also reflected on the responsibility of pharmacist to be involved in pharmaceutical care (e.g. hiv medications adherence and side effects) and education with all stakeholders. these values have been described in terms of collective responsibility and volunteerism to improve the success of drug addiction treatment in the community holistically. ‘i really want all children that are suffering from hiv can be cured…to change the mentality of community which being so scared to socialize with them…we, as a person who know, need to educate them on how safe it is to other people…’ (s35) ‘i was touched…there are some children who are hiv positive asked the reasons why he or she needs to take certain medication while others not…i know it is very hard for them. this experience makes me want to do my very best to become a successful person in the future and help all these needy people…i plan to go on the weekend with some of my friends as volunteer helpers’ (s4) ‘…i feel eager to contribute something to them as a support…to create opportunity to them to rebuild their life…we can organize programme to teach various skills…does not necessarily in large scale but can give a big impact to residents’ (s41). 2. perspective on pharmacy knowledge application . students were exposed to pharmacy related knowledge which they were able to identify the importance of correct education and improved adherence about medications for hiv/aids, haart, tuberculosis, sexually transmitted diseases, hepatitis and assesses their application. they were aware of the challenges occurred at the shelter homes involving children, psychiatric patients, and the needs of identification drug-related problems (e.g. side effects, drugdrug interaction) with pharmacists' intervention. students appreciated the knowledge of medicinal chemistry application when it was asked by the staff at the centre. they identified various aspects for involvement of pharmacist to provide pharmaceutical care and health education. ‘..as a pharmacist, we can do a home medication review to the residents there to see whether they take the medications correctly, compliance…’ (s22) ‘…he keeps mentioning about this certain group of public people that recently consume antitussive medication, codeine and been arrested for a positive urine strip test of morphine. however, later lab test result shows a negative result…the officer basically got confused…our preceptor found out that the principle might be behind the metabolism of codeine into morphine in vivo…since the structure of codeine and morphine are more or less quite the same, the problem might lie within the sensitivity of the strips itself.’ (s38) ‘rvd patients are not the same with tb (tuberculosis) patients whom all their belongings cannot be shared with healthy people…it is difficult to ensure rvd (retroviral disease) residents take the medication punctually with various side effects such as excessive heat, fever, vomiting and loss of appetite...the staff also found uneaten medications when they clean the area…we should treat rvd as normal person...express our empathy’ (s6) 3. professional and societal engagement students acknowledged the lack of engagement from the society including health care professionals to contribute better services and activities to improve health care in the shelter homes. the societal exclusion and isolation with stigma had led to empathy among students to encourage support to this population. they reflected the importance of integration of different types of services and disciplines to collaborate and engage to improve services at the centre. the community engagement activities are needed to support mohd taufek et al. (2022) journal of pharmacy, 2(2), 36-44 page 39 the recovery of the residents. ‘i will be glad if the visit would include us to also involve with the clients’ activities or at least have a thorough insight, watching them conducting their daily activities…it made me realize that i also had some stigma and afraid of the ex-drug addicts before…people out there should also get a close insight to them like this visit to truly understand them’ (s11) ‘…we can increase the work to educate our community regarding hiv..to help our people fully understand on how hiv spread and believe that hiv are not spread through air and touch…we can organize programme held by neighbourhood, the mosque…’ (s19) ‘…every individual is responsible to give moral support to ex-drug addict to return to normal life…if they return to abuse drug, it is part of our mistake because we neglected their presence as part of community’ (s4) 4. understanding people's behaviour students understood that the behaviour of residents was shaped by their past experiences particularly addressing the segment of society with underprivileged environment. they were able to identify difference in individual behavior affected by different stories first-hand and understood about different approaches applicable in their interaction. these are important soft skills needed by health professionals to be tailored to individual needs in health services and reflecting from experiences could open opportunities for innovation. ‘…she had depression once she knows that she has hiv+ from her husband…they are also struggling to make a life, to have courage to adapt with the society...not all of them involved with drugs by their own will, some get involved due to pressure situation from family and working environment…’ (s13) ‘the other boy is socially awkward and did not want to talk to us even we tried our best to make him speak…i can clearly see that probably he has some bad memory with his family before entering this house and eventually traumatized…in the next visit, we plan to bring some chocolate or some playful stuff to be played with and these may make the boy to be socially comfortable with us…’ (s6) ‘…we did not have much opportunity to interact with clients because they seem scared to be approached by us….fortunately we were able to interview a volunteer whom once a victim of drug addicts…it was meaningful…she had adverse effects when taking efavirenz such as lethargy, insomnia, confusion and dizziness…her stories motivate me to become a future responsible pharmacist’ (s26) 5. personal inadequacy the communication skill has been identified as the main challenge when interacting with the residents who had history of trauma from various aspects. students suggested that it would be beneficial if they had the chance to be exposed to actual treatment programmes at the centre, which could improve their understanding and specific skills in addressing this population. a better training and exposure would help student to perform better in building trust and rapport with the residents or future patients. ‘i need to improve my communication skills…needed when dealing with people involved with drug abuse…they could be sensitive and easily touched…need to be very careful when interviewing them..i need to practice and be prepared of what to ask and how to ask…’ (s19). ‘i think it would be a nice experience if we could join them in whatever programme that they do if appropriate, to understand them better and see the intervention..activities modules and daily chores’ (s13) ‘it is difficult for the patient to take the medicine because it can cause pain during swallowing…it is better if we get to see how the medicines look like…we need to know about medication for hiv patients…if possible, we can watch during their medication taking session’ (s28) mohd taufek et al. (2022) journal of pharmacy, 2(2), 36-44 page 40 table 1: relevant excerpts from five themes emerged from reflective diaries of 43 students. 1. sense of responsibility and volunteerism • realised the struggles and was motivated to serve the community after first-hand observation with residents. • responsibility of community to give hope to build future, to help improve awareness in the community • to help children in terms of education (e.g. free academic tuition), improve interaction and social skills, build confidence. • to educate the public, universities, and schools and participate in volunteer works. • health care providers should support vulnerable residents with low self-esteem. • as future pharmacists, there is a responsibility to educate the community about the harms of drugs, risk of infection and human immunodeficiency virus (hiv) and remove the stigma. • to volunteer in an outreach programme with the residents to help unfortunate people involved with drug abuse. 2. perspective on pharmacy knowledge application • knowledge on screening procedure for diseases, aware of critical role pharmacist plays to help with compliance of medications for various diseases hiv, hepatitis, tuberculosis – challenges for young children taking highly active anti-retroviral therapy (haart) medications due to side effects and lack understanding. • to arrange seminars and talks to educate on medications adherence, conduct research to find better solutions to the problem. • the positive urine test for opioid in residents taking pholcodine ® aware of the importance of educating about the chemical structure and appreciating medicinal chemistry knowledge. • severe side effects of withdrawal symptoms during treatment due to poly-drugs. • pharmacists should raise awareness on drug addiction education since many people underestimate the effects of drugs on themselves. • the need to improve knowledge about amphetamine-type stimulants that are widely used where users are prone to psychotic disorders. • ensure continuous supply and follow-up of medications for hiv, tuberculosis, hepatitis b or c infection, and psychiatric illnesses. • pharmacists need to strictly monitor the sales of cough mixture in the community setting as it can be abused easily. • a pharmacist is responsible for discovering new treatments to achieve the highest success rate to recover from drug addiction. • pharmacist should participate to help hiv patients and sex workers to practice healthy lifestyle, together with the therapy adherence peer support for hiv/aids, sexually transmitted diseases. • immunisation to ensure minimise risk from infectious diseases, condoms distribution for harm reduction. mohd taufek et al. (2022) journal of pharmacy, 2(2), 36-44 page 41 3. professional and societal engagement • community should not isolate the residents but give them hope to build good character, build new future, get opportunity for education. • society to treat residents equally so that they can contribute to the country. • judgmental society could not provide supportive role that prevents drug relapse recovery process is more challenging when residents face harsh treatment from society. • community activities to increase understanding and help maintaining the abstinence. • not to judge different methods of treatment total abstinence 'cold turkey' or harm reduction 'methadone substitution therapy', religious approaches, psychological methods. • health care providers can contribute their expertise in helping drug abusers to quit drugs since they understand better the physiological changes and chemistry of substances. • combination of methods including psychological, social, functional, spiritual, family and medication can contribute to effective treatment. 4. understanding people's behaviour • empathy on the suffering of drug withdrawal symptoms and strict medication schedule for hiv treatment. • challenges in the behaviour of children who had trauma and fear from their past related to family with drug abusers. • difficult behaviour of people with psychiatric illnesses from previous abuse of polydrugs cannabis, ecstasy morphine and cocaine. • acknowledge the positivity and optimistic behaviour portrayed by the clients who are determined to manage their challenges and recover. • sense of inferiority due to known people's perception, avoidance of eye contact, admitted their mistakes. • psychological impact in residents was severe related to health, family and financial issues but residents were nice, friendly and welcoming. 5. personal inadequacy • to participate in the program at the centre e.g. motivational talk, treatment modules, counselling as first-hand experience. • should understand the feeling of residents, not to ask sensitive questions. • lacked preparation when interacting with children with difficulties in social skills. • need to improve communication skills and psychological aspect, did not know what to expect and struggled to start conversations. mohd taufek et al. (2022) journal of pharmacy, 2(2), 36-44 page 42 discussion the present study reported that reflective practice was a strategy to identify intangible components of learning among pharmacy students addressing patient care in marginalised population with high-risk behaviors. the students reflected that a one-day visit to drug rehabilitation centers was beneficial in identifying actual issues related to pharmaceutical care, community engagement and social justice, empowering underprivileged group with education and training. collective efforts need to be implemented to reduce inequality in health care received by residents at these shelter homes. the experiential visit and reflective reports have provided evidence that pharmacy education is an excellent platform to improve knowledge and training regarding the role of pharmacists in harm reduction services, prevention and treatments strategies associated with suds. other than didactic classes, practical learning aspect in terms of interaction with patients who are former drug users is important to pragmatically identify contextual issues. training pharmacy students about suds produced a positive impact in their attitudes and knowledge on this subject matter (muzyk et al., 2017). undergraduate pharmacy curricula must be designed to demonstrate a patient-centred approach to care, treat others with sensitivity, empathy, respect and dignity, and recognise the value and structure of a multi-professional team (keating et al., 2019). our findings demonstrated that reflective-thinking skills need to be a focus in pharmacy education to improve its prospects and create pathway to prepare competent pharmacists who meet the healthcare needs of the public. it was able to develop ability of students to view patient's situations from different angles to gain new perspectives (tsingos et al., 2015). pharmacy service delivery should sustainably strengthen pharmacists’ capacities to meet complex needs of population such as suds associated with infectious diseases and psychiatric illnesses. our context has revealed the needs for collaboration and integration of knowledge between different disciplines for effective suds treatment. students were able to recognise the important role of respective disciplines towards holistic approaches of an individual with complex issues of physical and psychological trauma. this finding could open to opportunities in interprofessional collaboration and learning from other disciplines including nursing, medical, physiotherapy and psychology. the reflective diary was an important tool to advance the knowledge regarding specific subject or experience when the description of the events was translated and reflected in the form of feelings, knowledge application, barriers, gaps and opportunities. the fink's taxonomy of significant learning described the domains of foundational knowledge, application, integration, human dimension, caring, and learning how to learn emphasising that human factors can have an important impact on health services outcomes (fink, 2013). substance use disorders is one of the key areas in public health of which pharmacy services are involved with harm reduction programme, prevention, and treatment intervention (international pharmaceutical federation, 2018). from students' reflection about the experiential visit, we were able to recognise the need for improved training in pharmacy graduates to address the gaps in learning such as communication skills, pharmacy knowledge application related to reviewing medications commonly used in this population (e.g. psychiatric, infectious diseases), as well as efforts in addressing attitudes of health professionals and community towards stigmatised patients to reduce gaps in health services (van boekel et al., 2013; sattler et. al. 2017). pharmacists need to proactively involve educating society regarding issues with suds for relapse prevention and sustainable treatment and recovery (livingston et al., 2012). previous studies reported on reflective writing within pharmacy and other health professions education (ganesh &ganesh, 2010; brooman & darwent, 2012; ramli, et al., 2013; keating et al., 2019; hughes et al., 2019). depending on respective discipline-specific outcomes, the formats for reflective writing have been varied. our application of real-time journal entries through low technology (pen and paper) has also been reported as an effective tool in reporting the experiential learning in pharmacy students (mamede, schmidt & penaforte, 2008). in comparison to our previously published study using one-on-one interview with pharmacy students (mohd taufek et al., 2021), reflective diary method required shorter time to analyse, higher number of students consented to the study, more detailed description of experiences and data saturation was quickly achieved. the limitation of this study includes biases which may occur since it was part of course assessment submitted within one week after the visit, when compared to researcheradministered face-to-face interviews. however, both methods have been proven effective in exploring the views of pharmacy students addressing people with suds by reflecting on knowledge, stigma, empathy, motivation to learn and other learning values to become better pharmacists (mohd taufek et al., 2021). other methods may be explored in the future to enhance the ability and convenience of students reflecting on their experience addressing various learning components. it is proposed that reflective diary should be used as part of the learning tools in experiential learning activities towards improvement of pharmacy curriculum and personal growth of students. mohd taufek et al. (2022) journal of pharmacy, 2(2), 36-44 page 43 conclusion reflective practice has permitted pharmacy students to describe their experiential learning effectively from individual perspective. experiential visit to drug rehabilitation centres has exposed students in learning to address and discuss different issues and needs of local community related to suds. this learning outcome opens the opportunities for improvement in pharmacy curriculum as well as innovations in providing pharmacy services to encourage community engagement activities. acknowledgements the authors would like to thank drug intervention community and national anti-drug agency kuantan pahang for their permission, cooperation and commitment during the educational visit. conflict of interest the authors declare that there is no conflict of interest. references boud, d., keogh, r., & walker, d. 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(1977). the diary: diary-interview method. urban life, 5(4), 479– 498. https://doi.org/10.1177/089124167700500406 introduction reflective practice is an essential component that integrates the theory and practice for students in health profession disciplines. students are exposed to various tools on reflective practice through writing, reflecting on experiences, observation, ... there are various self-reflection techniques and tools that allow students to improve written communication skills, think critically, and link the learned information to practical applications. examples include writing reflective diaries that require ... qualitative researchers have used diaries for data collection when there is limited access to individual subjects' experiences with a certain situation (elliot, 1997; zimmerman & wieder, 1997). the main advantages of using the 'diary-interview' method... in pharmacy education, the training methods provided to address substance use disorders (suds) at the undergraduate level vary in course structures, experiential visits, and average hours of didactic instructions (thomas & muzyk, 2018). however, only ... methodology ethics approval was received from the international islamic university malaysia (iium) ethics committee (id no. irec 2020-116). a retrospective qualitative study using thematic content analysis of data from reflective diaries was conducted to describe... the visit involved six settings which were private and government drug rehabilitation centres which included shelters for children and recovering drug users for adults. students were divided into groups for each setting, shelter homes for children, ma... the reflective diaries were de-identified from the participant information and analysed anonymously. two researchers conducted the thematic content analysis to compare and cross-check the themes identified from the diaries. one researcher independent... results table 1. describes the relevant excerpts from five themes emerged from reflective diaries of 43 students. discussion the present study reported that reflective practice was a strategy to identify intangible components of learning among pharmacy students addressing patient care in marginalised population with high-risk behaviors. the students reflected that a one-day... our findings demonstrated that reflective-thinking skills need to be a focus in pharmacy education to improve its prospects and create pathway to prepare competent pharmacists who meet the healthcare needs of the public. it was able to develop ability... substance use disorders is one of the key areas in public health of which pharmacy services are involved with harm reduction programme, prevention, and treatment intervention (international pharmaceutical federation, 2018). from students' reflection a... previous studies reported on reflective writing within pharmacy and other health professions education (ganesh &ganesh, 2010; brooman & darwent, 2012; ramli, et al., 2013; keating et al., 2019; hughes et al., 2019). depending on respective discipline-... conclusion acknowledgements the authors would like to thank drug intervention community and national anti-drug agency kuantan pahang for their permission, cooperation and commitment during the educational visit. conflict of interest references barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework ismaeil et al. (2023) journal of pharmacy, 3(1), 66-74 page 66 infection control in digital era: future or futile? rehab ismaeil1,, abdul rahman fata nahas1, mohamad haniki nik mohamed1*, norhidayah kamarudin2 and mohd basri mat nor3 abstract new technologies are being developed and marketed to healthcare facilities all over the world as a way to stop healthcareassociated infections. the internet of things and artificial intelligence have been created with a variety of capabilities to improve people's health, offer necessary services, and monitor their health the potential adoption of these technology in automated surveillance and automated hand hygiene compliance monitoring systems has a lot to offer health care systems. however, the success or failure of the use of technology will depend on the awareness of the challenge and the establishment of a strategy, goals, and processes to support technology deployment, maintenance, and training. system differences between nations and a lack of standardization in the application of digitalization in health care hinder this technology from providing the full range of potential benefits. in this review, we explore the use of technology in the areas of automated infection surveillance in healthcareassociated infection and hand hygiene compliance, with an emphasis on the difficulties in developing such technologies. article history: received: 12 november 2022 accepted: 16 january 2023 published: 31 january 2023 keywords: infection prevention and control, internet of things, artificial intelligence, healthcareassociated infections, automated hand hygiene compliance, automated surveillance. how to cite this article: rehab ismaeil, abdul rahman fata nahas, mohamad haniki nik mohamed, norhidayah kamarudin & mohd basri mat nor. infection control in digital era: future or futile? journal of pharmacy, 3(1), 66-74 doi: 10.31436/jop.v3i1.195 authors’ affiliation: 1department of pharmacy practice, kulliyyah of pharmacy, international islamic university malaysia (iium), jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia. 2 department of medical microbiology, kulliyyah of medicine, jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia. 3department of intensive care, kulliyyah of medicine, international islamic university (iium) jalan sultan ahmad shah, 25200 kuantan, malaysia. *corresponding author: email address: haniki@iium.edu.my ismaeil et al. (2023) journal of pharmacy, 3(1), 66-74 page 67 introduction digital technologies are becoming increasingly important in almost every aspect of life. a wide range of digital health technology (dht), including internet of things (iot) and artificial intelligence (ai), mobile health applications, telehealth services and big data are widely employed in health care service, and a slew of solutions have arisen to mitigate the impact of covid19 (vaishya et al., 2020; vidal et al., 2020). the covid-19 has greatly accelerated digitalization and introduced new challenges and potential for infection prevention and control (ipc) (javaid et al., 2020; kalhori, et al., 2021). it has prompted healthcare systems to use new ipc technology and approaches (vidal et al., 2020; wang et al., 2020). which can improve the system's usability, efficacy, and level of care (fitzpatrick et al., 2020; torous et al., 2020). growing healthcareassociated infections (hais) reporting rates make it clearer that patient safety, healthcare quality, and preparedness for infectious disease casualties all need to be improved. as a result, surveillance measures act as the first line of defense against hais, highlighting the need of implementing efficient surveillance (degeling et al., 2019; parreco et al., 2018; villamarín et al., 2020). the traditional hais surveillance approaches are considered a sort of passive monitoring, which depends on case reporting through manual screenings however, it has shown to be time-consuming and unreliable (du et al., 2014; streefkerk et al., 2019). meanwhile, with modern health care technology, monitoring can be aided by the use of the sophisticated algorithms machine learning (ml) and deep learning (dl) that built on data seek to early detection populations at-risk and keep track of an estimate of the prevalence of hai to improve the emphasis of preventative interventions (parreco et al., 2018; li et al., 2019; liao et al., 2019). by adopting automated monitoring and infectious disease detection methods, hospitals can improve the quality and safety of patient care (streefkerk et al., 2020; yesmin et al., 2022). handwashing is an easily accessible and costeffective infection control behavior to reduce hais. the covid-19 pandemic has renewed calls for increased handwashing to stop the virus's spread (stangerup et al., 2021). despite this the compliance is very low (stangerup et al., 2021). hand hygiene compliance (hhc) continues to be a global challenge, indicating that knowledge and awareness are insufficient to change behavior (sadule-rios& aguilera, 2017; clancy et al., 2021). a crucial component of multimodal techniques to enhance hand hygiene (hh) is monitoring hh. direct observation (do) is the gold monitoring standard for calculating hhc rates (gould et al., 2017). however, the process is still not standardized, hawthorne effect, which outlines how providers' behaviour changes when they realize that they are being monitored has sparked interest in new methods for checking hhc and prompted the creation of automated hh monitoring systems (ahhmss) (gould et al., 2017; kelly et al., 2021; wu et al., 2018). it can track hand hygiene compliance in "real time," avoiding the hawthorne effect and allowing for more efficient data collecting for large groups of people (kelly et al., 2021). in this review, we examine the potential advantages of ipc digitalization for automated infection surveillance, predication of healthcareassociated infections and hand hygiene compliance. highlighting the challenges associated in implementing such technology. applications of digital technology in infection control automated surveillance surveillance is crucial for infection control because it determines which prevention strategies should be given priority and enables programs to assess the success of their prevention efforts (degeling et al., 2019; cha & kim, 2020). technological developments and the gradual digitalization of health data enable more hospitals use electronic medical records (emr) for automated hais surveillance. it is an innovative way to lower the infection incidence and produce novel disease control because it has been demonstrated to be more effective, reliable, lower costing and safe time in detecting infections than traditional surveillance (streefkerk et al., 2019; kelly et al., 2021). the efficiency of automated surveillance was revealed in many studies for instance, real time nosocomial infection surveillance system (rt-niss) was developed and validated in china by du et al in 2014. the sensitivity and specificity of automatic hospitalwide hais surveillance system rt-niss were 98.8% and 93.0% respectively, when compared to a manual survey of nosocomial infections (nis) (du et al.,2014). study done by blacky et al, in vienna general hospital showed that automated moni-icu (monitoring of nosocomial infections in intensive care unit (icu) gives surveillance staff and physicians almost-real-time view of clinical markers for nis with sensitivity, 90.3% (blacky et al., 2011). moreover, the innocbr ismaeil et al. (2023) journal of pharmacy, 3(1), 66-74 page 68 system, which was developed between 2010 and 2013, is an automatic hai detection and categorization software that is commonly utilized at chuo's preventive medicine (ourense university hospital complex, spain). since its implementation at more hospitals in galicia (spain) in 2013, the innocbr system has become the standard system for hai surveillance. innocbr achieves a high level of sensitivity (81.73 percent), specificity (99.47 percent), and a good positive predictive value (94.33 percent) when tested against the gold standard (villamarín et al., 2019). the potential impact of using ai tools in numerous aspects of healthcare is becoming more generally recognized. ai systems will be able to analyses, diagnose, and provide decision support for prevention and early intervention (fitzpatrick et al., 2020; li et al., 2019). ml, a subset of ai technology, can be used in clinical microbiology labs to identify and forecast diseases, enhancing patient safety. dl, a recently developed area of ai, has boosted accuracy greatly by utilizing new strategies, specialized software, and vastly larger datasets to find more complicated correlation in the data (tobore et al., 2019). park et al. published a study in 2021 that attempted to develop prediction models that physicians might utilize in the hospital setting to make clinical decisions based on dl and ml using laboratory data. the study found that using dl and ml might produce more accurate diagnosis findings than physicians (park et al., 2021). ai usage for prediction or early detection of hais has a lot of potential in ipc (fitzpatrick et al., 2020). for instance, the risk of nosocomial clostridium difficile infection (cdi) has been predicted using ml technologies (oh et al., 2018; li et al., 2019). parreco et al. investigated the effectiveness of three different ml-based models for the prediction of central line-associated blood stream infection (clabsi). this study revealed that models for predicting patients with clabsi had the highest accuracy, precision, sensitivity, and negative predictive value (parreco et al., 2018). the quality, cost, and outcome are all impacted by the necessity of early diagnosis of these individuals (parreco et al., 2018). the study established a noninvasive examination and inspection approach for ventilator-associated pneumonia (vap) diagnosis using an electronic nose. the results show that an ml-based electronic nose can help patients gradually attain the idea of high-quality medical care while also improving their quality of life. 2019 (liao et al.). the application of ai can improve patient risk assessment, provide real-time detection for more focused surveillance, and enable the development of targeted ipc interventions. automated hand hygiene compliance hhc is one of the most important factors in reducing hais, and accurate hhc monitoring among health professionals is essential to delivering high-quality care. the gold standard method for evaluating hhc is do and feedback, as measured by hospital auditors (gould et al., 2017). however, the observation bias and the requirement for numerous observers over a long period of time limit its effectiveness. (gould et al., 2017; wu et al., 2018). with the help of automated hh-measuring technologies, iot has a lot of potential to improve hhc. xu and colleagues in 2021 investigate the impact of an iot-based management system on hhc in a critical care unit. they found that although there was no decrease in nis, the new method increased the rate of hhc among all workers (xu et al., 2021). in another study in a hospital setting in ontario, canada to investigate the impact of iot interventions on patient safety measures such as patient falls and hhc. it emphasized several key points about the use of iot in healthcare. the hhc rates were increase in the first year followed by a reduction in the second year (yesmin et al., 2022). similarly, a study done by marques et al, 2017 using iot based automated monitoring systems in conjunction with gamification to enhance hhc among hcws showed that there was an improving in the awareness of nurse hhc (marques et al., 2017). ai applications in hospitals have a significant impact on hhc. computer vision, a type of ai, could provide a novel approach for performing more accurate and privacy-protected hand hygiene assessments. depth pictures, which simply capture an image without allowing identification of the persons being watched or the ability to identify characteristics, are used to allay concerns about the use of video surveillance in places where ismaeil et al. (2023) journal of pharmacy, 3(1), 66-74 page 69 privacy is a concern (awwad et al., 2019). in the study by awwad et al., it was discovered that the system was more successful at identifying the supply of alcohol hand rub when it used computer vision and depth images for hand hygiene auditing. the study demonstrated that it could automate the direct observation of hand hygiene practice, which boosts clinical application and decreases privacy concerns (awwad et al., 2019). the simplicity and accessibility of the dispenser allows for a stronger habit of hand washing, which has substantial potential benefits. singh discovered that using a computer vision system to track use of hand sanitizer dispensers was equivalent to human observation. given its capacity to be passive, inexpensive, privacy-safe, and sensitive, it may be helpful in attempting to eliminate an apparent recurring source of healthcare-induced harm due to its capacity to provide ongoing monitoring and feedback to clinicians (singh & sittig., 2020). on the other hand, hand sanitizer uses, or automatic dispenser activation counts cannot be used to assess hhc. as a result, the importance of embedding real-time feedback into ai applications to promote behaviour change has increasingly been emphasized (lacey et al., 2020). lacey and coworkers deployed an autonomous video auditing (ava) system with real-time feedback at handwashing. the findings revealed that using ava in conjunction with electronic monitoring enables for simultaneous auditing of providers' handwashing quality and quantity. but when the feedback was taken away, performance went back to normal (lacey et al., 2020). furthermore, according to a study done to evaluate the influence of the sanibit electronic hh system on hhc and quality changes over time in icu, a sensor-based platform with automated hhc and real-time feedback increased providers' hhc in an icu (xu et al., 2021). recently, the potential health benefits of wearable hand hygiene technology have received a lot of attention in the medical community. some study has been done in hospital hand hygiene monitoring using wearable sensors (li et al., 2019). wearables-based systems does not require the installation of a camera, and it typically captures wrist movement data during a handwashing event using sensors, to detect handwashing steps in compliance with who recommendations. wrist wash (li et al., 2019) is a widespread procedure involving the use of a wrist-worn platform that allows offline analysis for assessing the stages using a hidden markov model-based method according to who criteria (li et al., 2019). the results showed that userdependent models had an average accuracy of 92 percent, whereas user-independent models had an average accuracy of 85 percent. however, when that assumption is relaxed, the results for relaxed performance drop considerably, from 85 percent to 69 percent (li et al., 2019). the accuracy of a sensor wristband in monitoring adherence to who hand rub and handwashing guidelines was also tested by wang et al (wang et al., 2020). the limitations of camera-based technology were all overcome by this study. however, these are unsatisfactory in terms of gaining accuracy, reminding individuals to wash their hands, and offering feedback on the effectiveness of their handwashing (wang et al., 2020). over the years, a number of hand hygiene assessment systems have been developed to evaluate the quality of handwashing. the study on how a wearable device affected hhc and the quality of hand rubbing revealed that while hhc did not improve, the quality of hh activity did, with a substantial increase in both the amount of alcohol-based hand rub (abhr) utilised and the amount of time spent rubbing hands. (pires et al., 2021). smartwatchbased automated systems for higher accuracy assessment handwashing quality have been developed. iwash is a smartwatch-based system for evaluating the effectiveness of handwashing that precisely identifies whether the user followed who guidelines or not. it uses voice to remind users to wash their hands frequently, especially as they enter the house, and to provide real-time feedback on the effectiveness of their handwashing (samyoun et al., 2021). challenges data accessibility and the availability of sufficiently large data sets with high-quality and reliable data for data analytics are the initial barriers to the effective implementation of dt (gianfrancesco et al., 2021). healthcare professionals have spoken about difficulties with the healthcare system's data quality, including a high workload, enormous amounts of unstructured ismaeil et al. (2023) journal of pharmacy, 3(1), 66-74 page 70 data, a lack of diagnostic code sensitivity, and data extraction, that have an impact on the quality of the data and lead to an inaccurate assessment of the patient's current condition (ni et al., 2019). non-standard reporting, a lack of clarity, and a lack of validation are further implementation obstacles (beam, manrai, & ghassemi, 2020). ml outcomes may become less capable of classifying or identifying comparable patterns in new data, depending on how the data was gathered and the learning algorithms were created (conway., 2016; park et al., 2021). liu et al in 2019, provided a users' guide to improving research methodologies and training healthcare professionals particularly clinicians, on the fundamentals of ml, the necessity of efficient ml model validation, and effective methods for integrating ml models into clinical practice. this will make studies more credible and understandable, which will increase user confidence (liu et al., 2019). software developers, hospital it employees, epidemiology experts, and ipc specialists must work closely together for the development and validation phases of data. this guarantees clinical applicability and permits the interpretation of results (sittig et al., 2020). the benefits of using technology must be evaluated against the serious ethical and legal concerns. large-scale patient medical record use and sharing raises concerns about confidentiality and privacy by increasing the possibility of unlawful use, accessibility, and potential abuse of personal data (kalkman et al., 2019; char et al., 2020). furthermore, even when patients have been given assurances of secrecy and privacy, there is a higher possibility that their personal information will appearing on social media when databases are breached (gilbert et al., 2019). digital tracking with the intention of conducting public health surveillance, have been found to be closely linked to serious privacy concerns (zhao et al., 2021). it has been recognized as posing a significant risk to further disclosures of sensitive information. the ethical responsibility of the patient must be respected when using technology in healthcare to bolster the value of privacy (zhao et al., 2021). online resources for infection prevention recommendations are abundant and easy to access. internet connectivity issues negatively impact data availability and quality (pollett et al., 2017). lack of internet connectivity, along with low internet quality and stability, greatly hinder the use of digital technologies for infection control. in lowand middle-income countries (lmics) with underfunded health systems and fewer providers, it might be crucial to concentrate on digitalization deployment (jones et al., 2021). hospitals frequently struggle with staffing and financial issues related to ipc programs. lack of ipc training for staff members and inadequate adherence to ipc principles could exacerbate these problems (lowe et al., 2021; jones et al., 2022). evaluating the potential and difficulties of implementing technology advancements is crucial to enhance assessments, development, ethics, usage, and monitoring technology techniques that might reduce the local burden in lmics (kruse et al., 2019; jones et al., 2022). discussion the potential use of digital technology in ipc could significantly reduce the risk of the spread of infectious diseases. however, it is limited by the awareness of the ipc challenge, objectives and processes that relate to the health system (singh & sittig, 2015; lowe et al., 2021). the effective use of technology to boost safety and infection control is essential for improving healthcare. therefore, when providing care for their patients, all healthcare professionals must use technology correctly and completely. inaccurate or inadequate data causes faulty predictions and improper outcomes. reliability depends on the surveillance system's accessibility and connectivity to the completeness, and validity of emr data (streefkerk et al 2020; gianfrancesco et al., 2021) in-addition to consideration of the ethical issues posed by applying ml in healthcare is necessary (char et al., 2020). a systems-level approach is required for the optimum suited digital system's strategy, one that connects infection detection with case isolation, follow-up, and monitoring while also selecting the best interventions to control the infection (beam, manrai, & ghassemi, 2020). automated surveillance systems require monitoring systems to find problems and their causes, as well as ongoing maintenance and quality control, to assure improvements. both the software and the methods need to be evaluated and perhaps updated in order to deliver safe and effective care (siting et al., 2020). the development of ahhmss provided reduction in observation bias and offer the chance to continuously monitor and enhance hand hygiene procedures (pires et ismaeil et al. (2023) journal of pharmacy, 3(1), 66-74 page 71 al., 2021; lacey et al., 2020). the ability of any technology to be successfully embedded into the healthcare system is crucial. however, concerns over privacy are known to have an impact on hcws' attitudes regarding automated monitoring (awwad et al., 2019; wang et al., 2020). they believe that these systems violate their right to privacy leads to refuse to change hh practices (conway et al., 2016). the limitations of the algorithm must be considered before implementing ahhms. the ml algorithms used to determine compliance with hand hygiene recommendations may produce inaccurate classifications that impair system accuracy (wang et al., 2020). moreover, the deployment of ahhms at a healthcare facility is expensive requires infrastructure upgrades and maintenance expenses (conway et al., 2016). the hospital system must be improved before addressing hhc to reduce hais. to accomplish this, future automated hh challenges should be addressed through additional research on the impact of systems on hh practices, the implementation of novel training and educational initiatives, and the determination of the best monitoring methods for improving monitoring outcomes. conclusion although the advent of digitalization in infection control holds out the prospect of real improvements in global public health, there are still many obstacles in the way, including issues with people's awareness of digital infection surveillance systems, their knowledge of hhc, and their concern for their privacy on maintaining data integrity and security. the healthcare system must weigh the benefits and drawbacks of these developing technologies to select the appropriate digital tools for their particular requirements and budgetary constraints additionally, the limitations of their infrastructure and organizational culture. moreover, research are needed to determine whether using clinicians and healthcare experience in the context of enhancing infection control with digital technology is appropriate. conflict of interest there are no conflicts of interest. references awwad, s., tarvade, s., piccardi, m., & gattas, d. j. 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(2022) journal of pharmacy, 2(2), 45-58 page 45 critical quality attributes (cqa) and critical process parameters (cpp) roles in the pilot scale up process and stability of a cosmetic cream sharifah shakirah syed omar1, hazrina ab hadi1,* and abd almonem doolaanea1 abstract introduction: the objective of this study was to scale up a natural based cream containing piper betle l. extract to pilot scale and asses it’s stability over time. method: during the scale up process, the critical process parameters (cpp) were monitored closely to ensure the critical quality attributes (cqa) are maintained within a desired range. to ensure the formulation was stable, a 6-months stability study was done where the organoleptic properties, particle size, zeta potential, viscosity, microbial limit and hydroxchavicol content were assessed. the formulation was characterized repeatedly at 0-month, 1-month, 2-month, 3-month and 6-month time point after being stored in 30°c±2 and 40°c±2 at 75%±5% rh. results: cpps in each process were maintained at certain values throughout to achieve cqas. time, speed and temperature during mixing and homogenization process were monitored and maintained. stability study over 6 months proved the ability of the product to remain stable with ph < 5, particle size < 2µm, low span value, zeta potential < -50mv, apparent viscosity < 1.2pa.s-¹ and no microbial growth. conclusion: in conclusions, cpps played a major role in enabling the formulation produced attain acceptable cqas. identifying cqa and cpp ensured the production of quality and stable product. article history: received: 1 december 2021 accepted: 11 may 2022 published: 31 july 2022 keywords: scale up, pilot scale, manufacturing, piper betle, cosmetic cream. how to cite this article: syed omar, s. s., ab hadi, h., & doolaanea, a. a. critical quality attributes (cqa) and critical process parameters (cpp) roles in the pilot scale up process and stability of a cosmetic cream. journal of pharmacy, 2(2)., 45-58. doi: 10.31436/jop.v2i2.119 authors’ affiliation: 1 department of pharmaceutical technology, kulliyyah of pharmacy, international islamic university malaysia, 25200 kuantan, pahang, malaysia *corresponding author: email address: hazrina@iium.edu.my syed omar et al. (2022) journal of pharmacy, 2(2), 45-58 page 46 introduction according to the federal food, drug and cosmetic act and article 2 of the eu cosmetics regulation the definition of cosmetics are products that are meant to be applied on body parts either by being rubbed, poured, sprinkled, or sprayed on or introduced in to, for the purpose of changing the appearance, enhancing beauty, improving attractiveness cleaning, perfuming and preserve the skin’s good condition (european comission, 2009; u.s congress, 1934). moving forward the use of cosmetics have widen its horizon to maintaining healthy skin, protecting from harmful hazards as well as prevent or remedy for the many skin issues faced by all races, genders, and age globally (arti et al., 2014). based on euromonitor, the skincare segment recorded a turnover of us$107 billion worldwide in 2013, where 65% of it is contributed by facial treatments alone and 30% of the facial treatments are from skin lightening products (euromonitor, 2016). in 2018, asia pacific lead the world's skincare sales with china followed by japan and korea at the forefront. sales of skincare in malaysia, indonesia and thailand is also expected to grow exponentially (lueng, 2019). malaysia is one of the countries that is showing growth in the demand of skin lightening products. these products garner attention in the management of hyperpigmentation (charles & mclean, 2017; desai, 2014). skin lightening cosmetic products offer solutions that are accessible and affordable with multiple choices. although with abundance of skin-lightening products already available in the market, many are opting for natural based skincare products due to the occurrence of adverse effects (desai, 2014; oudenhoven et al., 2015). piper betle l. was found to have skin lightening effect due to its ability to inhibit tyrosinase enzyme in the melanogenesis pathway (majeed et al., 2012). piper betle l. is an evergreen perennial plant native to peninsular malaysia from the piperaceae family (gibson, 2016; singtongratana et al., 2013). this skin lightening effect is believed to be contributed by a compound name hydroxychavicol or its chemical name 1-allyl-3, 4-dihydroxybenzene. however, the stability of natural based formulation is concerning especially the effects of scale up on the formulation if commercialization is considered. scale up is the process of increasing a batch size with the same procedures to produce a larger output. scaling up to pilot scale means that the batch size is increased to intermediate batch size which is representative of the procedures to be applied in manufacturing scale (gibson, 2016). scale up is important as it provides information on suitability of the equipments, identifying critical parameters of the process (fda, 2011) and plausibility to produce a stable product (ctfa, 2004). identification of critical quality attributes (cqa) and critical process parameters (cpp) that should be monitored since laboratory scale formulation and method development is indispensable (ema, 2016). methodology the formulation consists of aqueous phase, oil phase and cooling phase. aqueous phase compose of water, lecithin (tianjin hexiyuan lecithin technology co ltd, chin), glycerin (ioi acidchem sdn bhd, malaysia), chitosan acetate which the chitosan is from sigma-aldrich (missouri, united states) while the acetic acid from merck (new jersey, united states); disodium edetate (xi’an lyphar biotech co ltd shaanxi,china) and piper betle l. (pbl) extract. the piper betle leaves extract was extracted through subcritical water extraction using an accelerated solvent extractor (ase) with temperature of 100°c for 60 minutes. anti-tyrosinase assay of the extract has been quantitatively determined and found that crude piper betle leaves extract possess activity to confer skin lightening effect (omar et al., 2021). the oil phase comprises of beeswax (kahl beeswax, germany), xanthan gum (deosen biochemical ltd., unites states), refined coconut oil and olive oil from natures natural india (new delhi, india), cocamidopropyl betaine (haihang industry co, ltd., china) and squalane from kono chem co. ltd. (xi’an, china). finally, the cooling phase includes ascorbic acid from xi’an lyphar biotech co ltd (shaanxi, china), tocopherol acetate from natures natural india (new delhi, india), pectin from kono chem co. ltd. (xi’an, china), euxyl-pe (imcd malaysia sdn. bhd, malaysia) and chamomile essential oil (hunan nutramax inc., china). raw material identification identification test was done using attenuated total reflectance-fourier transform infrared (atr-ftir). a total of 16 scans were run at a wave region of 400 – 4000 cmˉ¹ at temperature of 25ºc using spectrum rx two instrument (perkinelmer inc., usa) complete with spectrum 3.5 software. scale up procedures the aqueous phase was placed in the aqueous pot, while the oil phase was placed in the main pot of zjr-30 vacuum emulsification machine (wuxi yk automation technology, china). both phases were heated to 75±5ºc with continuous agitation. the aqueous and oil phase were mixed and homogenized for 30 minutes then left to cool to room temperature before addition of cooling phase and homogenized again. the cpp were monitored closely to ensure that the cqa were maintained within a desired range. cqa are parameters physical, chemical, or biological that should be maintained within an acceptable limit to achieve products that are safe, effective and of high quality (fda, 2011). syed omar et al. (2022) journal of pharmacy, 2(2), 45-58 page 47 cpp on the other hand are factors whose inconsistency may affect cqa, hence should be monitored to produce product of good quality (ich, 2009). table 1 shows cqa and cpp involved. table 1: cqa and cpp cqa cpp risk assessment viscosity homogenization time, homogenization speed, temperature monitor and adjust the parameters accordingly. homogeneity homogenization time, homogenization speed, speed of pouring particle size homogenization time characterisation physical analysis the organoleptic properties as well as the physical appearance of the cream were assessed for acceptability to potential users. the properties include appearance, colour, odour and texture of the formulation (smaoui et al., 2012). forced centrifugation 10 g of the cream formulation was placed in a centrifugal tube and centrifuged at 5000 rpm for 10 minutes at 25°c (smaoui et al., 2013). centrifugation study was employed to study the resistance of emulsion towards being centrifuged (ghosh et al., 2013; rodrigues et al., 2016). ph ph of the formulation was measured using a calibrated ph meter (metler toledo, switzerland). the ph of the formulation was determined by diluting 1 g of the formulation in 9 ml of distilled water to form a concentration of 10% (w/w) (mahmood et al., 2015). particle size analysis particle size was analysed using a laser particle size analyser (bt-9300h, dandong baite instrument co. ltd., china). cream was diluted to approximately 1:1000 in the sample cell containing distilled water to prevent multiple scattering effect before running the machine (bahramparvar & goff, 2013). a refractive index of 1.46 and 1.33 was used for the fat and dispersing medium respectively (kirtil & oztop, 2016). zeta potential determination the zeta potential was measured using zetasizer (zen 1600, malvern, uk). samples were diluted with deionized water to 1:100 to avoid multiple scattering effects (khor et al., 2014). all measurements were carried out at temperature of 25 °c (esteban et al., 2016). rheological analysis the rheological parameter was measured using stresscontrolled rheometer (haake mars, thermo scientific, germany). sample was loaded on to the lower plate of the rheometer and allowed to equilibrate to temperature of 25°c (khor et al., 2014). a shear rate ramp with shear rates of 0.1 sˉ¹ 100 s ˉ¹ was applied to the sample (kirtil & oztop, 2016). microbial limit count 1 g of the sample was diluted to a 1 in 10 dilution using phosphate buffer solution ph 7.2. 10 ml of the sample was used for inoculation on the tryptic soy broth which was then incubated at 30-35°c for 18-24 hours. sample was sub-cultured on a mannitol salt agar for staphylococcus aureus and on cetrimide agar for pseudomonas aeruiginosa, then incubated for 18-72 hours at 30-35°c (usp, 2014b). for the determination of total aerobic microbial count (tamc) and total yeast and mold count (tymc) a dilution of 1:10 of the cream in phosphate buffer solution ph 7.2 was prepared. plate-count method was employed, specifically pour-plate method. 1 ml of the diluted sample was added in a petri dish and 15-20 ml of tryptic soy agar for tamc was added. plate was then incubated at 30-35°c for 5 days . similar steps were repeated on sabouraud dextrose agar for tymc. the plate for tymc was then incubated at 20-25°c for 5 days (usp, 2014a). stability study stability study provides evidence on how different environmental factors affect the formulation thus provide information on the shelf life and recommended storage conditions. an accelerated stability study was employed at temperatures of 30°c±2 and 40°c±2 at 75%±5% rh for 6 months (asean, 2013; ich, 2003). samples were assessed initially, then after 1 month, 2 months, 3 months, and 6 months, which is a total of 5 collection points (singh et al., 2016). data was analyzed using statistical package for the social science (spss) software version 20 applying repeated measures anova. syed omar et al. (2022) journal of pharmacy, 2(2), 45-58 page 48 hydroxychavicol content analysis with high performance liquid chromatography (hplc) a validated hplc method was used to identify the presence of hydroxychavicol in the formulation (omar et al., 2021). analysis of the samples was done using an isocratic system with agilent g1310a pump linked with diode array detector (agilent, stevens creek blvd santa clara, usa). the column used was a c-18 silica-based column (5 µm, 250 × 4.6 mm) (phenomenex, torrance, usa) which makes it a reversed phase chromatography. acetonitrile and 2% acetic acid in water with ratio of 25:75 was used as the mobile phase with a flow rate of 1.0 ml/min. results raw material identification all the raw materials were identified. shown in table 2 is the fingerprint for each raw material. critical quality attributes (cqa) and critical process parameters (cpp) the cpps played a major role in enabling the formulation produced attain acceptable cqas. as shown in table 3 the cpps in each process were maintained at certain values. table 3: cpp during scale up. process process parameter oil pot and aqueous pot mixing time 30 minutes speed 1400 rpm temperature 75 ± 5 °c main pot homogenization time 30 minutes homogeniser speed 3000 rpm agitatator speed 2700 rpm temperature off vacuum on characterisation physical analysis and forced centrifugation study the colour of the formulation was light beige while texture-wise, it appears smooth with a slight shine. as the formulation had chamomile essential oil, the odour of the cream was pleasant and showed no phase separation. the results are as reported in table 4. table 4: physical observations of cream containing piper betle l. (pbl) extract stored in 30°c/75%rh and 40°c/75%rh at 0 month, 1 month, 2 months, 3 months, and 6 months characteristic storage pbl cream 0m 1m 2m 3m 6m colour 30°c/75%rh lb lb lb lb b 40°c/75%rh lb lb lb lb b texture 30°c/75%rh s s s s s 40°c/75%rh s s s s s odour 30°c/75%rh p p p p p 40°c/75%rh p p p p p phase separation 30°c/75%rh x x x x x 40°c/75%rh x x x x x abbreviations: 0m is day 0 month; 1m is 1 month; 2m is 2 months; 3m is 3 months; 6m is 6 months; lb is light beige; b is beige; s is smooth; p is pleasant; x is absent ph as shown in figure 1, ph of the formulation throughout the 6 months period ranged between 4.3-4.7. the change in ph was found to be significant over time (p=0.000). particle size figure 2 shows both formulations have particle size ranging from 0.99 µm 1.92 µm like most commercialized emulsions having sizes of 0.3-2 µm (terjung et al., 2012). although the changes of particle size over time is statistically significant (p=0.000), the size is still considerably small. the formulation maintained a span value below 2, except for at 3 months and 6 months as shown in figure 3. despite the significant changes (p=0.000), the cream did not show any observable changes or signs of instability. zeta potential zeta potential also known as electro kinetic potential is the measure of repulsive or attractive charge between particles (honary & zahir, 2013). results in figure 4 show both formulations in both conditions having high zeta potentials mostly below -50. over 6 months, zeta potential showed significant change (p=0.000) but remain well below the agglomeration threshold. syed omar et al. (2022) journal of pharmacy, 2(2), 45-58 page 49 table 2: atr-ftir results for each raw material. name ir fingerprint name ir fingerprint acetic acid chitosan ascorbic acid cocamidopropyl betaine beeswax disodium ethylenediaminetetraacetic acid (edta) syed omar et al. (2022) journal of pharmacy, 2(2), 45-58 page 50 chamomile essential oil euxyl-pe glycerin refined coconut oil lecithin squalane olive oil tocopherol acetate syed omar et al. (2022) journal of pharmacy, 2(2), 45-58 page 51 pectin xanthan gum figure 1: ph values of cream containing piper betle l. (mean ± sd, n = 3). abbreviations: 0m is 0 month; 1m is 1 month; 2m is 2 months; 3m is 3 months; 6m is 6 months. syed omar et al. (2022) journal of pharmacy, 2(2), 45-58 page 52 . figure 2: particle size of cream containing piper betle l. (mean ± sd, n = 3). figure 3: span of cream containing piper betle l. (mean ± sd, n = 3). syed omar et al. (2022) journal of pharmacy, 2(2), 45-58 page 53 figure 4 zeta potential of cream containing piper betle l. (mean ± sd, n = 3). figure 5: rheological analysis graphs for pbl cream. blue line shows relationship of shear stress (ƭ) and shear rate (ɣ), while red line shows relationship of viscosity (ŋ) and shear rate (ɣ). syed omar et al. (2022) journal of pharmacy, 2(2), 45-58 page 54 figure 6: apparent viscosity (pa.s) at 100sˉ¹ (mean ± sd, n = 3). figure 7: hydroxychavicol content in cream containing piper betle l. (mean ± sd, n = 3). syed omar et al. (2022) journal of pharmacy, 2(2), 45-58 page 55 rheological properties rheology is the science related to the flow of fluids which is associated with processes such as mixing, pouring, filling containers and extrusion of product through container (niu et al., 2016). based on figure 5, formulations follow a non-newtonian flow, where the relation between shear stress (mpa) and shear rate (1/s) is not linear. this is shown by the red lines depict decreasing viscosity as a result of increasing shear rate. the blue line forms a hysteresis loop which is a characteristic sign of a thixotropic system. thixotropy is a time-dependent shear thinning property where with stress; fluid will flow as viscosity reduces. the property of the formulation remained the same throughout the 6 months stability study. apparent viscosity is the applied shear stress on a liquid divided by the shear rate. the apparent viscosity (pa.s) at shear rate of 100sˉ¹, as presented in figure 6 shows low values maintained below 1.2 pa.s, despite being significantly different over time (p=0.000). microbial limit study results of the study shows no growth was observed for all tests which included tamc, tymc and the specific tests for staphylococcous aureus and pseudomonas aeruginosa over 6 months. hydroxychavicol content analysis figure 7 shows the hydroxychavicol content throughout the 6 months stability study. formulation stored in both conditions showed a declining trend but differ in the extent of degradation. the final content for formulations stored in 30°c/75%rh and 40°c/75%rh were 61.36 ± 0.53% and 32.46 ± 0.12% respectively. storage condition caused significant (p=0.000) reduction in the hydroxychavicol content over 6 months’ time. the effect of the different conditions towards the formulation was also found to be significant (p=0.000). discussion the light beige colour of the piper betle leaves (pbl) cream was caused by the dark brown colour of the extract mixed with white base cream. after 6 months of storage, pbl cream became a tone darker. raised temperature is known to cause physical changes to formulation including its colour (deuschle et al., 2015). creaming, foaming, and phase separation was not exhibited in the pbl cream during stability study. the presence of emulsifying agent lecithin conferred stability to the cream (fernandes et al., 2012). in addition to that, emulsion stabilizers such as xanthan gum, beeswax and pectin helped prevent separation to its oil and water components (krstonošić et al., 2015). the acidic ph is considered suitable for skin application with potentially no irritation as it lies within ph of 3-9 and the narrower range of 4-6 of ideal ph for skin formulation (s. ali & yosipovitch, 2013; contri et al., 2014). an acidic ph is preferred because the human skin is covered with an acidic mantle that can be maintained at an average ph 5.5 with the help of an acidic moisturizer (khan et al., 2013). the particle size ranges from 0.99 µm 1.92 µm is considered small and is favoured as it confers higher stability (khor et al., 2014). the gradual increase in particle size which is especially evident at 6 months can be caused by ostwald ripening, flocculation, or coalescence of the droplets (ahmed et al., 2012; long et al., 2012). the span value remained below 2 except at 3 months and 6 months. a small span value <2 indicates monodispersity of particles in the formulation and is favoured as polydispersity can lead to instability (santos & gaspar, 2018: saberi et al., 2013). despite increase in particle size and span value, the emulsion remained stable as no visible, creaming or phase separation occurred. the further the value of the zeta potential from 0, negative or positive, the greater repulsive force between particles, which ensures emulsion stability. as the values obtain are below -50, the formulation remained stable although size and span increased over time. zeta potential values of -41 to -50 have good stability, compared to values of -11 to -20 which are less stable as it is near the agglomeration threshold (salkar & tembhurkar, 2016). although results show significant difference (p=0.000) in zeta potential values, no significant change in the formulation stability was observed as a difference of more than 10mv frombaseline to month 6 is required to exhibit a significant change (lu & gao, 2010). both formulations portray a pseudo plastic flow or also known as the shear-thinning system. this system shows that increase in shear stress will increase the shear rate hence reduce viscosity (rodrigues et al., 2016). the longer time the fluid undergoes stress, the lower the viscosity. the hysteresis loop which represents thixotropy is formed because the breakdown of the formulation does not return to its original state immediately after stress is removed. the increase in shear rate surpasses the brownian motion hence cause less resistance to flow (niu et al., 2016). topical application has been associated with shear rates of up to 120 sˉ¹ (henderson et al., 1961). lower viscosity at around these shear rates may represent the good spreadability of the cream for consumers as shown by pbl formulation (adeyeye et al., 2002). the outcome which showed reduction over time most apparent at 40°c/75%rh however is different from past research which found apparent viscosity to increase over time (goyal et al., 2015).the product is deemed stable as it can prevent microbial growth (ctfa, 2004). one of the contributing reasons is the natural antimicrobial activity conferred by piper betle leaves itself on top of the presence of antioxidant and preservative (pradhan et al., 2013). syed omar et al. (2022) journal of pharmacy, 2(2), 45-58 page 56 hydroxychavicol content in formulation stored in 30°c/75%rh shows degradation of hydroxychavicol content less than that of the ones stored in a higher temperature of 40°c/75%rh. a study on the stability of phenolic compounds in piper betle at 5ºc and 25ºc showed that temperature influences the stability of the compounds whereby higher temperature will lead to degradation of the compounds (a. ali et al., 2018). as hydroxychavicol is a part of the propenylphenol compound, it is also subjected to degradation with higher temperature (singtongratana et al., 2013). although the study found minimal effect on hydroxychavicol particularly compared to other compounds, that study was done at a temperature of 25ºc. conclusion as a conclusion, a stable cosmetic cream containing piper betle l. extract was able to be produced in large scale. identifying cqa and cpp were an integral step to ensure the production of quality product. stability study over 6 months proved the ability of the product to remain stable physically and in ph, zeta potential, particle size, viscosity also the hydroxychavicol content. acknowledgement this work was supported by the ministry of higher education malaysia under grant prgs15-009-0019. conflict of interest the authors declare that there is no conflict of interest. references adeyeye, m. c., jain, a. c., ghorab, m. k. m., & reilly, w. j. 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(2011). guidance for industry process validation: general principles and practices. 20852–21448. oil pot and aqueous pot main pot introduction according to the federal food, drug and cosmetic act and article 2 of the eu cosmetics regulation the definition of cosmetics are products that are meant to be applied on body parts either by being rubbed, poured, sprinkled, or sprayed on or introduce... based on euromonitor, the skincare segment recorded a turnover of us$107 billion worldwide in 2013, where 65% of it is contributed by facial treatments alone and 30% of the facial treatments are from skin lightening products (euromonitor, 2016). in 20... although with abundance of skin-lightening products already available in the market, many are opting for natural based skincare products due to the occurrence of adverse effects (desai, 2014; oudenhoven et al., 2015). piper betle l. was found to have ... however, the stability of natural based formulation is concerning especially the effects of scale up on the formulation if commercialization is considered. scale up is the process of increasing a batch size with the same procedures to produce a larger... methodology the zeta potential was measured using zetasizer (zen 1600, malvern, uk). samples were diluted with deionized water to 1:100 to avoid multiple scattering effects (khor et al., 2014). all measurements were carried out at temperature of 25 c (esteban et... the rheological parameter was measured using stress-controlled rheometer (haake mars, thermo scientific, germany). sample was loaded on to the lower plate of the rheometer and allowed to equilibrate to temperature of 25 c (khor et al., 2014). a shear ... 1 g of the sample was diluted to a 1 in 10 dilution using phosphate buffer solution ph 7.2. 10 ml of the sample was used for inoculation on the tryptic soy broth which was then incubated at 30-35 c for 18-24 hours. sample was sub-cultured on a mannito... for the determination of total aerobic microbial count (tamc) and total yeast and mold count (tymc) a dilution of 1:10 of the cream in phosphate buffer solution ph 7.2 was prepared. plate-count method was employed, specifically pour-plate method. 1 ml... a validated hplc method was used to identify the presence of hydroxychavicol in the formulation (omar et al., 2021). analysis of the samples was done using an isocratic system with agilent g1310a pump linked with diode array detector (agilent, stevens... pbl cream storage characteristic 6m 3m 2m 1m 0m b lb lb lb lb 30°c/75%rh colour b lb lb lb lb 40°c/75%rh s s s s s 30°c/75%rh texture s s s s s 40°c/75%rh p p p p p 30°c/75%rh odour results p p p p p 40°c/75%rh x x x x x 30°c/75%rh phase separation x disodium ethylenediaminetetraacetic acid (edta) x x x x 40°c/75%rh abbreviations: 0m is day 0 month; 1m is 1 month; 2m is 2 months; 3m is 3 months; 6m is 6 months; lb is light beige; b is beige; s is smooth; p is pleasant; x is absent process parameter process 30 minutes time mixing 1400 rpm speed 75 ± 5 °c temperature 30 minutes time homogenization 3000 rpm homogeniser speed 2700 rpm agitatator speed off temperature on vacuum rheology is the science related to the flow of fluids which is associated with processes such as mixing, pouring, filling containers and extrusion of product through container (niu et al., 2016). based on figure 5, formulations follow a non-newtonian ... microbial limit study results of the study shows no growth was observed for all tests which included tamc, tymc and the specific tests for staphylococcous aureus and pseudomonas aeruginosa over 6 months. hydroxychavicol content analysis figure 7 shows the hydroxychavicol content throughout the 6 months stability study. formulation stored in both conditions showed a declining trend but differ in the extent of degradation. the final content for formulations stored in 30 c/75%rh and 40 ... discussion the light beige colour of the piper betle leaves (pbl) cream was caused by the dark brown colour of the extract mixed with white base cream. after 6 months of storage, pbl cream became a tone darker. raised temperature is known to cause physical chang... the particle size ranges from 0.99 µm 1.92 µm is considered small and is favoured as it confers higher stability (khor et al., 2014). the gradual increase in particle size which is especially evident at 6 months can be caused by ostwald ripening, fl... the further the value of the zeta potential from 0, negative or positive, the greater repulsive force between particles, which ensures emulsion stability. as the values obtain are below -50, the formulation remained stable although size and span incre... both formulations portray a pseudo plastic flow or also known as the shear-thinning system. this system shows that increase in shear stress will increase the shear rate hence reduce viscosity (rodrigues et al., 2016). the longer time the fluid undergo... topical application has been associated with shear rates of up to 120 sˉ¹ (henderson et al., 1961). lower viscosity at around these shear rates may represent the good spreadability of the cream for consumers as shown by pbl formulation (adeyeye et al.... hydroxychavicol content in formulation stored in 30 c/75%rh shows degradation of hydroxychavicol content less than that of the ones stored in a higher temperature of 40 c/75%rh. a study on the stability of phenolic compounds in piper betle at 5ºc and ... conclusion as a conclusion, a stable cosmetic cream containing piper betle l. extract was able to be produced in large scale. identifying cqa and cpp were an integral step to ensure the production of quality product. stability study over 6 months proved the abil... acknowledgement conflict of interest the authors declare that there is no conflict of interest. references barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework jamalud-din et al. (2022) journal of pharmacy, 2(2), 141-148 page 141 types and costs of medications returned by outpatients at a malaysian teaching hospital: a one-year cross-sectional study. nor akilah jamalud-din1*, tengku karmila tengku mohd kamil2, mery hu wei ying3, mohamed hassan elnaem2, abdulkareem mohamed ahmed4, nor ilyani mohamed nazar2, and nor hidayah mohd taufek2 abstract introduction: medication wastage is attributable to various factors, including therapeutic failure, over-prescription, and poor compliance. data regarding the cost and types of returned medications are essential to estimate the financial burden that can be reduced locally through targeted intervention. this study aimed to investigate the types and cost of medications returned by outpatients at a teaching hospital in kuantan, pahang. materials and methods: this cross-sectional study collected data from the electronic medical record based on the list of returned medications at the outpatient pharmacy department at sultan ahmad shah medical centre (sasmec) @iium between january and december 2021. all medications with sasmec patients' label were included. the medications were classified according to the classification index based on the british national formulary. the data were analysed using microsoft excel version 16.52. the cost associated with returned medications was calculated, referring to the latest cost price of the respective medications. results: a total of 134 patients returned their medications in 2021 (mean ± sd age: 63 ± 13 years), with 52% females. the total quantities of returned medications were 11,054 units. these included pills, devices (inhalers, insulin pens), bottles, and plastic containers. the overall cost of these medications was ringgit malaysia (rm) 13,594.90 (mean per patient: rm 101.45). the highest number of returned medications was from the cardiovascular group (32.5%), followed by the endocrine group (26%) and musculoskeletal group (11.9%). the highest cost for returned medications was the endocrine group (49%), followed by the cardiovascular group (20%) and the musculoskeletal group (10%). conclusion: medications used for cardiovascular diseases were the most frequently returned at outpatient pharmacy, whereas those for endocrine diseases contributed to the highest cost. future research could focus on identifying modifiable factors that can contribute to the sustainability of health resources by reducing the cost of returned medications. article history: received: 22 april 2022 accepted: 24 june 2022 published: 31 july 2022 keywords: cost, returned medications, types, outpatients, factors. how to cite this article: jamalud-din, n. a., tengku mohd kamil, t. k., mery hu, w. y., elanem, m. h., mohamed ahmed, a. mohamed nazar, n. i. & mohd taufek, n. h. types and costs of medications returned by outpatients at a malaysian teaching hospital: a one-year cross-sectional study. journal of pharmacy, 2 (2), 141-148. doi: 10.31436/jop.v2i2.150 authors’ affiliation: 1department of pharmacy practice, kulliyyah of pharmacy, international islamic university malaysia, 25200, kuantan, pahang. 2quality use of medicine research group, kulliyyah of pharmacy, international islamic university malaysia, 25200, kuantan, pahang. 3department of pharmacy, sultan ahmad shah medical centre, international islamic university malaysia, 25200, kuantan, pahang. 4department of pharmacy practice & clinical pharmacy, faculty of pharmacy, picoms international university college, batu caves, kuala lumpur. *corresponding author: email address: akilah.jamaluddin97@gmail.com page 142 jamalud-din et al. (2022) journal of pharmacy, 2(2), 141-148 introduction medication wastage occurs when medications are expired and unused throughout the supply chain of the medications. it adversely affects the economy and the patients themselves, thus requiring appropriate education to minimise pharmaceutical waste (west et al., 2015; smale et al., 2021). in addition, it has been reported that expired or unused medications could threaten both the healthcare system and the environment (ebrahim et al., 2019). for example, multiple strains of antibiotic-resistant bacteria have also been identified in unused medications disposal sites (watkinson & costanzo, 2017). medication wastage could also lead to loss of resources (ali & ibrahim, 2009; alnahas et al., 2020). for example, in sultan qaboos university hospital, approximately three million omani rial of the hospital budget was spent on purchasing the medications, where 40% and 60% of the hospital pharmacy itself had spent on its medications budget for inpatients and outpatients, respectively (alsiyabi & al-riyami, 2007). in the united states, the average annual cost of wasted medications was usd30.47 per person, resulting in a total annual cost of over usd1 billion for adults over the age of 65 (morgan, 2001). in the united kingdom, about £300 million was spent on unused prescription medication by the national health services annually in 2009 (trueman et al., 2011). in addition, around €10,000 was spent in malta on 80 different types of medications handed in to government pharmacies by the public over three months (times of malta, 2012; west et al., 2015). in japan, the annual medical expenses were increased to more than jpy43 trillion (matsuda, 2019), and the prescription medication cost was estimated at jpy10 trillion (ueki et al., 2022). generally, the medication cost contributes to a large percentage of overall medical costs. it may be due to the rising price of new medications (ghinea, lipworth & kerridge, 2016). it was anticipated that the global spending on medications to reach almost $1.3 trillion in 2018, which is a 30% increment over 2013 (aitken et al., 2014). the increased price of medication affects the sustainability of the health insurance system (ueki et al., 2022), so the initiative to investigate and monitor the medical costs is crucial to prevent medication wastage. this has become a global issue and can potentially influence the cost associated with medication disposal (ebrahim et al., 2019). on top of that, wastereducing actions taken by pharmacists, such as improving stock management, improving the pharmaceutical preparation process, increasing dispensing expertise, and redispensing unused medications, may help to ensure the long-term viability of the healthcare system, where sustainable supply and use of medication can be achieved (smale et al., 2021). it has also been reported that the reasons of returning unused medications to the pharmacy include public awareness regarding environmental risks posed by inappropriate disposal (arkaravichien et al., 2014; ong et al., 2020). in malaysia, more than 80% of the study population in selangor were aware of the issue of medication wastage and its effects on patients and the economy (hassali & shakeel, 2020). ibrahim and colleagues were the first investigated the extent of medication wastage by malaysian consumers in 1996, where they collected 451 types of leftover medications from 101 houses (ibrahim, awang, razak, 1997). in 2016, a single public hospital in malaysia has allocated approximately rm 62 million for medications, indicating that medications accounted for a significant portion of a hospital's revenue and expenditure (sim et al., 2018). the malaysian ministry of health claimed that between 2014 and 2016, rm 2 million was spent on the disposal of spoiled medications, which included medications from government hospitals' "medication take-back" services (rahim, 2016). there have been several reports of medication waste in hospitals in malaysia. for instance, at hospital tuanku jaafar seremban, the total cost was rm 4,362.28, and the average wastage per patient was rm 42.35 (hassali et al., 2012). an analysis of unused medications can help to identify patients who are stockpiling their medications, which could result in unnecessary health care costs, so intervention can be made to address this issue. therefore, this study aimed to examine the types and costs of medications returned by outpatients at a teaching hospital in pahang, malaysia. methodology this study received ethical approval from the international islamic university malaysia (iium) research ethics committee (irec) (id no. irec 2021-205). this study was performed in the outpatient pharmacy department at sultan ahmad shah medical centre (sasmec) @iium between january 2021 and december 2021 (a cross-sectional study). pharmacists collected returned medications, and data were recorded in the system. the data included patient registration number, age, gender, drug name, strength, dosage form, date of returned medications and quantities. each drug was counted as one dosage unit regardless of the dosage form (e.g. pills, pens, syrup). for the current study, data were collected from the electronic medical record based on the list of returned medications at the outpatient pharmacy. only medications with sasmec patients' labels were included, excluding items from external sources. all the returned medications were considered as waste and could not be re-used for other patients (alhamad et al., 2017). the medications were classified according to the classification index of the british national formulary. the data were analysed using microsoft excel version 16.52 programme. the cost associated with returned medications was calculated descriptively, referring to the latest cost price of the respective medications and per unit according to the dosage page 143 jamalud-din et al. (2022) journal of pharmacy, 2(2), 141-148 form (e.g. price per pill/tablet, pen, bottle). the ringgit malaysia (rm) amount that is stated throughout this manuscript refers to per unit of dosage form. results a total of 134 patients (mean ± sd age: 63 ± 13 years) returned their medications at the outpatient pharmacy throughout the study period. female patients were higher (52%) than males (48%), with most of the participants (99%) were elderly (60 years and above). a total of 11,054 units of returned medications were collected throughout 2021, with an average per patient of 82 units. the medications that were excluded were 22 units because they were from other sources like other private clinics or hospitals; thus, they were not under the cost that was covered by sasmec @iium. the total number of returned expired medications were 883 units. the average wastage was 921 units per month, consisting of pills, devices (inhalers, insulin pens), bottles, and plastic containers. the top six quantities of returned medications were calcium carbonate, metformin, perindopril, prazosin, atorvastatin, and celecoxib (table 1). table 2 shows the top six highest costs of returned medications, and most of them were devices. overall, endocrine group contributed to the highest cost of returned medications, although the percentage quantities were lower than the cardiovascular group (table 3). the most common pharmacological group of all returned medications was the cardiovascular medications (table 3). table 1: top six returned medications with the highest quantities at the outpatient pharmacy. table 2: top six returned medications with the highest cost at outpatient pharmacy. item quantity rm novorapid® insulin 95 2,653.00 levemir® insulin 65 2,600.00 clexane 40mg injection 77 1,463.00 celecoxib 200mg tab 362 818.48 novomix® insulin 26 719.68 tramadol 37.5mg and paracetamol 325mg tab 342 519.26 table 3: percentage distribution of pharmacological groups with quantity and cost. pharmacological classes percentage of medication quantities (%) percentage of medication cost (%) cardiovascular 32.5 19.24 respiratory 1.1 2.16 immuno and malignant 1.8 1.42 anti-infectives 0.1 2.16 central nervous system 4.4 2.8 peripheral nervous system 5.2 4.44 endocrine system 26 48.67 musculoskeletal 11.9 10.37 genito-urinary 1.4 0.85 eye-ear 4.4 5.13 gastrointestinal 5.1 1.1 nutrition 6.2 1.69 discussion our observations indicated that the return of unused medications was the most common among the elderly population. in the united states, medication wastage was amounted to more than usd1 billion among the elderly population (over the age of 65 years old), accounting for 2.3% of total medication costs (morgan, 2001; el-hamamsy, 2011). this might occur because the percentage of the elderly population has increased gradually in developed societies (chiatti et al., 2012). it was estimated that americans aged 65 and above to nearly double over the next three decades, rising from 48 million to 88 million by 2050 (national institutes of health, 2016). the health and economic burden of the elderly population was related to undesirable medication effects, improper drug prescribing, and poor adherence, all of which resulted in huge cost of unused medications (chiatti et al., 2012). we report in the current study about the types, quantities and costs associated with returned medications that were collected at the outpatient pharmacy at a teaching hospital in pahang state. a total of 11, 054 units of medications were returned in 2021, with an average per month of 921 units consisting of various dosage forms. from this, the total cost of the returned medication was rm 13,594.90, with an average of rm 1,132.90 per month. in comparison, a government hospital in a different state negeri sembilan reported a higher number of pills returned to outpatient pharmacy between june and november 2007, which was 131,098 pills, with an average of 21,850 pills per month (hassali et al., 2012). the total cost of medications reported in a government hospital in negeri sembilan between june and november 2007 was rm 59,566.50. the average wastage for each month was rm 9,927.75 the estimated total cost of the wastage for that year was rm item quantity rm calcium carbonate 500mg tab 2,070 103.50 metformin 500mg tab 1,998 159.84 perindopril 4mg tab 649 39.46 prazosin 2mg tab 475 20.93 atorvastatin 40mg tab 405 57.31 celecoxib 20 mg tab 362 818.48 page 144 jamalud-din et al. (2022) journal of pharmacy, 2(2), 141-148 119,133.00 (hassali et al., 2012). a different study in oman reported that 1,171 medications were returned to the pharmacy (mean per patient: 3.1 medications) (al-siyabi & al-riyami, 2007). our findings indicate a relatively lower return rate of returned medications by the outpatients at sasmec @iium. the present study also found that calcium carbonate, metformin, perindopril, prazosin, atorvastatin, and celecoxib were the top six highest in quantities of returned medications at the outpatient pharmacy. on the contrary, hassali et al. (2012) have reported that the highest quantities of medications returned to the pharmacy within five months were isosorbide dinitrate 10 mg tab, metformin 500 mg tab, lovastatin 20 mg tab, frusemide 40 mg tab, trimetazidine 20 mg tab and potassium chloride 600 mg tab. in egypt, the top five medications returned to 20 community pharmacies were antibiotics, gastrointestinal, cardiovascular system, respiratory system and nervous system medications (elhamamsy, 2011). among the reasons reported by elhamamsy (2011) were because the patient felt better or died, and medications reached the expiry date. in australia, 60% of the returned medications were pharmaceutical benefits scheme (pbs) medications, which were subsidised prescription medications (bettington et al., 2018). the topsix pbs-listed medications in australia in 2016 were paracetamol, salbutamol, glyceryl trinitrate, cefalexin, metoclopramide and doxycycline (wheeler et al., 2016). most of them were considered "if necessary" medications for acute diseases such as nausea, acute infections, asthma, and angina attacks (bettington et al., 2018). metformin was among the common medications of returned medications in our study. this could be due to nonadherence issues with the frequency to take metformin three times daily (hassali et al., 2012). similar results were reported where diabetic patients who took oral hypoglycemic agents tend to have adherence rates of 74.8% for once-a-day doses and 38% for three times daily doses (paes & barker, 1997; hassali et al., 2012). one-daily dosing was a strategy of reducing regimen complexity and thereby increasing adherence rate (roca, lapuebla, & vidal-tegedor, 2005; kuna et al., 2006; laliberte et al., 2013; hernández arroyo et al., 2016; oh et al., 2020). therefore, simplifying the dosing regimen to a once-daily dosage might be the most effective strategy to improve patient adherence (ma et al., 2010; rubio et al., 2010; fabbiani et al., 2014; elnaem et al., 2020). analgesics were found to be commonly returned medications in our study. analgesics indicated for chronic and acute pain were commonly prescribed in excessive amounts (bettington et al., 2018). for example, a study has highlighted that between 42% and 71% of opioid tablets prescribed after surgery remain unused (bicket et al., 2017). this could happen due to unawareness of healthcare providers about unused analgesics (chou et al., 2016). in hospital settings, the development of evidence-based prescription recommendations is complicated by diverse patient groups and treatment types (bicket et al., 2017). however, prescribing smaller quantities was not recommended even though it was a commonly proposed intervention to prevent medication wastage. this is due to non-adherence towards the medication and leads to more frequent dispensing and prescribing that later can increase the costs instead (bettington et al., 2018). future research on economic analysis can be explored to overcome this issue. in our study, cardiovascular medications were the most common pharmacological group of all returned medications (32.5%). it was similar to a study that reported cardiovascular medications were the highest quantity (24%) of returned medications (al-siyabi & al-riyami, 2007). it has been reported previously that patients did not believe in the necessity of statins (40%), some of them were not convinced (48%), and did not sure of statin's efficacy (47%), did not believe that statins can prevent cardiovascular disease (49%) or believed that the efficacy of the statin is limited (71%) (wouters et al., 2016). there were concerns about side effects associated with statins including ache, stiffness, muscle pain, swelling, or joint inflammation (newman et al., 2019). in our study, atorvastatin was prescribed once a day, and it depends on the patient to take it at any time, but there was a possibility that the patient took it at night, leading to non-adherence. the lack of convenience in taking medication might become the barrier to patient’s adherence. for example, lovastatin that was suggested to be taken at night, contributed to patients' non-adherence since they tended to forget to take the medication (hassali et al., 2012). non-adherence to anti-hypertensive medications could be the cause of wastage, as it was reported globally ranging from 10% to 80% (wijekoon et al., 2020). our study showed that the endocrine category of medications contributed to the highest cost (48.67%) due to the high cost of insulin pen. nevertheless, a previous study has reported that anti-infective group had the highest cost (61%) due to the expensive price of anti-viral drugs (valaciclovir) (al-siyabi & al-riyami, 2007). a study in australia found that cardiovascular group medications possessed the highest cost of disposed medications which amounted to about aud 2923 (guirguis, 2010). the highest cost of specific category of medications depicted the large quantities of returned medications, and its high utilised especially among the elderly (jörgensen et al., 2001). in addition, the high cost of the medications contributes to the high wastage cost even though the quantity is small. we found that the quantity of returned novorapid® insulin was 95 pens, but it contributed to the highest cost which was rm 2,653.00, followed by levemir® insulin (rm 2,600.00), and clexane 40 mg injection (rm 1,463.00). the quantities of these three medications were much lower than tablet celecoxib 200 mg, page 145 jamalud-din et al. (2022) journal of pharmacy, 2(2), 141-148 tablet tramadol 37.5 mg, and tablet paracetamol 325 mg. on the other hand, another study demonstrated that the highest cost of medication wastage was contributed by tablet donepezil 5 mg with rm 6,129.00 with 690 units, compared to 1920 tablets of amlodipine 5 mg, with the cost of rm 1,996.00 (hassali et al., 2012). it has been highlighted that the four main factors of medication wastage were the diagnosis (37.4%), perceived ineffectiveness (22.6%), change of prescriptions (15.8%) and perceived adverse effects (14.4%) (morgan, 2001; hassali et al., 2012). from an economic perspective, the behaviour of patients when using subsidised and non-subsidised medication could vary in particular to compliance. in brunei, the health care system was mainly subsidised, and medication wastage has been reported to be high as it affected the increment of brunei's government health expenditure by about 3.5% from 2013 to 2016 (nurolaini, 2016). in malaysia, a study found that self-paying patients had better medication adherence (63.8%) than subsidised patients (50%) (aziz et al., 2018). on the contrary, a different study reported different findings where subsidised patients had high adherence rates of 84.6%, compared to patients who paid 50% of medication cost (adherence rate of 71.6%), patients who paid for 75% of medication cost (adherence rate of 72.3%) and patients who fully paid the medication cost (adherence rate of 79.2%) (batavia et al., 2010; aziz et al., 2018). nevertheless, among the selfpaying patients, the adherence rate was higher in patients who paid more (batavia et al., 2010). in our study, subsidy or discount was available for pensioners, students, and staff of iium, which might influence non-adherence to a certain extent. it is the limitation of our study that this information was not investigated. future studies exploring this aspect would be able to develop tailored cost-effective strategies for different categories of outpatients to reduce the cost of unused medications. conclusion the most returned medications at our outpatient hospital setting were from the cardiovascular group, followed by endocrine group and musculoskeletal group. the highest cost for returned medications was the endocrine group, followed by cardiovascular group and musculoskeletal group. future studies should explore modifiable factors that contribute to the unused medications returned to the pharmacy. acknowledgements the authors would like to thank financial assistance received from sultan ahmad shah medical centre (sasmec) research grant 2021 and research clusterresearch initiative grant scheme (rc-rigs21). both research grants were provided by the international islamic university malaysia. the funders had no role in the study design and data interpretation. conflict of interest all the authors declare no conflict of interest. references al-siyabi, k., & al-riyami, k. 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(2016). understanding statin non-adherence: knowing which perceptions and experiences matter to different patients. plos one, 11(1), 1–12. https://doi.org/10.1371/journal.pone.0146272 introduction medication wastage occurs when medications are expired and unused throughout the supply chain of the medications. it adversely affects the economy and the patients themselves, thus requiring appropriate education to minimise pharmaceutical waste (wes... medication wastage could also lead to loss of resources (ali & ibrahim, 2009; alnahas et al., 2020). for example, in sultan qaboos university hospital, approximately three million omani rial of the hospital budget was spent on purchasing the medicat... generally, the medication cost contributes to a large percentage of overall medical costs. it may be due to the rising price of new medications (ghinea, lipworth & kerridge, 2016). it was anticipated that the global spending on medications to reach al... in malaysia, more than 80% of the study population in selangor were aware of the issue of medication wastage and its effects on patients and the economy (hassali & shakeel, 2020). ibrahim and colleagues were the first investigated the extent of medica... methodology this study received ethical approval from the international islamic university malaysia (iium) research ethics committee (irec) (id no. irec 2021-205). this study was performed in the outpatient pharmacy department at sultan ahmad shah medical centre (sasmec) @iium between january 2021 and december 2021 (a cross-sectional study). pharmacists collected returned medications, and data were recorded in ... for the current study, data were collected from the electronic medical record based on the list of returned medications at the outpatient pharmacy. only medications with sasmec patients' labels were included, excluding items from external sources. all... results a total of 134 patients (mean ± sd age: 63 ± 13 years) returned their medications at the outpatient pharmacy throughout the study period. female patients were higher (52%) than males (48%), with most of the participants (99%) were elderly (60 years an... discussion our observations indicated that the return of unused medications was the most common among the elderly population. in the united states, medication wastage was amounted to more than usd1 billion among the elderly population (over the age of 65 years ... we report in the current study about the types, quantities and costs associated with returned medications that were collected at the outpatient pharmacy at a teaching hospital in pahang state. a total of 11, 054 units of medications were returned in 2... the present study also found that calcium carbonate, metformin, perindopril, prazosin, atorvastatin, and celecoxib were the top six highest in quantities of returned medications at the outpatient pharmacy. on the contrary, hassali et al. (2012) have r... metformin was among the common medications of returned medications in our study. this could be due to non-adherence issues with the frequency to take metformin three times daily (hassali et al., 2012). similar results were reported where diabetic p... analgesics were found to be commonly returned medications in our study. analgesics indicated for chronic and acute pain were commonly prescribed in excessive amounts (bettington et al., 2018). for example, a study has highlighted that between 42% and ... in our study, cardiovascular medications were the most common pharmacological group of all returned medications (32.5%). it was similar to a study that reported cardiovascular medications were the highest quantity (24%) of returned medications (al-si... our study showed that the endocrine category of medications contributed to the highest cost (48.67%) due to the high cost of insulin pen. nevertheless, a previous study has reported that anti-infective group had the highest cost (61%) due to the expen... in addition, the high cost of the medications contributes to the high wastage cost even though the quantity is small. we found that the quantity of returned novorapid® insulin was 95 pens, but it contributed to the highest cost which was rm 2,653.00, ... from an economic perspective, the behaviour of patients when using subsidised and non-subsidised medication could vary in particular to compliance. in brunei, the health care system was mainly subsidised, and medication wastage has been reported to be... conclusion the most returned medications at our outpatient hospital setting were from the cardiovascular group, followed by endocrine group and musculoskeletal group. the highest cost for returned medications was the endocrine group, followed by cardiovascular... acknowledgements the authors would like to thank financial assistance received from sultan ahmad shah medical centre (sasmec) research grant 2021 and research clusterresearch initiative grant scheme (rc-rigs21). both research grants were provided by the internati... conflict of interest all the authors declare no conflict of interest. references barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework sidi omar et al. (2021) journal of pharmacy, 1(1), 8-18. page 8 human immunodeficiency virus (hiv)/ acquired immunodeficiency syndrome (aids): knowledge, perception, and attitude among pharmacists in kelantan, malaysia. muhammad munzir sidi omar1*, nur amalina wahida ab wahab1, ong ann gie1, nurul izyan mohamed azam1, aqilah muhammad1 and siti nor asiah ab ghani1 abstract introduction: although pharmacists’ role in the care of human immunodeficiency virus (hiv)infected and acquired immunodeficiency syndrome (aids) patients is well established, studies had reported pharmacists’ negative attitudes towards people living with hiv/aids (plwha), with negative impact on hiv management. this study aimed to explore pharmacists’ knowledge of hiv/aids, perception towards the changing of treatment regimens of hiv/aids, attitudes towards plwha, and to identify factors affecting the pharmacists’ attitudes towards plwha in the state of kelantan, which reportedly have the third highest number of hiv/aids patients in malaysia. materials and method: a validated online 43-item questionnaire was distributed to 400 pharmacists in kelantan. multivariate logistic regression was performed to identify factors associated with pharmacists’ negative attitude towards plwha. results: a total of 170 respondents (response rate 42%) completed the questionnaire. respondents had knowledge on the potential causes of hiv infection [median (iqr) score=13.00(1); maximum score=14.00], and preventive measures of hiv/aids transmission [median (iqr) score=12.00(1); maximum score=12.00]. on decision in changing regimens, the respondents agreed on the need to change treatment regimen for hiv/aids when required [median (iqr) score=7.00(2); maximum score=8.00]. up to 40.6% of respondents thought that the treatment regimen should not be switched based on cost. two-thirds of the respondents had negative attitudes towards plwha (67.1%). respondents who worked in community pharmacy reported positive attitudes towards plwha (adj or=0.125; 95%ci=0.025-0.623; p=0.011). conclusion: pharmacists in kelantan had several misconceptions towards the causes of hiv/aids, preventive measures of hiv/aids transmission, and necessity in changing treatment regimen when required. despite good disease related knowledge, most pharmacists had negative attitudes towards plwha, while pharmacists working in community settings presented more positive attitudes towards plwha. article history: received: 18 august 2020 accepted: 11 november 2020 published: 8 january 2021 keywords: pharmacists, people living with human immunodeficiency virus/acquired immunodeficiency syndrome (plwha), knowledge, perception, attitude. how to cite this article: sidi omar, m. m., ab wahab, n. a. w., ong, a. g., mohamed azam, n. i., muhammad, a. & ab ghani, s. n. a. (2021). human immunodeficiency virus (hiv)/acquired immunodeficiency syndrome (aids). journal of pharmacy, 1(1), 8-18. authors’ affiliation: 1 unit of pharmacy, hospital tanah merah, 17500 tanah merah, kelantan, malaysia *corresponding author: email address: munzir66@yahoo.com tel:+6013 4579535 sidi omar et al. (2021) journal of pharmacy, 1(1), 8-18. page 9 introduction human immunodeficiency virus (hiv) remains a major global public health issue. approximately 36.9 million people globally (world health organisation, 2019), and 87,122 people in malaysia was living with hiv (ministry of health malaysia, 2018) in 2017. almost 33 million lives were claimed by hiv infection by 2019. however, with the increasing efforts by the healthcare sector in hiv prevention and treatment, the infection has become a manageable chronic health condition (world health organisation, 2019). since 2015, malaysia has adopted the vision of world health organisation (who) in the new national strategic plan in ending aids 2016-2030 to end the acquired immune deficiency syndrome (aids) epidemic as a public health threat by year 2030, with the goal of “three zeros: zero new hiv infections, zero hiv-related deaths and zero hiv-related discrimination” (ministry of health malaysia, 2015). antiretroviral therapy (art) is the main treatment in management of hiv infection. high levels of adherence towards arts of greater than 95% is needed to adequately suppress hiv-viral replication, produce long-lasting response, and halt disease progression (paterson et al., 2000). poor patient and health care providers relationship was found to be among one of the significant causes of poor adherence to art (schaecher, 2013). involvement of pharmacists in the care of people living with hiv/aids (plwha) has been proven to be associated with better treatment outcomes through art adherence enhancement and pill burden reduction, resulting in improved cd4 cell counts, rates of viral suppression, and reduced medication errors (tseng et al., 2012). hiv-related discrimination is defined as the act of treating plwha differently than those without hiv; while hiv stigma is defined as the negative beliefs, feelings, and attitudes towards plwha (centers for disease control and prevention, 2019). hiv-related stigma and discrimination is a recognised barrier in combating hiv burden. in thailand, it was found that plwha were being denied health care services, provided with poor quality and delayed care, and were treated discriminatorily with violated rights due to the hiv-related stigmatization and discrimination (pudpong et al., 2014). these negative experiences may hinder plwha to seek or to access healthcare services. in malaysia, pharmacists play significant roles in providing education and counselling to plwha. in clinical settings, specialised medication therapy adherence clinics run by pharmacists were set up to monitor therapy adherence and outcomes, assess medication-related issues, and provide quality services for plwha (pharmaceutical services division ministry of health malaysia, 2014). although pharmacists’ involvement in hiv-positive patients care is well-established and recognised, one study conducted among hospital pharmacists in kedah, malaysia reported that hospital pharmacists had negative attitudes towards plwha (khan & baig, 2013). this may indicate hindrance towards health-care accessibility of plwha which can further impair the ministry of health’s efforts to curb the disease. however, these findings may not be generalised to kelantan, a different state in malaysia with the third most hiv case prevalence (10.3%). distribution of hiv-positive cases were lower in kedah (6.1%) (ministry of health malaysia, 2018), and the pharmacists’ exposure and awareness towards hiv may be lesser. in addition, the population and cultural distribution of kedah and kelantan is very different. in terms of ethnicity groups, kedah consists of 78% malays, 13.6% chinese, and 7.2% indians; while kelantan consists 95.6% malays, 3.4% chinese, and 0.3% indians (city population, 2017). these significant differences between the states may lead to differences in cultural beliefs which may further affect attitude towards plwha. furthermore, since the initiation of who’s national strategic plan in ending aids in 2015, there has been increased educational activities to promote awareness and reduce hiv-stigma in the recent years (ministry of health malaysia, 2015). therefore the findings by khan and baig (2013) may not reflect the current scenario among pharmacists in malaysia. hence, this study was conducted to explore pharmacists’ knowledge towards hiv/aids, perceptions towards changing of treatment regimen in hiv/aids, attitudes towards plwha, and investigate the factors affecting pharmacists’ attitudes towards plwha in the state of kelantan, malaysia. methodology study design and study process this cross-sectional self-administered online questionnaire-based study was conducted among pharmacists who worked in kelantan, malaysia. ethical approval was obtained from the ministry of health medical research ethics committee (nmrr-19-26-45683). all fully registered pharmacists and provisionally registered pharmacists who practiced in kelantan were recruited. pharmacists who were non-malaysian, retired, and on study leaves were excluded. the samples were included by using convenience sampling. emails of pharmacists who worked in kelantan were obtained through pharmaceutical services division of kelantan with permission. softcopies of formal invitations were distributed through valid emails to 400 pharmacists. emails were not sent to pharmacists who had no record of emails or with invalid emails, and pharmacists who met the exclusion criteria. two reminder emails were sent on two weeks intervals to increase the number of participants. participation by pharmacists into this study was voluntary. after informed consent form was signed, study participants were required to fill in respective pharmacist’s registration number as a measure to ensure that only sidi omar et al. (2021) journal of pharmacy, 1(1), 8-18. page 10 pharmacists were included into this study, and to avoid duplication of responses. instructions on filling the questionnaire were provided at the start of the questionnaire. all questions were displayed in the online questionnaire. for open-ended questions, participants were required to type in their answers. for closed-ended questions, participants were required to choose only one answer from the options displayed. the participants were required to answer all the questions before submitting the questionnaire. all the data were synchronised into a password-protected online subject data sheet automatically. study sample traced data from pharmaceutical services division of kelantan had shown that there were 334 pharmacists working in government facilities, 109 pharmacists working in private sectors, and 40 pharmacists working in academic institution. by using raosoft® sample size calculator, assuming the margin of error = 5%; confidence level = 95%; population size = 483; and response distribution = 50%, the minimum sample size required was 215. study instrument permission for the adoption of the validated questionnaire was obtained from khan and baig (2013). the questionnaire was developed into an online questionnaire. the questionnaire consisted of six sections. compared to the questionnaire by khan and baig (2013), collection of demographic data were modified to include academic qualifications and place of practice, and to exclude marital status. there were no modifications done on the questionnaire from section two to section six, which consisted of 43-items. a pilot test was carried out among 20 pharmacists working in other states of malaysia. respondents who were involved in the pilot study were not included in this study sample. section one was designed to obtain the respondents’ demographic information including age, gender, race, academic qualification, job experience, number of hiv encountered in the past twelve months, and place of practice. section two was designed to assess respondents’ general knowledge regarding hiv/aids. three open-ended questions were included in this section, including “what does the abbreviation of aids stands for?”, “is hiv a transmittable disease?”, and “can hiv/aids be cured at this moment?”. nominal scale “yes/no” was applied for the respondents to answer the statements from sections three and four. section three was designed to assess respondents’ knowledge towards the causes of hiv/aids, which consisted of 14 items. “yes” were the correct answers for statements one to eight. “no” were the correct answers for statements 9 to 14. one point was awarded to the respondent’s score for every correct answer. section four was designed to assess respondents’ knowledge towards the preventive measures of hiv transmission, which consisted of twelve items. “yes” were the correct answers for statements one to six. “no” were the correct answers for statements seven to twelve. one point was added to the respondent’s score for every correct answer. section five was designed to assess respondents’ perceptions on the decision in changing therapeutic regimen of hiv-positive patients, which consisted of eight items. nominal scale “yes/no” was applied for the respondents to answer the statements in this section. section six was designed to evaluate the respondents’ attitudes towards plwha, which consisted of six questions. nominal scale “yes/no” was applied for the respondents to answer the statements in this section. for all the statements, every “yes” contributed one point to the respondent’s total scores; while every “no” contributed two points to the respondent’s total scores (khan & baig, 2013). the maximum possible score on this section was 12. respondents who scored 6 were classified as having positive attitudes towards plwha, who scored 7-10 were classified as having negative attitudes towards plwha, and who scored 11-12 were classified as having very negative attitudes towards plwha (khan & baig, 2013) statistical analysis data were cleared and analysed using ibm spss version 20.0 (ibm corp, armonk, ny). kolmogorovsmirnov test was used to determine the normality of analysed data, with p>0.05 were considered as normally distributed. as all the numerical data were not normally distributed, the numerical variables were presented as median and interquartile range (iqr). categorical data were presented as frequency and percentage. the demographic information and response statements were analysed by using descriptive statistics. logistic regression was used to explore the independent factors contributed to attitudes of pharmacists towards plwha. the variables with p<0.200 in simple logistic regression were included into the variable selection for multiple logistic regression model. all the tests were two sided and p<0.05 were considered as statistically significant. results a total of 170 pharmacists (79.1% of the recommended minimum sample size) completed the questionnaire with a response rate of 42%. the baseline demographic data of respondents participated in this study are summarised in table 1. more than half of the respondents were aged 24-30 years old (57.1%). majority of the respondents were females (78.8%). most of respondents were malay (92.4%). almost one-third of the respondents had job experience of 4-7 years (37.1%), and almost two-thirds of the respondents sidi omar et al. (2021) journal of pharmacy, 1(1), 8-18. page 11 encountered 1-20 patients in the past twelve months (62.4%). most of the respondents worked in government hospitals (61.2%). table 1: demographic data of study respondents demographic data (n = 170) n (%) age (years) 21-30 97 (57.1) 31-40 60 (35.3) 41-50 11 (6.5) 51-60 2 (1.1) gender male 36 (21.2) female 134 (78.8) race malay 157 (92.4) chinese 11 (6.5) indian 0 (0.0) others 2 (1.1) academic qualification bachelor 159 (93.5) master 11 (6.5) job experience < 1 year 14 (8.2) 1 – 3 years 37 (21.8) 4 – 7 years 63 (37.1) 8 – 11 years 25 (14.7) 12 – 14 years 13 (7.6) > 14 years 18 (10.6) number of hiv patients encountered for the past 12 months none 39 (22.9) 1 – 20 patients 106 (62.4) 21 – 50 patients 14 (8.2) > 50 patients 11 (6.5) place of practice government hospital 104 (61.2) government health clinic 43 (25.3) pharmaceutical services division 10 (5.9) community pharmacy 9 (5.3) academic institution 2 (1.1) private clinic/ hospital 1 (0.6) pharmaceutical industry 1 (0.6) pharmacists’ general knowledge about hiv/aids it was revealed that 87.6% of the respondents were aware of the abbreviation of aids, 98.8% agreed that hiv is a contagious disease, and 95.9% of the respondents understood that hiv/aids is unable to be cured at this moment. pharmacists’ knowledge towards the causes and preventive measures of hiv/aids majority of the respondents aware of the potential causes [median (iqr) score = 13.00 (1.0); maximum score = 14.00] and preventive measures [median (iqr) score = 12.00 (1.0); maximum score = 12.00] of hiv/aids transmission. the respondents showed most erroneous perceptions on the causes of hiv through kissing an hivinfected person (31.8%), followed by tattoo or body piercing (27.6%), and breast-feeding from an hiv-infected mother (26.5%) (table 2). one-sixth of the respondents believed that treating sexually transmitted diseases (stds) promptly did not prevent transmission of hiv (14.1%), and 10.6% of respondents believed that avoiding mosquitoes’ bites can prevent transmission of hiv (table 3). table 2: pharmacists’ knowledge towards the possible causes of hiv infection statements correctly answered, n (%) sexual intercourse without a condom with hiv-infected person 166 (97.7) sharing needle with hiv-infected person 169 (99.4) transfusion of hiv-infected blood or receiving hiv-infected organ 169 (99.4) having sex with multiple sexual partners with unknown hiv status 167 (98.2) from an hiv positive mother to her foetus 160 (94.1) sharing personal items such as shaving blades 141 (82.9) breast-feeding from an hiv-infected mother 125 (73.5) having tattoo or body piercing 123 (72.4) kissing an hiv-infected person [mouth to mouth/ french kissing] 116 (68.2) mosquito bites 156 (91.8) sharing/ eating a meal with an hivinfected person 158 (92.9) using a public swimming pool 166 (97.7) by using a public toilet 168 (98.8) casual contacts (hugging or touching) with an hiv-infected person 167 (98.2) sidi omar et al. (2021) journal of pharmacy, 1(1), 8-18. page 12 table 3: pharmacists’ knowledge towards the preventive measures of hiv infection statements correctly answered, n (%) avoid taking illicit drugs/use of intravenous drug 158 (92.9) by avoiding sharing needles and syringes 170 (100.0) having sex with only one faithful, uninfected partner 167 (98.2) using condoms during sexual intercourse 167 (98.2) treating stds promptly 146 (85.9) screening donated blood before transfusion 169 (99.4) not sharing swimming pools or toilet with an infected person 157 (92.4) not sharing food with an infected person 158 (92.9) isolating people living with hiv/aids 164 (96.5) do not stay with infected person in the same house 164 (96.5) do not have casual contact with infected person 163 (95.9) avoiding mosquito bites 152 (89.4) pharmacists’ perceptions towards the decision to change treatment regimen for hiv/aids patients the median (iqr) score of respondents on decision to change regimens were 7.00(2.0) [maximum score = 8.00]. nearly all respondents agreed that treatment regimen should be changed when patient is unable to tolerate adverse reactions of the regimen (97.1%), and when hivviral load increases (95.3%). two-fifth of the respondents thought that the treatment regimen should not be switched based on the factor that the patient could not afford the treatment cost (40.6%) (table 4). pharmacists’ attitudes towards people living with hiv/aids two-thirds of the respondents had negative (61.8%) and very negative attitudes (5.3%) towards plwha (figure 1), with median (iqr) score = 8.00(3.0) [maximum score = 12.00]. half of the respondents revealed that they were not willing to live with hiv/aidspositive people in the same house (51.8%). almost twofifth of the respondents were not willing to take care of hiv/aids-positive patients (41.2%). about one-third of the respondents confessed that they did not feel comfortable working together with a colleague who is hiv-positive (31.2%) (table 5). table 4: pharmacists’ perceptions towards the decision to change treatment regimen for hiv/aids patients statements yes, n (%) patient's compliance is poor 154 (90.6) patient cannot tolerate adverse reactions of the regimen 165 (97.1) viral load increases 162 (95.3) cd4 t-cell count decreases 144 (84.7) patient experiences opportunistic infections 143 (84.1) drug interactions with other medicines 160 (94.1) patient cannot afford the treatment cost 101 (59.4) patient is found to be pregnant 146 (85.9) table 5: pharmacists’ attitudes towards people living with hiv/aids statements yes, n (%) do you feel comfortable about counselling hiv/aids patients? 135 (79.4) do you feel comfortable to work together with a colleague who is a hiv/aids patient? 117 (68.8) are you willing to live with people having hiv/aids in the same house? 82 (48.2) do you feel empathetic towards people living with hiv and aids? 161 (94.7) are you willing to take care of patients who have hiv/aids? 100 (58.8) do hiv/aids patients deserve free treatment? 133 (78.2) figure 1: pharmacists’ attitudes towards plwha sidi omar et al. (2021) journal of pharmacy, 1(1), 8-18. page 13 multivariate logistic regression was performed to identify the significant independent factors associated with respondents’ negative attitudes towards plwha (table 6). respondents worked at community pharmacies were 0.87 times less likely to have negative attitudes towards plwha than respondents who practiced in other workplaces (adj or=0.125; 95%ci=0.025-0.623; p=0.011). discussion in the general knowledge assessment section, the potential of hiv transmission through kissing is the most misunderstood. this trend of misconceptions were similar to the study by khan and baig (2013) where up to 89.3% of respondents believe that kissing hiv-infected person contributed to hiv transmission. who stated that “hiv can be transmitted through the exchange of body fluids from infected people, such as blood, breast milk, semen, and vaginal secretions. individuals cannot become infected through kissing, hugging, shaking hands, or sharing personal objects, food or water” (world health organisation, 2019). the hypotonicity of saliva is believed to be protective against hiv transmission by inactivating hiv-transmitting leucocytes, preventing attachment of the hiv-transmitting leucocytes to the mucosal epithelial cells, and lastly hiv production (baron, poast, & cloyd, 1999). another highly prevalent misconception on hiv prevention among recruited respondents is that hiv cannot be prevented by treating sexually transmitted diseases (stds) promptly. this is different from the findings of khan and baig (2013), where most pharmacists’ misconception was avoiding taking illicit drug/ use of intravenous drug to prevent hiv transmission. nevertheless, up to 29.3% of respondents in kedah also believed that treating stds promptly will not prevent hiv transmission (khan & baig, 2013). the risk of hiv transmission through sexual contact were reported to increase by five-to-ten folds in people with ulcerative stds (sahasrabuddhe & vermund, 2007). disruption of the integrity of epithelial mucosa by ulcerative stds, facilitates the contact of hiv with lymphatic and circulatory systems (cohen, 2004; wasserheit, 1992). for inflammatory and exudative stds, the risk of hiv transmission through sexual transmission is increased by two-to-five folds (sahasrabuddhe & vermund, 2007), as the infection and inflammation is associated with recruitment of urethral or cervical discharge filled with hiv-susceptible leucocytes in large volume (cohen, 2004; wasserheit, 1992). management of stds was reported to achieve reduction of 38% in incidence of hiv-infection in tanzania over two years period (grosskurth et al., 1995). another study in malawi had also shown that treatment of stds reduced the genital tract hiv-viral loads, thereby leading to infectiousness index reduction and lowering of transmission probability (cohen et al., 1997). most respondents agreed that the treatment regimen of hiv/aids patients should be modified only in the event of intolerable adverse reactions. again, our finding is consistent with the earlier study conducted by khan and baig (2013). in instances of moderate to severe adverse events, the malaysian consensus guidelines on antiretroviral therapy 2017 (malaysian society for hiv medicine, 2017) recommend substituting the causative drug with another of the same art class, but of different toxicity profile. in our study, we also found that the statement that most respondents disagreed on was changing of treatment regimen is necessary if the patients were unable to afford the treatment cost. in the study by (khan & baig, 2013), it was found that up to three-quarter of respondents did not agree to change treatment regimen on the grounds of treatment cost. however, cost of medication is a true obstacle in hiv treatment and medication adherence as reported in a qualitative study conducted in south africa (hardon et al., 2006). in a metaanalysis, ivers, kendrick, & doucette (2005) found cost of treatment to be an important determinant of adherence towards arts in resource-poor settings. these studies provide insights that cost of treatment should be considered when deciding on treatment regimen. in malaysia, hiv-positive patients who seek treatment in government healthcare facilities have free access to firstline arts. in the state of kelantan, the free-of-charge treatments are extended to alternative choices or secondline treatments. therefore, the affordable healthcare system in malaysia may have led to the local practicing pharmacists to have such perceptions on treatment costs. we found that two-thirds of the respondents in kelantan had negative attitudes towards plwha, similar to the findings in the study by khan and baig (2013), which reported that a majority of the hospital pharmacists in kedah also had negative attitudes towards plwha. discriminatory attitudes of pharmacist against plwha were also being reported in nigeria (ubaka, adibe, & ukwe, 2014). nevertheless, pharmacists in iraq showed positive attitude towards plwha in providing care and treatment (allela, shareef, & ismael, 2017). we observed that our respondents were reluctant to have social and casual contact with plwha, but generally had empathy towards them. despite having high scores of knowledge in the causes and preventive measures of hiv/aids, and awareness that casual and social contact with plwha will not cause hiv transmission, the respondents were unable to apply their theoretical knowledge in daily practices towards plwha with negative attitudes. in fact, we found that there was no significant association between knowledge scores and attitudes towards plwha in this study. this finding is in contrast to another study by balfour et al. (2010) which reported that pharmacists and health science students with better hiv knowledge were associated with lower hiv stigma in south america. sidi omar et al. (2021) journal of pharmacy, 1(1), 8-18. page 14 table 6: independent factors affecting pharmacists’ attitudes towards people living with hiv/aids variables positive (n=56) n (%) negative (n=114) n (%) univariate logistic regression multivariate logistic regression or (95%ci) p value adj or (95%ci) p value age (years) 21 30 31 40 41 50 50 60 28 (50.0) 21 (37.5) 6 (10.7) 1 (1.8) 69 (60.5) 39 (34.2) 5 (4.4) 1 (0.9) 1.533 (0.805-2.921) 0.936 (0.479-1.829) 0.495 (0.137-1.792) 0.515 (0.032-8.398) 0.194 0.847 0.284 0.641 gender male female 11 (19.7) 45 (80.3) 25 (21.9) 89 (78.1) 1.149 (0.519-2.544) 0.870 (0.393-1.926) 0.732 0.732 race malay chinese 50 (89.3) 4 (7.1) 107 (93.8) 7 (6.2) 1.834 (0.586-5.741) 0.545 (0.174-1.706) 0.297 0.297 academic qualification degree master 53 (94.6) 3 (5.4) 106 (92.9) 8 (7.1) 1.333 (0.340-5.233) 0.750 (0.191-2.943) 0.680 0.680 job experience (years) < 1 1 3 4 7 8 11 11 13 > 14 2 (3.6) 10 (17.8) 24 (42.9) 7 (12.5) 3 (5.4) 10 (17.8) 12 (10.5) 27 (23.7) 39 (34.2) 18 (15.8) 10 (8.8) 8 (7.0) 3.176 (0.686-14.71) 1.359 (0.604-3.061) 0.693 (0.360-1.336) 1.312 (0.514-3.355) 1.679 (0.448-6.425) 0.347 (0.129-0.936) 0.139 0.459 0.274 0.574 0.436 0.037 sidi omar et al. (2021) journal of pharmacy, 1(1), 8-18. page 15 variables positive (n=56) n (%) negative (n=114) n (%) univariate logistic regression multivariate logistic regression or (95%ci) p value adj or (95%ci) p value place of practice government hospital government health clinic pharm. service division community pharmacy academic institution private hospital/ clinic pharm. industrial 28 (50.0) 16 (28.6) 3 (5.4) 7 (12.5) 2 (3.5) 0 (0.0) 0 (0.0) 76 (66.7) 27 (23.7) 7 (6.1) 2 (1.7) 0 (0.0) 1 (0.9) 1 (0.9) 2.000 (1.041-3.841) 0.848 (0.408-1.764) 0.981 (0.236-4.078) 0.125 (0.025-0.623) 0.037 0.660 0.979 0.011 >0.999 >0.999 >0.999 0.125 (0.025-0.623) 0.011 no of hiv patients encountered past 1 year none 1 – 20 21 – 50 > 50 12 (21.4) 35 (62.5) 5 (9.0) 4 (7.1) 27 (23.7) 71 (62.3) 9 (7.9) 7 (6.1) 1.138 (0.527-2.459) 0.991 (0.512-1.918) 0.874 (0.279-2.743) 0.850 (0.238-3.036) 0.742 0.978 0.818 0.803 knowledge of hiv/aids [median (iqr)] 24.00 (2) 24.00 (2) 0.862 (0.703-1.056) 0.152 knowledge on causes of hiv/aids [median (iqr)] 12.50 (1) 13.00 (2) 0.861 (0.641-1.156) 0.319 knowledge on preventive measures of hiv/aids [median (iqr)] 12.00 (1) 12.00 (1) 0.788 (0.559-1.111) 0.788 sidi omar et al. (2021) journal of pharmacy, 1(1), 8-18. page 16 here, it seems that education on scientific matters may not be sufficient to achieve practices changes, and that attitudes and cultural beliefs should also be addressed in future educational programs (reis et al.,2005). hiv-related discriminations and its related problems is prevalent throughout the world, especially in developing countries with rich cultural, moral, and religious values such as malaysia. hiv/aids cases were often thought to be linked to moral improprieties, such as drug use, prostitution, heterosexual promiscuity, and homosexual behaviour. hiv is also believed to be highly contagious and may pose a threat to the community (wong & nur syuhada, 2011). the life-threatening nature of hiv/aids may also lead to the negative attitudes among pharmacists towards plwha (allela et al., 2017). in addition, as majority of our respondents were malays, and up to 68.2% (n=107) of the malays had negative attitude towards plwha, these negative attitudes may be associated with cultural-related conception. the burial rite of muslim is done with bleach for known hiv-infected people, which may reinforce the societal stigma and individual anxiety towards plwha (fadzil, othman, & mustafa, 2016). with these concerns, our respondents may refuse to take any unnecessary risks that may expose oneself in hiv transmission risk through accidental injuries or accidental transmission of infected body fluids through daily contact. in this study, respondents working in community pharmacies had significant positive attitudes towards plwha. in the study that only focused in hospital pharmacists, khan and baig (2013) reported that 86.6% of the hospital pharmacists in kedah had negative attitudes towards plwha, but the reasons for such findings were not further investigated. a study conducted among community pharmacists in india by gupta et al. (2010) found that majority of the pharmacists had senses of professional obligation towards plwha and were not worried about potential hiv exposure during medications dispensing to hiv-positive patients. however, the surveyed community pharmacists were not actively involved in hiv services as about two-thirds of pharmacists revealed that they did not frequently encountered hiv-positive patients (gupta et al., 2010). this similar situation may occur in our community pharmacy settings, where there is minimal involvement of the community pharmacists in providing care towards plwha, as most of the hiv-positive patients were managed in hospitals. as most of the hiv/aids patients with presence of opportunistic infections were treated in hospitals, this may have led to the fear of hospital pharmacists in contacting with hiv/aids patients compared to the community pharmacists, for example concerns for risk of exposure to contagious opportunistic infections such as occupationally-acquired tuberculosis (engelbrecht et al., 2019). there are some limitations and bias in this study. some degree of social desirability bias, which may affect participants’ response according to ethics of pharmacy practices, may be present in our study. the environment to complete the questionnaires were beyond our control. we were also unable to ensure that the respondents did not search for references to answer the questionnaires, or whether their responses were influenced by peers when answered in groups. a positive response bias is also likely to be present, as pharmacists with more knowledge on hiv/aids were more likely to fill in the questionnaire, which may yield non-representative high scores (domnich et al., 2015). as the number of respondents included in this study were unable to achieve the recommended sample size, our results may not statistically represent the pharmacist population in kelantan. lastly, this study was conducted in kelantan, which made up 10.3% of the national hiv population (ministry of health malaysia, 2018); free arts were provided to all hiv-positive patients; lesser distribution of community pharmacies compared to other states (pharmaceutical services division ministry of health malaysia, 2011); and due to inherent geographical distribution of the malay race, there were also more malay pharmacists in the state, as reflected in the high proportion in the study respondents. hence, the findings on this study may not necessarily be generalizable to other regions and settings in malaysia. further studies may also be expanded to the whole malaysia to identify the pharmacists’ knowledge, perception and attitudes towards plwha in malaysia. in the future, education and sharing sessions may be carried out to enhance the pharmacists’ knowledge on hiv/aids, focusing on the common misconceptions. as pharmacists’ high knowledge scores on causes and prevention of hiv/aids were not associated with the high rates of negative attitudes, it is important to investigate further the concerns of the pharmacists towards plwha. as stigma and discrimination reduces the quality of treatment (yang, zhang, chan, & reidpath, 2005) and affect the self-esteem of hiv-positive patients (surlis & hyde, 2001), efforts to assist pharmacists in overcoming their uneasiness when dealing with plwha will contribute positively towards future hiv disease control strategies. conclusion in conclusion, several key misconceptions towards the causes of hiv/aids, preventive measures of hiv/aids transmission, and the necessity in changing treatment regimen when needed were identified among pharmacists in kelantan, malaysia. despite having good disease related knowledge, most pharmacists in kelantan present negative attitudes towards plwha while working in a community setting was significantly associated with more positive attitudes. future studies should identify the causes of such sidi omar et al. (2021) journal of pharmacy, 1(1), 8-18. page 17 negative attitudes; and feed educational program strategies as part of the efforts to improve existing disease management measures attitudes. acknowledgements we thank the director of health malaysia for permission to publish this paper; ms lim xin yi, institute for medical research, for guiding and proof-reading the article; pharmaceutical service division of kelantan, ms azura binti musa, and ms noraniza binti mohamad zalik for delivering help in distribution online questionnaire; ms siti nurul zuliana binti abdullah for reviewing the online questionnaire. conflict of interest none. references allela, o. q. b., shareef, n. s., & ismael, b. s. 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(2005). institutional and structural forms of hivrelated discrimination in health care: a study set in beijing. aids care, 17(suppl 2), s129–140. doi:10.1080/09540120500119874 microsoft word 226-article text-original_galley proof.docx page 129 determination of zinc, copper, selenium, and manganese in human milk using acid digestion by icp-ms and its application in biological trace element monitoring nor hidayah mohd taufek1*, awis sukarni mohmad sabere2, ummi syahidah mohamad jamahari1, nur balkhis amran1, abdul rahman fata nahas1 and joseph bidai3 *corresponding author: email address: hidayahtaufek@iium.edu.my authors’ affiliation: 1 department of pharmacy practice, kulliyyah of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia. 2 department of pharmaceutical chemistry, kulliyyah of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia. 3 south china sea repository & reference center, institute of oceanography and environment (inos), university malaysia terengganu, 21030, kuala terengganu, terengganu, malaysia. abstract introduction: human milk contains essential trace elements which support healthy development of infants. previous studies have reported various analytical methods using different instruments to measure trace elements in human milk. this study aimed to determine trace element concentration in human milk using a validated acid digestion method and its application in biomonitoring among postpartum mothers. method: human milk samples were collected from three postpartum mothers and prepared using acid digestion method. all samples were analysed using inductively coupled plasma massspectrometry (icp-ms) and all validation parameters were measured. results: four trace elements which were zinc, copper, manganese and selenium were found to have good linearity (r² > 0.99), limit of detection in µg/l (0.06, 0.0001, 0.005, 0.00003, respectively) and limit of quantification in µg/l (0.18,0.0003, 0.02, 0.0001, respectively). good accuracy (83.4 – 112.7%), inter-day, and intra-day repeatability were obtained. method application on trace element monitoring over postpartum period of three participants showed the median concentration of zinc, copper and selenium in human milk gradually decreased with slight variation, whereas manganese remained stable. positive significant correlations were observed for most of the elements (r > 0.40, p < 0.001) except for copper-manganese. conclusion: acid digestion method is sensitive, accurate and precise to analyse and quantify zinc, copper, manganese and selenium concentrations in human milk simultaneously by icp-ms. it can be applied to monitor trace elements concentration in human milk in clinical and public health settings. article history: received: 9 may 2023 accepted: 13 july 2023 published: 31 july 2023 keywords: trace elements, human milk, icpms, acid digestion, validation how to cite this article: mohd-taufek, n. h., mohmad sabere, a. s., mohamad jamahari, u. s., amran, n. b., fata nahas, a. r., & bidai, j. (2023). determination of zinc, copper, selenium, and manganese in human milk using acid digestion by icp-ms and its application in biological trace element monitoring. journal of pharmacy, 3(2), 129-139 doi: 10.31436/jop.v3i2.226 page 130 introduction human milk has been reported to contain essential trace elements such as zinc, copper, iron, iodine, and selenium (mohd-taufek et al., 2016). the presence of these trace elements despite only in minute amount is very crucial to support infants’ development and prevent trace element deficiencies. determining the optimum trace elements concentration in human milk will ensure that infants get the most benefits and prevent deficiencies. trace elements play vital roles in growth and metabolisms, and the concentration have been reported to vary in human milk of different study populations and at different lactational stage according to infant’s requirement (hunt & nielsen, 2009). it is important to monitor trace elements concentration in human milk and identify reference range at different stages of lactation. previously, numerous analytical instruments have been reported to measure trace element analysis in human milk. some of the reported techniques were using inductively coupled plasma–optical emission spectrometry (icp-oes) (durović, et al., 2017), flame atomic absorption spectrometry (faas) (kim et al., 2012), inductively coupled plasma sector field mass spectrometry (icp-sfms) (ecsedi-angyal et al., 2019) and graphite furnace atomic absorption spectroscopy (ait lhaj et al., 2021). currently, the inductively coupled plasma massspectrometry (icp-ms) has been widely utilised to determine multi-element concentration in human milk due to its capability to detect many elements simultaneously at very low concentrations (mohd-taufek et al., 2016; rovira et al., 2022; tahboub et al., 2021). another advantage of icp-ms is that it involves simple preparation of samples. for example, acid digestion and alkaline dissolution methods have been used to prepare human milk samples prior to analysis (jagodic et al., 2020; levi et al., 2018; mohd-taufek et al., 2016). nitric acid and hydrochloric acid were the most often used acids in acid digestion method, whereas ammonium hydroxide and tetramethylammonium hydroxide (tmah) were the most frequently used alkali in alkaline dissolution method (wilschefski & baxter, 2019). nitric acid also has been reported to be the most optimum acid media for icp-ms analysis due to its strong oxidising ability that is able to extract the trace element from the sample by forming soluble nitrate salts (hu & qi, 2014). although there were studies which reported that the alkaline dissolution method was better than the acid digestion method, some elements were reported to be accurately analysed by the acid digestion method and there were also elements that produced the consistent results regardless of using alkaline dissolution or acid digestion method (levi et al., 2018; mohd-taufek et al., 2016). in human milk studies, there is no certified reference material for human milk available. therefore, various certified standard reference materials in the form of milk powder such as nist 1549, crm 8534, and nist 8435 and spiked samples have been used as the reference material to validate a developed analytical method (alves peixoto et al., 2019; baranowska-bosiacka et al., 2016; jagodic et al., 2020; mohd-taufek et al., 2016; pekou et al., 2022). currently to our knowledge, there is no study available that has reported about a validated method to measure trace elements concentration in human milk in malaysia probably due to limited access to analytical instruments and ethical issues about human milk samples. this study aimed to validate a simple acid digestion method to measure simultaneously the concentration of trace elements in human milk by icp-ms and determine its application in biological monitoring among postpartum mothers. methodology this study received international islamic university malaysia (iium) research ethics committee (irec) approval (id no.: irec 2021-053). study participants a total of three postpartum women were recruited to provide milk samples. participant information sheet and informed consent form were provided. the mothers were informed about the study and voluntarily provided human milk samples for analysis at their convenience. data collection form was filled in by the participants for demographic information, medical and medication history, and their lifestyles. the participants provided expressed breast milk (ebm), which were collected in 2 ml syringes or the ebm plastic container, labelled with date of milk collection. all samples were frozen at -21ºc prior to analysis. sample analyses were conducted at the institute of oceanography and environment (inos), university malaysia terengganu. sample preparation the frozen human milk samples were thawed at room temperature and manually shaken until the samples were completely homogenised. for the acid digestion method, 15.4 ml of 65% (v/v) nitric acid (merck suprapur) was diluted with deionised water up to 1 l to prepare 1% (v/v) nitric acid. then, 1 ml of samples was then added to 9 ml of 1% (v/v) nitric acid in a 15 ml polypropylene conical tube (corning falcon) and was shaken manually until a homogenous solution was formed. the prepared sample were then analysed using icp-ms. blanks preparation a total of 10 sets of blanks were prepared by adding 10 ml of 1% (v/v) nitric acid into a 15 ml polypropylene conical tube. the blanks were used to calculate the limit of detection (lod) and limit of quantification (loq) for page 131 method validation purpose. wash solutions the milli-q water was used for wash purposes between samples analysis. acid washout using 1% (v/v) nitric acid was done after analysing ten to twenty samples. standards preparation for the preparation of standards, a multi-element standard solution (perkinelmer pure viii) with 100 g/ml of al, b, ba, be, bi, ca, cd, co, cr, cu, fe, ga, k, li, mg, mn, na, ni, pb, se, sr, te, tl, and zn (merck certipur) were used. to make a standard stock solution, 1 ml of multi-element standard solution was added to a polypropylene conical tube that contained 49 ml of 1% (v/v) nitric acid. for calibration purposes, six calibration standard solutions were prepared by mixing 1 ml of milk sample with 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, and 0.6 ml of multielement stock solution. all the standard solutions were then further diluted up to 10 ml with 1% (v/v) nitric acid to produce the following final concentrations: 0.02 µg/ml, 0.04 µg/ml, 0.06 µg/ml, 0.08 µg/ml, 0.1 µg/ml and 0.12 µg/ml for all elements. quality control skimmed milk powder erm-bd150 was used as a certified reference material for quality control in this study. a total of 0.02 g of erm-bd150 was weighed and diluted up to 10 ml with 1% (v/v) nitric acid in a polypropylene conical tube. the solution was manually shaken until all milk powder was fully dissolved. three replicates of certified reference material were prepared daily for method validation purpose. the milk samples were analysed together with the blanks, reference materials, and spiked samples. samples analysis sample analysis was conducted using perkin-elmer sciex icp-ms model elan 9000 connected with dell pc equipped with elan instrument control session software (perkinelmer inc., massachusetts, usa). the working condition for this instrument is presented in table 1. during analysis, the samples were nebulised with a pneumatic nebuliser and then transported into the plasma for ion production using argon gas. ions were brought into a quadrupole, where they were separated according to their mass/charge ratio (m/z), after passing through several focusing lenses and a reaction cell. on an electron multiplier detector (simulscan™), specific isotopes of single-charged ions were identified, and the resulting electrical current was intensified to produce an intensity value in counts per second (cps). the system software was then used to translate the intensities for various isotopes in the tested samples into concentrations and compared them to those obtained from calibration standard solutions. each sample underwent analysis for a total of one minute and forty seconds. table 1: icp-ms working conditions (elan 9000, perkinelmer sciex) nebulizer gas flow (l min-1) 0.94 rf power (w) 1100 analog stage voltage (v) -1700 lens voltage (v) 6 pulse stage voltage (v) 900 ac rod offset (v) -6 discriminator threshold (v) 70 scan mode peak hopping speed of peristaltic pump (rpm) 26 detector pulse sweeps/reading 50 replicates 2 sampler/skimmer cones nickel dwell time (ms) 2.5 spray chamber ryton® double-pass scott-type spray chamber nebulizer gem-tip cross-flow pneumatic nebulizer linearity, lod, loq, accuracy and repeatability the validation parameters that were assessed and calculated included linearity, lod, loq, accuracy and repeatability. to assess the linearity of the method, calibration graphs for each element were generated by the system software at final concentrations of 0.02 g/ml, 0.04 g/ml, 0.06 g/ml, 0.08 g/ml, 0.1 g/ml, and 0.12 g/ml. the linearity was assessed using the correlation coefficient value (r2) derived from the calibration graph given by the system software. ten series of blank solutions were employed to calculate lod and loq. the ich harmonised tripartite guideline (2005) formula were used in this study to determine lod and loq based on the slope and the standard deviation of the blank: lod = 3.3 σblank b page 132 loq = 10 σblank b *σblank: standard deviation of 10 series of blanks *b: slope of calibration curve accuracy which was reported in the form of recovery percentage was calculated using microsoft excel 2019 by comparing the result obtained from the analysis of ermbd150 to the certified reference value provided by the manufacturer. repeatability was assessed on the same day (intra-day) and three different days (inter-day). inter-day repeatability was determined by analysing three erm-bd150 samples and five human milk samples on three different days, whereas intra-day repeatability was determined by analysing three erm-bd150 samples and five human milk samples on the same day. repeatability was reported as %rsd and calculated using the following formula: %rsd = standard deviation mean ×100% the inter-day repeatability was computed using the pooled relative standard deviation formula derived from the formula provided by mc naught & wilkinson (2012). the inter-day repeatability was calculated as follows: rsdpooled= (n1−1)rsd1 2+ (n2−1)rsd22+⋯+(nk−1)rsd!2 n1+ n2+⋯ +nk −! *k: different series of measurements *n: number of analysed samples the acceptance range for accuracy and repeatability are 70-120% for recoveries with an rsd% ≤20% based on the 2017 codex alimentarius commission. results calibration data obtained from this analysis were tabulated in table 2 that showed good linearity (r2 > 0.99) calibration curve for four trace elements which were zinc (zn), copper (cu), selenium (se) and manganese (mn). zinc had the highest lod value (0.06 µg/l), whereas mn had the lowest (0.00003 µg/l). the concentration of zn, cu, se and mn were also analysed in certified reference material erm-bd150 in µg/l. the concentrations in µg/l were multiplied by the dilution ratio for the erm-bd150 solution, then divided by 1000 to get milligrams per kilogram for the purpose of comparison with the manufacturer's certified value to measure the accuracy. the accuracy values for all four trace elements from the analysed erm-bd150 were between 83.4% and 112.7%. all trace elements achieved satisfactory percentage recoveries within the acceptable range (table 3). except for the inter-day repeatability value of se (18.37%), the intra-day and inter-day repeatability values for other elements in the erm-bd150 samples were less than 10%. table 4 showed the repeatability values for intra-day and inter-day measurements for human milk samples. it was notable that the very low concentration of mn and se found in human milk demonstrated significant variability that lowered the method precision. nevertheless, this method is considered precise as all repeatability values for all four trace elements were less than 20% which is considered acceptable by the guideline of 2017 joint fao/who codex alimentarius commissions. application in biological trace element monitoring: case studies the validated method was then applied to analyse a total of 105 milk samples donated by three postpartum mothers. the data were reported as case studies in this research and demographics of the mothers were presented in table 5. participant a was a 30-year old mother who delivered a male neonate at 41 weeks of gestation with a birth weight (bw) of 3.04 kilograms. she voluntarily donated a total of 64 breast milk samples, collected at different times of the day over six months, at her convenience. the infant was healthy and fully breastfed during the study period. participant b was a 27-year old mother who gave birth to a term male infant with a bw of 3.04 kilograms at 38 weeks of gestation. a total of 30 breast milk samples table 2: lod, loq, slope and correlation coefficient for four trace elements measured in acid digestion methods. trace elements lod (µg/l) loq (µg/l) slope correlation coefficient (r2) zn 0.06 0.18 y= 937x 5877 0.9976 cu 0.0001 0.0003 y= 3747x 21033 0.9992 se 0.005 0.02 y= 203x 967 0.9993 mn 0.00003 0.0001 y= 10617x 47720 0.9992 page 133 table 3: accuracy, inter-day (3 days), and intra-day repeatability of erm-bd150. erm-bd150 (n=3) trace elements certified value [mg/kg] observed value [mg/kg] accuracy [%] inter-day [%rsd] intra-day [%rsd] zn 44.8 ± 2.0 50.5 ± 3.7 112.7 4.79 4.39 cu 1.08 ± 0.06 0.92 ± 0.04 85.2 3.52 2.56 se 0.188 ± 0.014 0.188 ± 0.086 100 18.37 3.71 mn 0.289 ± 0.018 0.241 ± 0.042 83.4 9.94 9.48 table 4: inter-day (3 days) and intra-day repeatability (n = 5) of human milk samples.. trace elements inter-day [%rsd] intra-day [%rsd] zn 5.66 5.03 cu 6.74 3.78 se 14.10 16.24 mn 18.10 12.09 table 5: demographics of participants (n = 3) demographic a b c race malay malay malay body mass index 23.19 24.0 21.83 bw pre-pregnancy (kg) 55 51.5 56 bw pre-delivery (kg) 64 62 62 bw post-delivery (kg) 55 54 58 no. of pregnancies 4 1 5 no. of deliveries 2 1 5 no. of miscarriages 2 0 0 birth method caesarean normal normal dietary intake rcbv rcbv rfcvfr medical history no no gestational diabetes, asthma supplement no yes (se, mn, zn, cu) yes (zn, fe) bw: body weight; r: rice; c: chicken; b: beef; f: fish; v: vegetable; fr: fruit were collected from 12 months until 15 months postpartum at her convenience. at birth, the infant presented with high fever and jaundice 10 days after birth. he was fully breastfed only until the first six months and then continued with partial breastfeeding and solid food. there were no abnormalities in the growth and development of the infant except he was underweight and had mild eczema. participant c was a 27-year old mother who delivered a male infant with a bw of 3.08 kilograms at 37 weeks of gestation. she was able to donate a total of 11 human milk page 134 samples that were expressed at different times of the day in the 21st month postpartum only, at her convenience. the infant showed normal growth and development with no other health problems except for jaundice after birth. he was fully breastfed only until the first six months and then continued with partial breastfeeding and solid food. table 6 showed the concentrations of zn, cu and se were found to be the highest at one month postpartum compared to the later months in participant a. however, only se and cu gradually decreased wheras zn showed some fluctuations. on the other hand, mn exhibited no significant changes in concentration over the time period. the concentration of zn and cu also showed relatively higher concentration than mn and se. the correlations between trace elements in human milk also were examined with p < 0.001 was considered as significant. the spearman’s correlation coefficients and p-value between essential trace elements in human milk of participant a were: zn-se (r =0.607, p = <0.001), zn-cu (r = 0.615, p = <0.001), zn-mn (0.471, p = <0.001), cu-se (r=0.735, p = <0.001), mn-se (r=0.467, p = <0.001). all trace elements showed significant positive correlations (r > 0.40, p < 0.001) except for cu-mn. table 7 showed the concentrations of zn, cu, se and mn in participant b at 12 to 15 months postpartum and participant c at 21 month postpartum. for participant b, the median zn concentrations were slightly decreased from 430.5 µg/l at 12 months to 353 µg/l at 15 months postpartum. for se, the median concentration was increased from 7.6 µg/l at 12 months to 10.9 µg/l at 13 months and remained around 10 µg/l at 14 and 15 months postpartum. there was a fluctuation in the levels of cu during 12 to 15 months postpartum. the median concentration of mn increased from 5.8 µg/l to 7.1 µg/l at 12-13 months and later declined to 4.5 µg/l and 4.7 µg/l at 14-15 months. for participant c, the median values of zn and mn were similar but se and cu seemed to be higher than participant b despite later lactation period. table 6: the concentrations of trace elements in the human milk of participant a over a period of 6 months postpartum (µg/l). trace elements mean/ median month postpartum 1 2 3 4 5 6 n = 15 n = 8 n = 10 n = 15 n = 6 n = 10 zn (µg/l) mean±sd 2698±1010 1211±304 1480±355 1296±773 967±221 1254±541 median (range) 2640 (1550-5870) 1110 (907-1810) 1495 (841-1960) 1190 (352-3780) 886 (716-1250) 978 (713-2350) cu (µg/l) mean±sd 403 ± 84 180 ± 20 164 ± 26 116 ± 46 93 ± 29 77 ± 18 median (range) 406 (265-621) 188 (147-200) 166 (114-215) 104 (61-223) 89 (61-143) 75 (50-111) se (µg/l) mean±sd 24.8 ± 7.2 11.9 ± 4.7 14.8 ± 4.5 12.3 ± 6.8 9.8 ± 4.0 11.3 ± 4.1 median (range) 23.6 (14.6-43.1) 11.9 (2.7-18.3) 14.2 (9.4-22.9) 12.2 (4.4-32.0) 10.7 (4.4-15.0) 10.4 (5.9-19.7) mn (µg/l) mean±sd 4.7 ± 1.2 3.6 ± 1.1 4.7 ± 1.8 4.7 ± 3.1 3.3 ± 0.5 5.1 ± 3.0 median (range) 4.4 (3.5-7.6) 3.6 (2.2-5.9) 4.3 (1.8-7.7) 3.8 (2.6-14.8) 3.2 (2.9-4.0) 3.5 (2.7-10.5) page 135 table 7: the concentration of trace elements in human milk of participants b and c at 12 months postpartum and above (µg/l). trace elements mean/ median b c 12 13 14 15 21 n = 16 n = 4 n = 6 n = 4 n = 11 zn (µg/l) mean±sd 450±117 1178±1557 373±126 390±86 606±532 median (range) 431 (243-642) 454 (292-3510) 354 (203-585) 353 (338-518) 450 (179-2150) cu (µg/l) mean±sd 8.4±2.3 12.8±5.4 10.1±1.7 9.8±2.0 15.3±4.3 median (range) 7.6 (5.1-14.0) 10.9 (8.9-20.7) 10.3 (7.9-12.6) 10.2 (7.4-11.5) 16.4 (7.2-21.7) se (µg/l) mean±sd 76.7±23.9 84.2±22.2 82.4±31.2 105.3±40.3 155±33 median (range) 72 (47-136) 84 (57-111) 72 (62-144) 106 (56-154) 166 (83-200) mn (µg/l) mean±sd 5.7±2.3 7.1±1.7 20.8±34.7 4.6±0.4 3.5±1.3 median (range) 5.8 (3.2-11.2) 7.1 (5.0-9.1) 4.5 (3.8-90.8) 4.7 (4.2-5.0) 3.5 (1.1-6.3) discussion we report a method using acid digestion to measure total elements of zn, cu, se and mn concentration in human milk. previous studies that reported acid digestion method have used heat application during sample preparation (alves peixoto et al., 2019; jagodic et al., 2020; tahboub et al., 2021). theoretically, by raising the temperature of the samples, the average kinetic energy will rise, thus increase the chances of acid and biological sample collisions and enhance trace element dissolution (mohammed et al., 2017). our method did not require heat application since human milk is a biological matrix that contains proteins such as mucins, caseins and whey proteins that can be affected by high temperature. an increase in temperature has been reported to increase the amount of protein adsorbed onto solid surfaces resulting in a lower trace element content in the pasteurized milk solution possibly due to protein precipitation (da costa et al., 2003; rabe et al., 2011). our method was simple and produced good results for the required validation parameters to determine four trace elements simultaneously in 1 ml of human milk for biological monitoring purpose. homogenisation of milk samples before the digestion process is also a crucial step during sample preparation prior to the analysis. after being thawed, human milk is known to form two different layers, the fatty and aqueous layers. a study has reported that the concentration of iodine in the fatty layers was significantly different than those in the aqueous layers (huynh et al., 2015). therefore, sample homogenisation prior to mixing with nitric acid would ensure that all trace elements in the samples uniformly dispersed. information about amounts of trace elements in two different layers of human milk have not been available to date. further studies are needed to investigate the differences in the amount of trace elements in two distinct layers of human milk after thawing. all validation parameters measured in this study produced good measurements which were within the acceptable range specified by the guideline, indicating that this method performed satisfactorily. the employed method in this study had a lower loq value for all four trace elements compared to other studies (alves peixoto et al., 2019; mohd taufek et al., 2016). the fact that the loq values of zn, cu, se and mn obtained using this method were also significantly lower than the anticipated range concentrations in human milk reported in other countries showed that this method may be used to determine the levels of trace elements in human milk over a wide variety of populations. this method did not utilise any internal standard. it has been demonstrated that the implementation of internal standards that had the mass numbers almost the same as tested trace elements could enhance the precision of the methods used (vanhaecke et al., 1992). although still within the acceptable range, mn and se in human milk samples had slightly lower precision in the present study. therefore, the precision of this page 136 method may be further optimised by utilising internal standards in the future. for the purpose of method application in biological monitoring, we measured the concentration of zn, cu, se and mn in human milk collected at different postpartum period from three mothers as case studies. the median zn concentration in participant a was high (2640 µg/l) at the first month postpartum and rapidly decreased (978 µg/l) at 6 months postpartum. this pattern was found to be similar to a previous report that highlighted a rapid decline of zinc concentration in term breast milk from 3000 µg/l at one month to 1200 µg/l at six months postpartum (hunt & nielsen, 2009). we found the mean±sd of zn concentration at 1 month was 2698 ± 1010 µg/l and 1480 ± 355 µg/l at 3 months postpartum. a study by motoyama et al. (2021) involving 78 japanese mothers reported higher mean zn concentration at 1-month and 3-months postpartum with levels of 3000 ± 1300 µg/l and 1680 ± 950 µg/l respectively. similarly, another study by dumrongwongsiri et al. (2015) also reported higher zn levels with a range of 500-3200 µg/l at 4-6 months postpartum than the present case study (352-2350 µg/l). however, lower mean zn values were found in term milk of 70 mothers in spain of 1237.76 ± 949 µg/l at 1 month postpartum (mandiá et al., 2021). in table 7, the median levels of zn for both participant b at month 12-15 and participant c at month 21 appeared to be relatively similar. further monitoring on zinc concentrations in human milk over postpartum period may be needed to understand the relevance and factor behind its variation relative to the needs of infant’s growth and development. in our study, the median cu values were 406 µg/l in the first month postpartum and 166 µg/l at 3 months, which were lower compared to the values reported by motoyama et al. (2021) with values of 500 µg/l and 330 µg/l respectively. however, the mean cu concentration in the first month postpartum in our case study was 403 ± 84 µg/l, which was higher than the reported value by mandia et al. (2021) of 250.11 ± 163 µg/l. in iran, a study also reported a higher mean cu concentration of 1070 ± 1140 µg/l in the breast milk of 160 lactating mothers at 6 months postpartum (sadeghi et al., 2020). a previous report also highlighted the decline of mean cu levels from 250 µg/l in the first six months to 100-200 µg/l in seven to 12 months postpartum (hunt & nielsen, 2009). currently, optimal levels of cu in human milk have not yet been determined at different lactation stages. the median se concentration found in the current study was 23.6 µg/l in the first month then decreased to 14.15 µg/l at 3 months and 10.41 µg/l at 6 months postpartum. another study also found similar level of se at 1-month which was 22 (17-29) µg/l but higher level at 3 months postpartum, of 21 (16-25) µg/l (motoyama et al., 2021). however, a previous study by mandiá et al. (2021) reported lower mean se values of 8.87 µg/l in the mature milk compared to the current study which was 24.82 µg/l. in a previous study involving 470 lactating women from slovenia, the mean se levels found in the human milk at 6 to 8 weeks after delivery was 12.6 µg/l (snoj tratnik et al., 2019). based on the findings by han et al. (2019), the adequate intake of se for 0-3 months chinese infants was 15.29 µg/day, which is almost the same with the levels reported in the current study (14.15 µg/l at 3 month). it is important to monitor selenium status in infants receiving different amounts of selenium from human milk to prevent deficiency or toxicity. we also observed median mn concentration that were relatively stable with 4.44 µg/l in the first month postpartum, 4.29 µg/l at three months and 3.54 µg/l at 6 months. these results were lower than those reported by a previous study by motoyama et al. (2021) which found median mn levels of 8 µg/l in the first month and 7 µg/l at 3 months postpartum. similarly, li et al. (2016) also reported higher mn levels of 9.33 to 11.53 µg/l in the first 2 months and then declined at 4 to 6 months to a mean value of 7.69 µg/l. our study suggests that mn concentrations in human milk are relatively stable over the postpartum period. human milk was reported to provide an adequate amount of mn to prevent deficiency at about 3– 10 µg/l (horning et al., 2015). this study observed that trace elements concentration in human milk varied over postpartum period. the variations in the trace elements levels might be due to several factors such as lactation stages, geographical regions, dietary intakes or trace element supplements that were taken by some of the participants. consistent with the previous reports (li et al., 2016; motoyama et al., 2021), we found positive significant correlations for most of the elements (r > 0.40, p < 0.001) except for cu-mn. the limitation of our study includes the small population size due to challenges in recruiting participants. however, the milk samples provided by these participants were sufficient to validate the method and its application in biomonitoring of trace elements in human milk. future studies with large sample size would be able to produce a thorough investigation on the factors that may influence the trace element concentrations in human milk. this study did not seek to evaluate the association of factors affecting trace elements in human milk. however, the findings from this study are important since this is the first study reported in malaysia using a validated acid digestion method that is simple, accurate and sensitive to measure zn, cu, se, and mn. it is applicable and relevant for clinical and public health settings by using minimal amount of sample for biomonitoring to detect potential deficiency or toxicity relative to the needs of infants. previous studies reporting data of malaysian population have examined the concentration of a single trace element (pb and fe) in human milk in the past few decades (huat et al., 1983; loh & sinnathuray, 1971). new data can be produced for malaysian population using a simple and robust method reported in the present study. page 137 conclusion acid digestion method was simple, sensitive, accurate, precise, and robust to quantify zinc, copper, manganese and selenium simultaneously in human milk by icp-ms. human milk analysis is challenging due to high variability of trace elements contents between individuals and across postpartum period. there were significant correlations between concentration of zn-se, zn-cu, zn-mn, cu-se and mn-se. future studies may utilise this method to determine reference range of trace elements concentration in human milk in larger study population. author contribution n.h. mohd-taufek, a.s. mohmad sabere & j. bidai conceived and designed the experiment, conducted sample and data collection, data analysis, n.b. amran, u.s. mohamad jamahari and a.r. fata nahas analysed the sample and data, all authors wrote the manuscript. acknowledgements the authors would like to thank institute of oceanography and environment (inos), university malaysia terengganu for their cooperation and assistance with method development and validation. data availability the datasets analysed in the current study are available from the corresponding author upon reasonable request. competing interest n.h. mohd-taufek, a.s. mohmad sabere, j.a. bidai, n.b. amran, u.s. mohamad jamahari and a.r. fata nahas declare that there is no competing interest in this research. funding this work was supported by research management centre grant 2020 (rmcg20-046-0046) of international islamic university malaysia. references ait lhaj, f., elhamri, h., ait lhaj, z., zarrouk, a., el abidi, a., el hajjaji, s., & bellaouchou, a. 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(2021) journal of pharmacy, 1(2), 87-96 page 87 a review of black seed extract as an agent in the wound healing process muhammad hakeem mohd zaid1, muhammad faezuddin hanafi1 and muhammad salahuddin haris1* abstract introduction: nigella sativa or black cumin is an annual flowering plant that can be used for wound treatment. in the treatment for wound healing, this plant contains many beneficial components that can help with the treatment but this plant utilisation in the medical field is still limited. this review will provide an overview of the advantages when this plant is used in the wound healing treatment. method: the review was based on the roses protocol, and the databases used were sciencedirect, pubmed, and google scholar. after the search, only 11 papers had been chosen to be included in the results and they were divided into four main themes that were used for the analysis of the results. results: the themes were gross analysis, microscopic analysis, biochemical analysis and antimicrobial analysis. conclusion: in conclusion, the black seed extract contains several valuable properties, such as antimicrobial and antioxidant properties, which help improve the wound healing process. further study needs to be done to discover more potential of nigella sativa in treating the wound. article history: received: 29 january 2021 accepted: 14 june 2021 published: 31 july 2021 keywords: nigella sativa, black seed, wound healing. how to cite this article: mohd zaid, m. h., hanafi, m. f. & haris, m. s. (2021). a review of black seed extract as an agent in the wound healing process. journal of pharmacy, 1(2), 87-96. doi: 10.31436/jop.v1i2.67 authors’ affiliation: 1 department of pharmaceutical technology, kulliyyah of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia. *corresponding author: email address: solah@iium.edu.my tel:+6014 3373960 mohd zaid et al. (2021) journal of pharmacy, 1(2), 87-96 page 88 introduction nigella sativa and its bioactive constituents new plant-based medicine has emerged and been investigated extensively by lots of pharma companies and researchers. nigella sativa or black cumin is an annual flowering plant that located in the eastern europe (bulgaria, cyprus, and romania) and western asia (turkey, iran, and iraq). it has been spread into more locations like northern africa and east to myanmar (yimer, 2019). this plant in the family of ranunculaceae can grow to 20-30 cm tall and has large, inflated fruits containing lots of seeds. these seeds are usually used as a spice in the food, then gained world recognition as new herbal medicines. even though the recognition is new, the evidence regarding the usage of nigella sativa seeds as traditional medicines can be tracked from the ancient egypt era (tutankhamun's era). another evidence showed that the great islamic scholar, avicenna, the persian physician described nigella sativa as a treatment for dyspnoea in his book, canon of medicine (razavi, 2014). nigella sativa seeds have a lot of benefits due to their bioactive constituents. the most significant bioactive component in nigella sativa is thymoquinone (tq) which consist of 30-48% of the total compounds followed by pcymene, carvacrol, thymohydroquinone (thq), dihydrothymoquinone (dhtq), α-thujene, thymol, tanethole, β-pinene, α-pinene, and γ-terpinene (sahak et al., 2016). these bioactive compounds can be used as a diuretic, antidiabetic, analgesic, antihypertensive, anticancer and immunomodulatory, anthelmintic, antimicrobial, antiinflammatory, spasmolytic, gastroprotective, hepatoprotective, renal protective, bronchodilator, and antioxidant properties (ahmad et al., 2013). however, this review paper will only focus on the wound-healing effects that have been produced by the usage of nigella sativa and its bioactive constituents. wound healing cascade part of the human body can self-regenerated when that area damaged due to internal or external causes and this process is called wound healing cascade. the cascade consists of four stages of wound healing which are the haemostasis phase (blood clotting), inflammatory phase, proliferative phase (tissue growth), and maturation phase (tissue remodelling) (singh et al., 2017). each of the stages will differ in terms of the time taken to complete the phase according to the condition of the body, nutrients available, and the surroundings. the first stage is the haemostasis phase. it begins right after our body is exposed to the injury. the haemostasis from the words “'heme' and 'static' means that the blood and stay, respectively. it means that the haemostasis phase will keep the blood within the blood vessel. this phase will involve the blood coagulation process by recruiting and initiating the platelets to form the blood clot and stop the bleeding (davies, 2000). after that, the second stage, which is the inflammatory phase, will start. the purpose is to destroy the bacteria involved and remove the debris from the surroundings (sinno, 2013). to complete this stage, lots of white blood cells (wbc), mostly neutrophils, will be recruited into the wound area. after about three days after injury, another specialised cell called macrophages will continue the work to remove more debris, preparing the wound bed for new tissue growth. macrophages also will secrete growth factors to facilitate wound healing. after all pathogens and debris are removed, the proliferative phase will begin. this 'stage's objective is to fill, contract, and cover the wound with new tissues. this stage will undergo from day four until day 24. lastly, after the wound has been closed, the maturation phase will start. during this stage, the wound will be susceptible to interruption caused by both internal and external factors. therefore, the right healing environment is needed to speed up and optimise the healing process (mayet, 2014). wound healing parameters the wound healing process is dynamic. therefore, to assess wound healing, it needs to be evaluated consistently. there are multiple ways to evaluate the wound healing process: gross observation and monitoring, microscopic evaluation, and biochemical or biomechanical analysis. the gross observation and monitoring are done by comparing gross observation, geometrical assessments of percentage wound contraction, percentage wound closure, and percentage wound re-epithelialisation, recorded at a specific time interval, between the samples and the control (singh et al., 2017). however, the gross observation itself does not prove the wound healing as the evaluation can only be done by nothing more than things that the naked eye can see. on the other hand, the microscopic evaluation can assess the wound in a more precise manner. for example, evaluation in histological perspective can be made on the wound healing cascade based on the scab formation from the four stages of the wound healing process (etulain, 2018, p. 561). finally, analysis on the biochemical and biomechanical can be used as an assessment for the wound healing process. recent findings suggested the wound healing impairment is associated with oxidative stress (dunnill, 2017). oxidative stress occurs when there are excess free radicals compared to antioxidant enzymes (e.g., superoxide dismutase, glutathione peroxidase, and reductase, catalase.) produced by our body (rowiński, 2013). as a result, these free radicals will interrupt the wound healing process. research gap most of the reviewed paper only focused on the overall ability and advantage of taking the black seeds only. not much review had been done for the black seed's properties mohd zaid et al. (2021) journal of pharmacy, 1(2), 87-96 page 89 in the human model and the animal model. therefore, this review was done to combine the most recent experiments and research on the black seeds to prove their suitability for wound healing usage. based on figure 1, it is depicted the targeted objectives that the authors wanted to emphasise. in the figure, it shows that the black seeds which had many active chemical components can be utilised in the wound healing treatment development due to the properties of the black seeds which can assist and bring many positive effects for the healing process of the wounded area. this review will help to provide some overview of the black seeds’ effects on the wound healing process and also provide some evidences for the black seeds to be used in the wound healing treatment. the utilisation of this plant may provide a new solution for the current treatment and improve the outcome of the treatment especially for the special groups that need extra precaution when using certain chemical such as diabetic patients. figure 1: the authors’ insight regarding the black seeds’ advantages in the wound healing treatment methodology the study was based on reporting standard for systematic evidence syntheses (roses) review protocol. the authors start their systematic literature review (slr) by constructing appropriate research questions for the review. the authors then used the systematic searching strategy, which consists of three-step continuous processes: identification process that used search strings in the selected data bases (table 1), screening process that used the inclusion and exclusion criteria as mentioned in table 2 and eligibility process. then, the authors proceed to the quality appraisal of the selected articles using a specific strategy to ensure the quality of the reviewed articles and the summary of this filtering process is depicted in the figure 2. then, the reviewed papers will undergo a data abstraction process that divided the data into four main themes as depicted in the table 3, and those data will be analysed and validated by both authors to improve the quality of the slr. table 1: databases and search strings database search strings google scholar (black seed extract or black seed cumin extract or nigella sativa extract) and (antioxidant or oxidative antagonist or oxidative competitor) and (burns wound or blaze wound) and (healing process or curing process or repair process or regenerating process or rebuild process) pubmed ("black seed" or "black caraway" or "nutmeg flower" or "roman coriander" or "nigella sativa" or "fennel flower" or "black cumin") and ("wound healing" or "wound regeneration" or "wound repair" or "wound remedy" or "cut healing" or "cut regeneration" or "cut remedy" or "laceration healing" or " laceration regeneration" or "laceration repair" or "laceration remedy" or "trauma healing" or "trauma remedy" or "lesion healing" or "lesion remedy" or "angiogenesis") sciencedirect table 2: criteria for the papers to be included and excluded from this review before the eligibility process criterion inclusion exclusion literature type research paper, book, journal literature review, encyclopedia, magazines language english languages other than english timeline 2016-2020 2015 and before focus of review agent of wound healing other application other than wound healing mohd zaid et al. (2021) journal of pharmacy, 1(2), 87-96 page 90 figure 2: flow diagram depicted the process for the filtering the papers that will be used for the review table 3: data abstraction of the 11 papers chosen for the review divided into four categories related to wound healing (g: gross, m: microscopic, b: biochemical, a: antimicrobial) authors g m b a (kadhim, 2017) / / (ali et al., 2020) / / / (mastuti et al., 2017) / (yalçın turhan, mehmet arıcan, zekeriya okan karaduman, ozan turhal & ozkan, 2019) / (abd elrahman et al., 2019) / / / (rohmah & suprayitno, 2020) / (trisnawati, 2018) / (apaydin & gedikli, 2019) / (nourbar et al., 2019) / (sallehuddin et al., 2020) / / (rajabian & hosseinzadeh, 2020) / / results among the 11 selected papers, the results were extracted and divided accordingly to the respective themes. in the gross and microscopic analysis of wound healing, there are four papers used in each theme, while in the biochemical analysis of wound healing, only five selected papers that the results can be put under this theme. only one paper had results on the antimicrobial analysis of the black seeds. information extracted from sallehuddin et al. (2020) and rajabian & hosseinzadeh (2020) will be used in the discussion because these two papers only described the research that other researches had done. gross analysis based on ali et al. (2020), it had stated about the black seeds extract that had been combined with pva to produce a nanofibrous mat for wound healing purposes. they had observed that the treatment using the nanofibrous mat indeed showed the wound healed without any infections detected on the wound during the execution of the experiment for seven days. apart from that, abd. elrahman et al. (2019) had conducted a study on the healing of teeth sockets in rabbits and had portrayed that the newly formed bone surface area percentage was higher in the rabbits' group which used black seeds oil than the control group. these results showed that the black seeds had better healing properties than the other group, which did not use the oil of black seeds. another paper that had studied the effect of the black seeds oil with albumin depicted the fastest wound closure for the first three days was observed when 12% of the black seeds oil was with the albumin while the longest was observed in the negative control group. these results remained the same on the 5th and the 7th day of the study. the mixture also showed the shortest average wound length during the study. the study showed that the black seeds oil did enhance the wound healing process, and it also assisted in the closure of the wound (rohmah & suprayitno, 2020). apart from that, an experiment done on diabetic male rats, it was observed that black seeds extract with a concentration of 20% had an increase in the wound area for the first day of the experiment but kept reducing later in the next few days. the wound was healed completely on the 18th day. as for the extract with 40% concentration, the observations were the same, but the wound healed on the 15th day which was much quicker than the previous concentration. during the experiment, they found that the rats treated with the black seeds extract had a thicker epidermal layer than the other two groups: the untreated group and phenytoin-treated group. besides that, they had also found that the wound treated with the extract had much more regularly arranged collagen fibres and fibroblasts than the other groups (nourbar et al., 2019). mohd zaid et al. (2021) journal of pharmacy, 1(2), 87-96 page 91 microscopic analysis in terms of observation under the microscope, khadim et al. (2017) found that the application of black seeds in combination with propolis and honey showed healing properties when tested using the rabbits' oral mucosa. during the experiment, they found that signs showed the wound started to close with new epithelium on the third day of the investigation. in the dermis layer, they had also observed that the collagen fibres and the fibroblasts had begun to organise within that layer of skin. on the 7th day of the experiment, they managed to capture images of the wound under the microscope, and they found that the epithelium had sealed the wound site. moreover, the fibroblasts and the connective tissue were already organised in the dermis layer. another observation had been done on the 10th day of the experiment and they had discovered that the wound site had a thick epithelium layer. the connective tissue was observed to be organised in a good manner. apart from that, an experiment that used black seeds extracts in a nanofibrous mat depicted that the proliferation phase had been started on the second day of the experiment. this can be observed by the formation of granulation tissue on the wound site that helps increase the production of interleukins. moreover, the researchers stated that fatty acids from the black seeds in the nano-fibrous mat helped make collagen that can promote the wound healing process (ali et al., 2020). besides that, a study used black seeds oil to treat the socket after removing the teeth in rabbits. it was portrayed that the socket applied with the oil had dilated blood vessels while the control group had limited blood supply in the bone marrow. the increase in blood was the indication that active bone formation was occuring when the black seeds oil was used in the treatment. moreover, the researchers also observed thicker trabecular bone and higher vascular formation in the socket area that had been applied with the black seeds oil. (abd elrahman et al., 2019). other than that, apaydin & gedikli (2019) had done a study on diabetic rats where the rats were infected with streptozocin and tested with the black seeds essential oil for its wound healing properties. in this study, they emphasised the parts related to the inflammation development, deposition of collagen regeneration of the epithelium, and the formation of new blood vessels. during the 3rd day of the experiment, the researchers had not seen any epithelial formed yet in the three groups of this experiment which were normal rats for the control group, the stz-dm group (sd) that contained diabetic rats with streptozocin infection, and the stz-dm+nes group (sdn) that used essential oil on the wound of diabetic rats infected with streptozocin bacteria. they also observed more inflammation, haemorrhagic sites, granulation tissue in the sd group than any other group. except for the sd group, other groups portrayed a good level of improvement in the wounded areas. besides that, the sdn group had been observed to have the greatest level of regeneration for the dermal and epidermal layer on the 14th day of the experiment. the wound of the rats was closed completely and there was a formation of sebaceous garland together with the hair follicle at the site of the wound. there was also epithelial, and keratin formed in the sd group, but the level of healing improvement in the group was poorer than the control group. on the other hand, a study that used the black seeds extract in an ointment revealed that the fibroblasts were at the highest quantity when 40% of the extract (ns-40) had been included in the ointment followed by the 60% of the extract (ns-60) contained in the ointment. from the analysis using the mann whitney statistical, they did confirm that the ns-40 had a significantly higher number of fibroblast than the negative controlled group (cn) together with the ns-60. as for collagen present at the wound site, it was found that ns-60 had the highest amount while the lowest was found in the cn (mastuti et al., 2017). biochemical analysis in one of the papers reviewed, the researchers had seen tgf-beta 3 localised and produced by the progenitor cells at the wound site on the third day of the experiment that had used rabbits' mucosa at the oral area. the experiment used black seeds mixed with honey and propolis to be applied at the wound site. instead of that, other components such as fibrous connective tissue, blood vessels endothelial lining, and fibroblasts had also been seen localised at the wound site. after seven days had passed, they had found that tgfbeta 3 was still detected in the epithelium. besides that, there were still endothelial cell lining of the blood vessels and fibroblasts observed at the wound site. on the 10th day of the experiment, they detected the tgf-beta 3 localised in the epithelial cells and collagen fibres in the dermis layer (kadhim, 2017). in another study, the researchers had divided the number of rat specimens into four groups. the 1st group, which was the control group, had used a dressing that had been wet with a saline solution, and the 2nd group was using black seed oil to wet the dressing. the 3rd and 4th group had used a nano-silver solution as the wetting solution with 50 % of the black seeds oil was mixed with the solution in the 3rd group and 50 % concentration of the silver solution was used the 4th group. the study results showed that the highest mean density of collagen was found in the 4th group while the lowest mean vertical scar thickness was observed in the 2nd group. the had deduced this might happen in the 2nd group results due to the black seeds oil had the effect of reducing the free radicals when the wound was inflicted on the specimens (yalçın turhan, mehmet arıcan, zekeriya okan karaduman, ozan turhal & ozkan, 2019). apart from that, the usage of black seeds oil in the treatment of sockets after tooth removal in rabbits showed that vascular endothelial growth factor (vegf) significantly increased the experiment and the von willebrand factor in the experiment was lower than the controlled experiment. these two factors present at the mohd zaid et al. (2021) journal of pharmacy, 1(2), 87-96 page 92 wound site can indicate the black seeds oil to be a potential inducer for the formation of new vascular in the wounded area that can help with the wound healing process (abd elrahman et al., 2019). the increase in the vegf had also been seen in an experiment that used the tq extract from black seeds in treating teeth sockets for diabetic-induced rat specimens (trisnawati, 2018). in the experiment, group k was the control group, while the p1 group used the extract mixed with olive oil. another group which was called the p2 group used 100 mg/kg of metformin that had been dissolved in distilled water. the early stage of the experiment showed that the p2 group had shown higher vegf levels than other groups but, on the 7th, and 10th day of the experiment, the p1 group was the one with the highest vegf level (trisnawati, 2018). antimicrobial analysis one of the findings in a paper mentioned that the antibacterial resistance was rising as the percentage of black seed extract increased in the nano-fibrous mat made through the electrospinning technique. the researchers stated that this could happen due to thymoquinone (tq) and thymohydroquinone (thq) in the extracted samples from the black seeds. they had also mentioned several chemicals in the black seeds, such as carvacrol, thymol, and terpenoids, that can possess potent antimicrobial properties. these chemicals can contribute to the destruction of bacterial cell walls and cause the inside of the bacterial cells to spill out, leading to bacterial death (mohammed et al., 2019). some of the bacterial strains such as s. aureus and enterococcusfaecalis cannot form biofilm due to the existence of the tq while the gram negative bacteria such as e. coli were less susceptible to tq and thq (halawani, 2009). the chemical can prevent the oxidative activity of the bacteria and reduce the number of bacteria (ali et al., 2020). discussion each of the main themes chosen contributed for the elements targeted for the wound healing treatment. based on the gross analysis, it can be concluded that black seeds can help with the time taken for the wound to be healed. most of the results shown that the black seeds can speed up the healing process and making the wound to be closed at faster rate than any other conditions that had been set up in the experiments. for examples rohmah & suprayitno (2020) had founded that black seeds oil with albumin had the fastest wound closure when compared with the negative control group. besides that, nourbar et al. (2019) mentioned their research paper that the wound healing was faster as the concentration of the black seeds increasing. as for the microscopic and biochemical analytsis, it is mainly to see the biocompatibility of the black seeds when used as wound healing agent. the observation was mainly to see if there is any negative changes that can alter the normal cells to regenerated normally. in the analysis, it can be seen that the black seeds can help in reducing the free radicles that can slow down the healing process as founded by yalçın turhan, mehmet arıcan, zekeriya okan karaduman, ozan turhal & ozkan (2019). moreover, it portrayed that the black seeds can also help in the fast regeneration of cells at the wound site by increasing the blood flow to the site and this can be observed based on the research done by abd elrahman et al. (2019). instead of that, there were several other review paper that mentioned the same results as the paper reviewed in this review paper. in the paper written by sallehuddin et al. (2020), the author had mentioned the black seeds contained tq that had many medicinal benefits such as antioxidant, antiinflammatory, antibacterial, and providing protection for the renal as well as for the nerve function. the black seeds also had several good outcomes when they are used as the agent for wound healing. this outcomes happens because the black seeds can induce angiogenesis, the proliferation of fibroblasts, and even increase collagen production. besides that, some of the papers reported that the black seeds lower down the number of white blood cells and reduce the extent of tissue damage and a bacterial infection. sallehuddin et al. (2020) also included many relevant papers that depicted the action of black seeds in wound treatments. one of the papers included in the study written by selçuk et al. (2013) used tq extracted from the black seeds to treat burn wounds. the results were compared with the burn wound's results treated with silver sulfadiazine (ss) in the same model as previous treatment. the researchers found that tq had the same rate of wound closure as the ss when administered intraperitoneally. still, tq exhibited better healing outcomes than ss when it was administered topically. apart from that, a study that had been done by elgohary et al. (2018) was comparing the black seeds wound healing properties with moist-exposed burn ointment (mebo) and chinese traditional herbs when used in the burn wound caused by chemical substances. they had found that all treatments can reduce the wound size with the smallest area found to be treated by black seeds oil. han et al. (2017) also had obtained the same results when black seed oil was made into a cream and used in the normal wound. in this study, the cream of black seeds oil was compared with hypericum perforatum (hp) oil cream, and it was observed that black seeds oil cream had almost the same effect as hp oil cream in reducing the wound area and increasing the contraction process of the wound healing. in excisional wounds, javadi et al. (2018) had observed that black seeds in combination with honey showed the smallest area of necrotic tissue 20 days after the wound was inflicted. the second smallest area was the wound treated by phenytoin, followed by the group that used honey or black seeds. as for kumandas et al. (2019), mohd zaid et al. (2021) journal of pharmacy, 1(2), 87-96 page 93 they had observed that the black seeds oil cream used in this type of wound had portrayed a slower rate of healing than zinc-silver cream. besides that, black seeds had also been experimented with in the chronic delayed wounds by yusmin and ahmad (2017). their study used petroleum jelly as the carrier to administer the black seeds extract on alloxan-induced diabetic wounds. it was seen that contraction of the wound was the greatest for the black seeds group only for the first three days of the experiment. on the 7th and 14th day of the experiment, they had seen the wound contraction was greater in the control group than in the black seeds group. sari et al. (2018) used the same model, but the black seeds oil was compared with aloe vera (av) oil gel during the experiment. the gel containing black seed oil affected the wound size after seven days, but it was considered insignificant compared with other groups. although the results were like those in those experiments, nourbar et al. (2019) had found the opposite of those results. they observed the fastest rate of healing when black seeds extract was used in the streptozotocin-induced diabetic rats during the experiment. the wound-healing effect was better than phenytoin treatment. the author deduced that the results were varied due to the difference in the mechanism for the death of beta cells in both experiments. in terms of the microscopic observation, yaman et al. (2010) observed that vascularisation and epithelialisation were better in the group that used black seed oil for burn wounds than in the ss and control groups. moreover, granulation tissue formation and the response of antiinflammatory cells were also better in the black seeds group than the rest of the other groups. besides that, there was a study done by shahani et al. (2013) on excisional wounds by using rabbits as the test subjects, and the black seeds had produced better angiogenesis and fibroblast proliferation at the wound site. furthermore, selçuk et al. (2013) had observed tq's good outcomes when applied to burn wounds. the wounds were showing great response for anti-inflammatory cell activity and the good formation of blood vessels, granulation tissue, and epithelial layer. in another study, han et al. (2017) found that granulation tissue formation and collagen synthesis happens more in the black seeds group rather than in the hp group and the control group but the results for angiogenesis, epithelialisation, and infiltration of inflammatory cells became insignificant when compared with the control group on the 14th day of the study. apart from that, sari et al. (2018) observed less inflammation in diabetic wounds when the test subjects were treated using the black seeds. the wound had less infiltration of polymorphonuclear neutrophil when the black seeds group was compared with the control group. they also observed more fibroblasts had infiltrated into the wound site and more epithelialisation to happen on the 7th day of the study. with regards to biochemical analysis, it was stated by cui et al. (2018) total antioxidant state (tas) and total oxidant stress (tos) values can be used as the determinants for observing whether oxidative stress was occurring or not. malondialdehyde (mda) can also become the determinants for oxidative stress. as for the antioxidant level, glutathione is one of the markers that can be used for observation. dunnill et al. (2017) had stated that the increase in tos value and reduction in tas level could indicate oxidative stress occurrence. oxidative stress is vital due to its association with burn wounds (nielson et al., 2017). moreover, vigani et al. (2017) mentioned that antioxidants could reduce the mortality caused by burn injury. three studies which are done separately by han et al. (2017), kumandas et al. (2019), and selçuk et al. (2013) had observed low tos level and high tas level when black seeds and tq were used for the rat model that a burn and excisional injuries had inflicted. the mda level was also decreased significantly and both, black seeds and tq, had greater outcomes in reducing the oxidative stress when compared with ss, hp, and zinc-silver treatments. instead of that, another review done by rajabian & hosseinzadeh (2020) had reported that ahmad et al. (1995), together with sarkhail et al. (2011) and mandal et al. (2015) mentioned in their paper about the black seeds oil could help to reduce the time for the healing process of the wound when applied topically. besides that, rajabian & hosseinzadeh had also reported that abu-zinadah (2009) found the usage of black seed oil can improve the state of burn wounds on rabbits' skin. moreover, in the review, they had written about the study done by ab rahman et al. (2014) about the aqueous extract of black seeds potential in healing wounds. the researchers had observed an increase in the fibroblast's proliferation as well as wound closure activity by the extract. the extract was also observed to be able to scavenge on the free radicals at the wound site. another study by abu-al-basal (2011) was included in the review. he had mentioned that extract of black seeds obtained by using petroleum ether can provide great healing properties in balb/c mice infected with staphylococcal bacteria. moreover, the researcher stated that tissue impairment prevention and decreased white blood cell count can be achieved in the study due to the immune-modulating, anti-inflammatory, antioxidant, and bactericidal properties of the extract. mohd zaid et al. (2021) journal of pharmacy, 1(2), 87-96 page 94 conclusion the black seeds indeed contain several properties that can help in enhancing the wound healing process. this statement is supported by the results which showed the black seeds is compatible with human body when apply to the wounded area and it can also help to shorten the time for the wound to heal. both of these outcomes can be observed based on the result analysis from the research papers that were included in this review paper. moreover, the black seeds also contained antimicrobial properties and anti-inflammation properties that become the advantage of using them in wound treatment. this property may not be found in certain types of synthetic chemicals and it also can prevent any complications that may happen due to unwanted infection or pain cause by the inflammation. hope that there will be effort to bring this plant into the medicinal world and more research being done to establish its safety in long-term treatment and the usage of this plant's seeds in the patient with comorbidities. acknowledgements this review was conducted without any funds. conflict of interest there was no conflict of interest in this review. references ab rahman, m. r., abdul razak, f., & mohd bakri, m. 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(2017). effect of thymoquinone on wound healing in alloxan-induced diabetic rats. asian journal of pharmaceutical and clinical research, 10(9), 242–245. https://doi.org/10.22159/ajpcr.2017.v10i9.18951 https://doi.org/10.29322/ijsrp.10.07.2020.p10371 https://doi.org/10.3390/ijerph17114160 https://doi.org/10.1055/s-0031-1282650 https://doi.org/10.1097/bcr.0b013e31827a2be1 https://doi.org/10.1016/j.mpsur.2017.06.004 http://rpdemos.net/clients/hendunadmin/admin/uploads/source/dcm/dcm-18-2-110.pdf http://rpdemos.net/clients/hendunadmin/admin/uploads/source/dcm/dcm-18-2-110.pdf https://doi.org/10.5812/ircmj.84650.research https://doi.org/10.1155/2019/1528635 https://doi.org/10.22159/ajpcr.2017.v10i9.18951 introduction search strings database (black seed extract or black seed cumin extract or nigella sativa extract) and (antioxidant or oxidative antagonist or oxidative competitor) and (burns wound or blaze wound) and (healing process or curing process or repair process or regenerating process or rebuild process) google scholar pubmed sciencedirect methodology table 2: criteria for the papers to be included and excluded from this review before the eligibility process table 2: criteria for the papers to be included and excluded from this review before the eligibility process results discussion conclusion the black seeds indeed contain several properties that can help in enhancing the wound healing process. this statement is supported by the results which showed the black seeds is compatible with human body when apply to the wounded area and it can als... acknowledgements conflict of interest references barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework mohamad hamdi et al. (2023) journal of pharmacy, 3(1), 53-65 page 53 an insight into the use and advantages of carbopol in topical mucoadhesive drug delivery system: a systematic review nurul ain mohamad hamdi1,*, nurul atifah azmi1, nurul hanis mohd sabari1, ahmad fahmi harun2, muhammad salahuddin haris1,3,*, abstract introduction: in recent years, mucoadhesive topical application of mucous membrane has gained considerable interest among formulation researchers in advanced drug delivery systems. it has been identified as a potential route for both local and systemic drug delivery. a mucoadhesive agent is usually incorporated in the formulation to overcome the disadvantages associated with the conventional topical formulation. these disadvantages include low residence time of the medication on the site of application due to tongue movement and salivary washout in the intraoral formulation, mucociliary clearance in the intranasal application, and rapid precorneal elimination in the intraocular formulation. carbomer or known as carbopol is a mucoadhesive polymer that is widely studied for topical delivery of pharmaceutical agents to the mucous membrane. the use of carbopol and its advantages in the mucoadhesive topical application has gained considerable interest with several published studies and is available in various grades. in this study, a systematic review was performed on the available literature that investigates the carbopol application in mucoadhesive topical drug delivery. method: a systematic searching strategy was performed in scopus, proquest, and pubmed databases using predetermined search strings. a total of 778 articles were retrieved, however, only 25 articles met the inclusion criteria and were used for data synthesis. results: the results showed that incorporation of carbopol as mucoadhesive polymer hold multiple advantages in drug delivery namely excellent mucoadhesion effect, the prolonged residence time of the formulation, enhanced drug permeation, prolonged release of drug, pseudoplastic behaviour of the formulation, ph compatibility with all mucosal site, and biocompatible. conclusion: this suggests that the incorporation of carbopol can be an effective mucoadhesive agent for topical drug delivery systems. article history: received: 12 may 2022 accepted: 11 october 2022 published: 31 january 2023 keywords: polyacrylic acid, carbomer, mucoadhesion, benefits, roles how to cite this article: mohamad hamdi, n. a., azmi, n. a., mohd sabari, n. h., harun, a. f. & haris, m. s. an insight into the use and advantages of carbopol in topical mucoadhesive drug delivery system: a systematic review. journal of pharmacy, 3(1)., 53-65 doi: 10.31436/jop.v3i1.156 authors’ affiliation: 1 department of pharmaceutical technology, kulliyyah of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, 25200 kuantan, malaysia. 2 department of physical rehabilitation sciences, kulliyyah of allied health sciences, international islamic university malaysia, jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia. 3 ikop pharma sdn. bhd., jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia. *corresponding author: email address: aenhamdi@gmail.com; solah@iium.edu.my mailto:aenhamdi@gmail.com mohamad hamdi et al. (2023) journal of pharmacy, 3(1), 53-65 page 54 introduction mucoadhesive topical application is an interesting field among formulation researchers in advanced drug delivery systems (duarah, durai, & narayanan, 2017; kapileshwari et al., 2020; matos et al., 2020). it is an external drug’s introduction to the mucous membrane of the body part that exploits the property of bioadhesion of certain polymers (kore, shete, desai, & dnyanpeeths, 2013). mucous membranes are found in many body parts including the eyes, respiratory tract, gastrointestinal tract, and reproductive tract (netsomboon & bernkop-schnürch, 2016) that serves as a potential route for both local and systemic drug delivery (kumar, naik, pradhan, ghosh, & rath, 2020; srikrishna et al., 2017). mucoadhesive drug delivery is adopted to resolve the disadvantages associated with the conventional topical formulation including low residence medication time on the site of application due to tongue movement and salivary washout in the intraoral formulation, mucociliary clearance in the intranasal application, and rapid precorneal elimination in the intraocular formulation (netsomboon & bernkop-schnürch, 2016; pagano, giovagnoli, perioli, tiralti, & ricci, 2020; saisree et al., 2019; sheshala, ming, kok, singh, & dua, 2019). carbomer or is typically called carbopol and and polyacrylic acid is a mucoadhesive polymer with the formula (ch2-chco2h)n that is widely incorporated for topical delivery of pharmaceutical agents to the mucous membrane (arun karthick, ramya devi, & vedha hari, 2018; m. n. a. rahman, qader, sukmasari, ismail, & doolaanea, 2017; sheshala et al., 2019; suzilla, izzati, isha, zalina, & rajaletchumy, 2020). figure 1. general structure of carbopol polymer. the most attractive properties of carbopol as a mucoadhesive agent for topical application are biodegradable, bioadhesive, non-irritant, not absorbed into the body, and less expensive (bera, mazumder, & khanam, 2016; suzilla et al., 2020). carbopol possesses several grades and is classified according to the degree of crosslinking, viscosity, and manufacturing condition, which offers flexibility in drug release profile as well as mucoadhesion (singla, chawla, & singh, 2000). it is incorporated in various dosage form and various strength for topical oral, nasal, ophthalmic, and vaginal drug delivery. the main aim of this study is to systematically review the use of carbopol according to its grade and formulation dosage form as mucoadhesive topical drug delivery particularly on the oral mucosa, nasal mucosa, ophthalmic mucosa, and vaginal mucosa based on recent studies. this review also aims to discuss the merits of carbopol in topical mucoadhesive drug delivery. methodology this section discusses the chosen method used to select articles related to the use of carbopol in topical drug delivery. the reviewers used the prisma method that includes scopus, proquest, and pubmed to perform a systematic review in identifying, screening, eligibility, quality appraisal and data abstraction, and analysis. 1. the review protocol – prisma this systematic review was guided by the prisma statement. 2. formulation of research question the formulation of the research question for this review was based on pico. it is a tool that facilitates the authors in generating relevant research question for the review. pico is based on three key concepts namely problem, interest, and context. based on these concepts, the authors chose three primary aspects for the review namely carbopol grade and strength based on the formulation dosage form (problem), advantages of carbopol in the topical application on mucous membrane such as oral, nose, eye and vagina (interest), and drug delivery system (context). these concepts have guided the authors to formulate the key research question namely what is the grade, strength, and advantages of carbopol in topical mucoadhesive drug delivery system? 3. systematic searching strategies there are three main processes in the systematic searching strategies that include identification, screening, and eligibility. 3.1 identification identification of the keywords followed by the process of searching for the synonym of the keywords was performed. the search string was developed including ( ( carbopol or caborpol or cabormer or cabormer or carbapol ) and ( mucosa* or "mucosa layer" or "mucosa membrane") and (eye* or ophthalmic* or ocular* or optic* or oral* or mouth* or lingual or periodontal or gum* or vagina ) and ( mucoadhesive or mucoadhesion or bioadhesive or bioadhesion ) ). the search string was performed on three databases in which 300 articles were mohamad hamdi et al. (2023) journal of pharmacy, 3(1), 53-65 page 55 retrieved in scopus, 468 articles in proquest, and 10 articles in pubmed. 3.2 screening screening of the articles was conducted automatically based on the sorting function available in the databases according to the inclusion and exclusion criteria as shown in table 1. a total of 615 articles was excluded after the sorting function whereas 3 duplicate articles were removed. table 1: inclusion and exclusion criteria of this systematic review criteria inclusion exclusion publication timeline 2016 2020 2015 and before document type original research articles conference proceeding, chapters in book, book series, books etc language english non-english nature of the study focus on topical drug delivery focus on other than topical drug delivery 3.3 eligibility eligibility is the third process whereby the authors read the full text of the articles to ensure that the articles met the inclusion criteria. a total of 160 articles were assessed and 87 articles were excluded due to lack of details regarding carbopol used on the mucous membrane in the topical application. this resulted in only 73 articles proceeded to the quality appraisal step. 4. quality appraisal quality appraisal was conducted to ensure the quality content of the articles. all 73 articles were assessed individually by 3 authors and these articles were ranked to either high, medium, or low-quality article based on the predetermined criteria. the criteria were established based on the research questions. mutual agreement between the authors was practised in this process to reduce bias. the authors concluded to only include high-quality articles hence, only 25 articles proceeded to the data abstraction and analysis step (figure 2). 5. data abstraction and analysis this study used a qualitative technique to analyse the data. data abstraction was conducted based on the research questions in which 2 of the authors categorised each article into the dosage design, the drug used, site of application, carbopol grade and strength, and the advantages of carbopol based on the summary of the article. figure 2. the prisma flow diagram. results 1. background of the selected articles the total selected articles were 25 of which 2 articles were published in 2020, 6 in 2019, 4 in 2018, 8 in 2017, and 5 in 2016. these articles were evaluated for carbopol application to the oral mucosa, nasal mucosa, eye mucosa, and vaginal mucosa. the results showed that 14 articles incorporated carbopol for oral mucosa application, 5 studies in the nasal mucosa, 5 studies in vaginal mucosa while only 1 study in eye mucosa. this review has identified 7 formulated dosages, which is in situ gel, gel, tablet, patches, film, aerosol, and wafer. there were 5 grades of carbopol polymer were documented namely carbopol 940, carbopol 974p, carbopol 934p, carbopol 971p, and carbopol 980. the most frequently used carbopol grade was carbopol 940 in which it was reported in 10 articles followed by 8 articles utilised carbopol 934p, 6 articles used carbopol 974p, and 1 article each used carbopol 971p and carbopol 980. table 2 summarises the included studies. mohamad hamdi et al. (2023) journal of pharmacy, 3(1), xx-xx page 56 table 2: summary of the included studies. year ref dosage design drug site carbopol type and strength advantages 2020 (li, bao, shen, lalla, & burgess, 2020) in situ gel bupivacaine oral 974p (0.08%) superior mucoadhesion and greater swelling 2019 (mohamad, abdelkader, elrehany, & mansour, 2019) tablets cyanocobalamin oral 971p (50% and 49% w/w) superior mucoadhesion, exhibit slow-release, good swelling rate 2016 (patel, prabhu, dubey, & kamath, 2016) buccal patches hydrochlorothiazide and atenolol oral 934p (100 mg – 300 mg) superior mucoadhesion, greater swelling, better permeation and biocompatible 2017 (marques, rocha, leal, estanqueiro, & lobo, 2017) buccal gel of lipid nanoparticle ibuprofen oral 980 (1.5% w/w) superior mucoadhesion, higher firmness and exhibit pseudoplastic behaviour 2017 (pham, van vo, tran, tran, & tran, 2017) microemulsion-based wafer prednisolone oral 940 (1.5%) exhibit slow-release and superior mucoadhesion 2017 (azeran et al., 2017) gel moxifloxacin oral 940 (0.3%) superior mucoadhesion, exhibit slow-release, and exert permeation enhancing effect 2017 (sadeq & rajab, 2017) patches captopril oral 934 (93.75mg and 18.75mg) superior mucoadhesion, greater swelling index, exhibit slow release and compatible 2016 (kumria, nair, goomber, & gupta, 2016) film prednisolone oral 940 (100mg, 75 mg and 50 mg) improved viscosity, superior mucoadhesion, slow and steady hydration, exhibit slowrelease, and great drug permeation 2019 (jain, gilhotra, & kori, 2019) hydrogel l-glutamine oral 934p (0.251%) good consistencies and homogeneity, compatible, and exhibit slow release 2020 (t. a. ahmed, bawazir, alharbi, & safo, 2020) film simvastatin oral 940 (5% and 10%) uniform distribution, compatible (drug and excipient) superior mucoadhesion and exhibit slow release. 2017 (ali, sabati, & ali, 2017) film baclofen oral 940 (1-5%) biocompatible, greater swelling, superior mucoadhesion, exhibit slow release and greater in vivo residence time 2018 (aslani, zolfaghari, & fereidani, 2018) gel herbs oral 940 (0.5% and 1%) superior mucoadhesion and exhibit slow release 2017 (calixto et al., 2017) liquid-crystaline system with in situ gelling peptide p1025 oral 974p (2.5%) superior mucoadhesion and good pseudoelasticity and elasticity 2018 (chaiprateep, khobjai, & noysang, 2018) film clinacanthus nutans oral 934p (7.5 to 24 g) superior mucoadhesion, increased elongation and tensile strength and greater swelling 2019 (mahajan, shende, dumore, & nasare, 2019) in situ gel tapentadol nasal 934p 0.1% w/v effective gelation viscosity and gel strength, good drug release and good mucoadhesive strength mohamad hamdi et al. (2023) journal of pharmacy, 3(1), xx-xx page 57 2019 (abdelnabi, abdallah, & elghamry, 2019) in situ nanovesicular gel buspirone nasal 974p (0.3% & 0.5 % w/v) increased muchoadhesiveness, sustained drug release, increased bioavailability and has penetration enhancing effect 2016 (ayoub, ibrahim, abdallah, & mahdy, 2016) microemulsion based gel (mbg) sulpiride nasal 940 (0.5% 2%) uniform spreadability, increased mucoadhesive force, enhanced nasal bioavailability, has penetration enhancing effect and prolong residence time 2017 (malekar, gondkar, bhairav, paralkar, & saudagar, 2017) in situ nasal gel naratriptan nasal 934 0.2% w/v prolong residence time, better mucoadhesive property and improved bioavailability 2016 (shelke et al., 2016) thermoreversible nanoethosomal gel eletriptan nasal 934 (0.4% w/v) increased mucoadhesive strength, prolong retention, increased absorption and better drug permeation 2. grade and strength of carbopol for oral mucosa application oral mucosa topical application reported 6 types of dosage forms namely in situ gel, oral gel, buccal mucoadhesive tablet, patches, film, and wafer. different concentration of carbopol was used according to the type of formulation. carbopol ranging from 0.25% to 1.5% was used in the oral gel formulation whereas 1.5% of carbopol 980 was used as a single gelling polymer in nanoparticle loaded gel formulation (marques et al., 2017). a lower concentration was reported in 1 study whereby 0.3% of carbopol 940 was used as a single gelling agent for oral gel formulation (azeran et al., 2017). 0.25% to 1% of carbopol 934p was used in combination with hpmc polymer and 0.5% to 1% carbopol 940 was used in combination with nacmc carbomer for intraoral gel formulation (aslani et al., 2018; jain et al., 2019). carbopol 974p was used in 2 articles for in situ gel formulation with 0.08% and 2.5% in combination with pluronic polymer and as single polymer, respectively (calixto et al., 2017; li et al., 2020). in addition, 3 articles utilised carbopol 940 in mucoadhesive film formulation ranging from 1% to 10% and 1 article used 7.5 g to 24 g of carbopol 934p with the combination of various polymers such as gum acacia, sodium alginate, polymethacrylates, and hpmc (t. a. ahmed et al., 2020; ali et al., 2017; chaiprateep et al., 2018; kumria et al., 2016). 1.5% of carbopol 940 was employed in microemulsion based mucoadhesive buccal wafer (pham et al., 2017) and 2 articles utilised 18.75 mg to 300 mg of carbopol 934 for buccal patches formulation (patel et al., 2016; sadeq & rajab, 2017). meanwhile, 1 study incorporated 49% and 50% of carbopol 971p in combination with hpmc and chitosan for mucoadhesive buccal tablet formulation (mohamad et al., 2019). 3. grade and strength of carbopol for nasal mucosa application for intranasal application, 0.5% to 2% of carbopol 940 was used as a single mucoadhesive polymer for gel formulation (ayoub et al., 2016). meanwhile, 3 studies used carbopol 934p with a concentration of 0.1% to 0.4% for in situ gel formulation with a combination of other polymers such as hpmc, gellan gum, xanthan gum, and poloxamer 407 (mahajan et al., 2019; malekar et al., 2017; shelke et al., 2016). furthermore, 1 study used 0.3% and 0.5% of carbopol 974p with a combination of hpmc for in situ intranasal formulation (abdelnabi et al., 2019). it can be observed that the incorporation of carbopol as the only gelling polymer requires a higher concentration of up to 2%. in contrast, a lower concentration of carbopol is needed below 0.5% when used in combination with other polymers. 4. grade and strength of carbopol for vaginal mucosa application in single gelling agent for intravaginal gel formulation, 2 articles incorporated 1% carbopol 974p (s. s. rahman & ahmed, 2019; takalkar & desai, 2018) while another study used 0.8% carbopol 940 (salah, awad, & makhlouf, 2018). an article studied 2 grades of carbopol namely carbopol 934 and carbopol 940 both ranging from 0.5% to 1% with the combination of hpmc polymer for vaginal gel formulation (choudhury & roy, 2016). mohamad hamdi et al. (2023) journal of pharmacy, 3(1), xx-xx page 58 5. grade and strength of carbopol for eye mucosa application 0.1% and 0.2% carbopol 940 was used in in situ gel ophthalmic application in combination with hpmc polymer (kouchak, mahmoodzadeh, & farrahi, 2019). based on the results, it can be concluded that hpmc was the most frequently used polymer in combination with carbopol for topical application on the mucosal membrane. table 3 summarises the carbopol use according to site of application and dosage form. table 3: summary of the carbopol use according to the site of application and dosage form. application site dosage form carbopol type concentration /amount oral mucosa in situ gel 974p 0.08%, 2.5% gel 980 1.5% gel 940 0.3% 1% gel 934p 0.25% 1% tablet 971p 49%, 50% patches 934 18.75 mg 300 mg film 940 1% 10% film 934p 7.5 g 24 g wafer 940 1.5% nasal mucosa gel 940 0.5% 2% gel 934 0.1% 0.4% in situ gel 974p 0.3% and 0.5% eye mucosa in situ gel 940 0.1% and 0.2% vaginal mucosa gel 974p 0.8% 1% gel 934/934p 0.5% -1% gel 940 0.5% 1% 6. advantages of carbopol used in mucoadhesive topical drug delivery system there were 7 advantages of carbopol in topical drug delivery namely excellent mucoadhesion effect, the prolonged residence time of the formulation, enhanced drug permeation, prolonged release of drug, pseudoplastic behaviour of the formulation, ph compatibility with all mucosal site, and biocompatible with mucous membrane. the most reported benefit is the excellent mucoadhesion property. discussion carbopol is a mucoadhesive polymer that is extensively used in drug delivery studies. there are several types of carbopol in which dominantly applied in a specific part of the body based on their rheological properties. the availability of the various grades of carbopol depends on the manufacturing condition namely polymerisation and degree of cross-linking of the polymer reflected by the viscosity. carbopol that carry the letter p after the number means that they are of high purity that makes them suitable for oral use (mariageraldrajan, 2007; panzade & puranik, 2010). the summary of findings of this systematic review is summarised in figure 3. 1. mucoadhesive effect mucoadhesion is a characteristic of a dosage form that can interact with the mucous layer covering mucosal epithelial cells (ahmed & bhaduri, 2017). it plays an important role in the drug absorption and bioavailability (ahmed et al., 2020). besides this property is also important to preserve high level of drugs at the application site and to prevent expulsion of the formulation. for example, in buccal patch or buccal film, adequate mucoadhesion is prerequisite for optimal performance because low mucoadhesion would result in spitting or ingestion of the formulation (kumria et al., 2016). strong mucoadhesion of formulation incorporated with carbopol were reported in various formulations types such as buccal patch, buccal film, buccal wafer, oral gel, nasal in situ gel, vaginal gel and in situ eye gel (ahmed et al., 2020; choudhury & roy, 2016; kouchak et al., 2019; kumria et al., 2016; mahajan et al., 2019; malekar et al., 2017; marques et al., 2017; patel et al., 2016; shelke et al., 2016). mucoadhesive effect of carbopol is attributed to the strong interaction that exists between carboxyl group (cooh) of carbopol and a component of mucous membrane called mucin. mucin are large glycoprotein expressed by epithelial membranes and are a component of the mucous secretions that covers epithelial. mucin has a protein core with carbohydrate side chain and is the target to improve drug retention. chemically, carbopol polymer having abundance of carboxyl groups mohamad hamdi et al. (2023) journal of pharmacy, 3(1), xx-xx page 59 figure 3. summary of findings of this systematic review. mohamad hamdi et al. (2023) journal of pharmacy, 3(1), xx-xx page 60 tend to form hydrogen bonding with amide group of mucin as proton accepting group (shelke et al., 2016). besides, studies showed that mucoadhesion of formulation were directly proportional with carbopol concentration present in formulations (ayoub et al., 2016). the plausible explanation is at higher carbopol concentration, more interactions would be formed between carbopol and mucous membrane leading to increase in mucoadhesion strength (shelke et al., 2016). however, too strong mucoadhesive would damage the mucosal membrane (kouchak et al., 2019). 2. prolonged residence time of the formulation at high carbopol concentration, more compact lattice structure and more hydrogen bonding will be formed led to an increase in mucoadhesive strength. high mucoadhesive strength prolongs drugs retention and eventually inprove the absorption of drug across the mucosal tissue (ayoub et al., 2016; malekar et al., 2017). in oral application, the retention time of prednisolone buccal wafer containing 1.5% carbopol in ex vivo study was longer which is about 5 hours compared to formulation with lower carbopol concentration (pham et al., 2017). similarly, another study found that the formulation of oral buccoadhesive films using carbopol possesses greater mucoadhesive retention compared to without carbopol content (kumria et al., 2016). dissolution of buccal film influences the retention time of the film. the high viscosity of carbopol retard the dissolution of the film and subsequently increasing the film retention time (kumria et al., 2016). in nasal application, adequate mucoadhesive strength is important as this could help improving nasal drugs delivery as it prevents drainage from the nose cavity (malekar et al., 2017). a pharmacodynamic study on the paw test was conducted to compare intranasal microemulsion based gel (mbg) over intranasal microemulsion (me) of sulpiride. it was found that mbg has higher hind limb refraction time (hrt) values compared to me of sulpiride. this demonstrated the role of carbopol 940 incorporated in mbg in reducing the mucociliary clearance (mcc) and subsequently prolonging the residence time of sulpiride (ayoub et al., 2016). in the ophthalmic preparation, the drug’s residence time plays a major concern in the formulation as it may influence the effectiveness of the drug. prolonged residence time could result in a long duration of intraocular pressure (iop) reduction and increase the efficiency of the in situ gel. the incorporation of carbopol 940 (0.1 w/v) and hpmc (0.1% w/v) for in situ gel of dorzolamide hcl showed a longer and higher intra ocular pressure-lowering activity compared to dorzolamide solution and marketed drop. the prolonged residence time of the drug is attributed to its high viscosity and mucoadhesive property of the polymers. this subsequently increases the bioavailability and reducing administration frequency (kouchak et al., 2019). 3. drug permeation enhancing effect several studies have shown that the concentration of carbopol improves the bioavailability and permeation of the drugs in certain formulations of oral and nasal application (ayoub et al., 2016; kumria et al., 2016; mohamad et al., 2019). the administration of cyanocobalamine buccoadhesive tablet with 20% 50% carbopol 971p in combination with hpmc polymer exhibited a significant increase in the total amount of cyanocobalamin that enter systemic circulation that reflected by the estimated area under the curve (auc) value (mohamad et al., 2019). similarly, enhancement in the rate and absorption of prednisolone have been reported in a study of prednisolone buccal film that use combination of hpmc and 50 mg of carbopol 940. higher auc value of 2 folds was observed with buccal route compared to oral suspension formulation of prednisolone (kumria et al., 2016). good permeation ability of drug also reported with combination of 300 mg carbopol 934 and hpmc in mucoadhesive buccal patch formulation of anti-hypertensive agents (patel et al., 2016). the penetration enhancing effect are mainly due to the mucoadhesive properties as the residence time of formulation on mucosa also improved with the drug’s permeation across the oral mucosa (azeran et al., 2017). the permeation enhancing effect of carbopol was also reported with nasal application. microemulsion based gel with 0.5% to 2% carbopol 940 showed greater permeation of sulpiride compared to microemulsion after 24 hours. carbopol exert the penetration enhancing effect by opening the tight junctions of the nasal mucosa thus promoting the transport of drugs via a paracellular pathway (ayoub et al., 2016). 4. prolonged release of drug carbopol plays important roles in sustaining the drug release for hours. prolonged release formulation is desirable as it allows reduction of medication administration frequency and improving patient compliance (da silva, ferreira, reis, cook, & bruschi, 2018). various studies reported that incorporation of carbopol polymer would retard the release of drugs from many formulations such as buccal tablet, buccal patches, buccal wafer, buccal film, in situ nasal gel and microemulsion based gel (ali et al., 2017; ayoub et al., 2016; mahajan et al., 2019; mohamad et al., 2019; patel et al., 2016; pham et al., 2017). it was found that, with mohamad hamdi et al. (2023) journal of pharmacy, 3(1), xx-xx page 61 increase in concentration of carbopol the release rates decrease gradually. theoretically, drug was trapped by higher carbopol concentration in which would exert resistance for the drug to travel through it. additionally, drug movement area also would be limited by the density of chain structure especially at higher carbopol concentration. subsequently producing slower drug release rate for a longer duration (ahmed et al., 2020; ayoub et al., 2016; mahajan et al., 2019; patel et al., 2016; pham et al., 2017). a slow-release rate of cyanocobalamine reported from buccal tablet up to 5 hours duration (mohamad et al., 2019). meanwhile, longer release duration of more than 6 hours were reported with the formulations of combination antihypertensive agents (hydrochlorothiazide and atenolol) and prednisolone from buccal patches (100-300 mg carbopol) and microemulsion based gel buccal wafer (1.5% carbopol) respectively (patel et al., 2016; pham et al., 2017). for nasal delivery, extended release of tapentadol for duration of 5 hours have been shown with in situ nasal gel formulation with 0.2% carbopol (mahajan et al., 2019). a longer release duration of naratriptan up to 8 hours was reported with formulation of in situ nasal gel that use combination of 0.2% carbopol with 0.1% xanthan gum (malekar et al., 2017). for vaginal mucosa application, formulation of fluconazole gel with combination of hpmc and 0.5% carbopol 940 show a constant and uniform drug release with around 80 – 85% drug release after 10th hour compared to formulation fabricated with sodium cmc and guar gum that complete the drug release within 10 hours (choudhury & roy, 2016). besides, a study of nanogel of nevirapine formulated with 1% carbopol 974p reported a zero-order kinetics suggesting the system release the drug at a constant rate for 6 hours duration (rahman & ahmed, 2019). 5. ph compatibility the ph of oral cavity is maintained by the saliva with the normal ph range of 6.2 to 7.6 (baliga, muglikar, & kale, 2013). it is advisable to keep the surface ph of the formulation similar to the buccal and salivary ph to minimise irritation to the oral mucosa (kumria et al., 2016). in this review study, various forms of topical oral formulations namely buccal tablets, buccal patch, wafer, oral gel and buccal film have been successfully developed within the saliva ph value (ali et al., 2017; jain et al., 2019; kumria et al., 2016; mohamad et al., 2019; patel et al., 2016; pham et al., 2017; sadeq & rajab, 2017). the normal nasal mucosa ph ranges between 5.5 6.5 (england, homer, knight, & ell, 1999). the ph of nasal formulation reviewed in this study were within the physiological ph of the nasal mucosa (ayoub et al., 2016; mahajan et al., 2019; malekar et al., 2017; shelke et al., 2016). the slight acidic ph is necessary for lysozyme activity (takalkar & desai, 2018). lysozyme is produced in nasal secretions and is responsible for killing bacteria at an acidic ph and is ineffective under alkaline ph and could promote the nasal tissue to be vulnerable to microbial infection (salah et al., 2018). similarly, the normal vaginal ph ranges between 3.8 to 5.0, which is moderately acidic. this slight acidic ph of vaginal mucosa is crucial to protect the vagina mucosa from pathogenic organism (lin, chen, cheng, & shen, 2021). nevirapine nanoparticle loaded 1% carbopol 974p gel and fluconazole gel (1.5% carbopol 934) were formulated with desired physicochemical characteristics within the vaginal physiological ph (choudhury & roy, 2016; rahman & ahmed, 2019). in contrast, another study of miconazole microsponge gel was formulated with slightly higher ph which is 7 to prolong the retention of the gel at the vaginal mucosa to allow complete drug release as the maximum viscosity of carbopol gel achieved at ph 6-7. the plausible explanation is that the neutral ph is not harmful to the vaginal mucosa as the ph of the semen ranged between 7.2-8 and also higher vaginal ph was reported in fungal infection (salah et al., 2018). meanwhile, the normal ph range of tear lies between 6.5 to 7.6 (abelson, udell, & weston, 1981). ph is an essential parameter of the eye's acceptance and tolerance with the formulation. with buffering capacities that tolerate the ph around 4-8, the ph of the tear is about 7.4. a ph value beyond this range will reduce the drug's bioavailability due to the stimulation of blinking and tearing (kouchak et al., 2019). ph triggered in situ gel under was successfully formulated with combination of 0.1% carbomer and 0.1% hpmc and desired viscous gel formed under the physiological tear ph (kouchak et al., 2019). 6. ph compatibility biocompatibility refers to the ability of a formulation in not causing toxicity or injury effects on living tissue (l. guy, 1988). carbopol is biocompatible to be administered since it does not irritate the mucous membrane. a study showed that after removal of buccal film containing (1-5% carbopol 940), visual inspection of the mucosal tissue showed no evidence of mucosal injury to any of the polymers. no discomfort was reported by the volunteers during or after in vivo study of baclofen buccal film (ali et al., 2017). besides, sheep’s nasal mucosa photomicrographs were observed for histopathological changes after permeation tests with in situ gel containing opioid for nose to brain delivery. no sign of remarkable destructive effect of formulations on the treated nasal mucosa was observed (mahajan et al., 2019). in another study, mohamad hamdi et al. (2023) journal of pharmacy, 3(1), xx-xx page 62 histopathological photographs were conducted where nasal mucosal membranes treated with thermoreversible gel revealed minor epithelial cell destruction, meanwhile, intact cellular integrity was seen in the untreated mucosal membrane. the physical impact caused by the application of gel and ph shock could be related to the cause of this injury. that may be attributed to the swollen aspect of carbopol resulting in mild damage to the intact columnar cells of the epithelial cells. the absence of damage to the glands that secrete mucus, cell necrosis, and columnar cells indicated that these ethosomal thermoreversible gels were healthy for nasal mucosa and could be used to treat migraine through intranasal path (mei et al., 2017). for the vaginal drug delivery, histopathological findings concluded an absence of vaginal mucosal irritation indicated by normal cell lining without any vaginal mucosa injury. another study also reported no clinical symptoms of irritation involving rash, inflammation, swelling, scaling, and irregular tissue formation hence suggesting that the carbopol-based formulation is free from any irritation (malekar et al., 2017). additionally, there were no signs of clinical irritation of rabbit’s eye with the application of ph triggered in situ gel in the ophthalmic drug delivery (kouchak et al., 2019). 7. pseudoplastic behaviour the incorporation of carbopol in the formulations exhibit a non-newtonian pseudoplastic behaviour with yield stress. pseudoplastic behaviour helps to facilitate liquid flow out from its container. in the formulation of in situ gel for dorzolamide, carbopol 940 nf and hpmc showed a lower pseudoplastic behaviour in the physiological condition compared to the nonphysiological condition (kouchak et al., 2019). force application on the buccal mucosa causes the breakdown of the gel network structure hence making it easier to spread on the mucosa (marques et al., 2017). in order to sustain the site-specific action for a longer period, pseudoplastic behaviour is desired for topical application (azeran et al., 2017). conclusion the result of this systematic review revealed that carbopol 940 is the most frequent carbomer grade incorporated in mucoadhesive topical drug delivery formulation. the concentration of carbopol polymer incorporated in a formulation varies according to the type of pharmaceutical dosage form and application site. carbopol polymer exhibits various advantages in mucoadhesive topical drug delivery systems apart from mucoadhesive behaviour alone. carbopol as a mucoadhesive polymer benefits drug delivery in various ways namely excellent mucoadhesion effect, the prolonged residence time of the formulation, enhanced drug permeation, prolonged release of the drug, ph compatibility with all mucosal sites, biocompatible and pseudoplastic behaviour of the formulation. thus, it is suggested that the incorporation of carbopol can be an effective mucoadhesive agent for topical drug delivery systems. acknowledgements this study was supported by the [international islamic university malaysia (iium) research] under acculturation grant scheme (irags18-026-0027). conflict of interest the authors declare that there is no conflict of interest. references abdelnabi, d. m., abdallah, m. h., & elghamry, h. a. 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(2018). nanolipid gel of an antimycotic drug for treating vulvovaginal candidiasis—development and evaluation. aaps pharmscitech, 19(3), 1297–1307. https://doi.org/10.1208/s12249-017-0918-7 microsoft word jop_article 76 galley word_final.docx page 16 *corresponding author: email address: norny@iium.edu.my a systematic review of the hospitals’ antimicrobial stewardship programs implemented to improve antibiotics’ utilization, cost and resistance patterns ovais ullah shirazi1, norny syafinaz ab rahman1,2,*, che suraya zin1,2 abstract introduction: the high reliance of the physicians and surgeons on the antibiotics since their discovery has led to an irrational antibiotic utilization which not only has raised the incidence of antimicrobial resistance (amr) but also increased the cost of treatment with antibiotics as high use of antibiotics has been found related to the occurrence of certain nosocomial infections which need extra antibiotic courses to be cured. in order to overcome these antibiotic utilization related problems an antimicrobial stewardship (ams) program being the set of various persuasive, restrictive and structural interventions is considered an effective tool to rationalize the in-patient antimicrobial utilization worldwide. method: the focus of this review is on the interventions that are being implemented during the inpatient ams programs and have been described effective in controlling the antibiotic utilization, their cost of treatment and an overall infection control. the literature containing the information about various ams interventions effecting the utilization and cost patterns along with the impact on amr was searched in various databases such as pubmed, google scholar, science direct, ovid (medline) and scopus. the categorical sorting of the published data is based on various ams interventions such as the guideline development, formulary restriction (pre-authorization), educative interventions, clinical pathway development and prospective (post prescription) audit. considering the objectives of the study such as the goal to curb overutilization of antibiotics, control of their cost of treatment for in-patients and infection control the sorted literature is presented in three different tables describing the ams impact on the said outcomes. results: the post ams changes in utilization patterns are described as fall of antibiotics defined daily doses (ddd) and days of therapy (dot) which resulted in the reduction of the cost of treatment with antibiotics. the reduction of the cost of treatment with antibiotics also resulted due to the ams impact on the control of various nosocomial and multi-drug resistant (mdr) infections. conclusion: it has been concluded that the ams program if implemented under the supervision of an expert ams team mainly comprising of an infectious disease (id) physician, clinical pharmacists and microbiologists with considerable support by the hospital authorities could be a highly efficient tool of the pharmacovigilance for rationalizing the in-patient antimicrobial practice. article history: received: 22 march 2021 accepted: 27 january 2022 published: 31 january 2022 keywords: antimicrobial stewardship (ams) program, interventions, utilization patterns of antibiotics, cost patterns of antibiotics; infection control, antimicrobial resistance (amr) study designs how to cite this article: shirazi, o. u., ab rahman, n. s., & zin, c. s. (2021). a systematic review of the hospitals’ antimicrobial stewardship programs implemented to improve antibiotics’ utilization, cost and resistance patterns. journal of pharmacy, 2(1), 16-30 doi: 10.31436/jop.v2i1.76 authors’ affiliation: page 17 introduction the antibiotics after their discovery have become an important shield against the deadly infections but a rapid development of the antimicrobial resistance (amr) or a decreased susceptibility of bacteria to the antibiotics has invited the researchers to address this serious healthcare problem (suwantarat & carroll, 2016). the most common bacteria resistant to the broad spectrum antibiotics such as third generation cephalosporins and fluoroquinolones are escherichia coli and klebsiella pneumoniae (phoon et al., 2018). staphylococcus aureus are the important bacteria that are resistant to the methicillin and are known as methicillin resistant staphylococcus aureus (mrsa) (mendem et al., 2016). over the past decades streptococcus pneumoniae have exhibited marked reduction in their susceptibility against various penicillins and this fact is elaborated in the published reports of world health organization (who) (cottagnoud et al., 2013). vancomycin resistant enterococci (vre), carbapenem resistant enterobacteriaceae (cre) and pseudomonas aeruginosa contain an effective gene modification capacity and plasmid equipped resistance that enabled them to challenge the efficacy of certain broad spectrum antibiotics namely vancomycin, carbapenems, ceftazidime and gentamicin etc. (o’driscoll & crank, 2015). the contemporary literature reveals that the overutilization of the antibiotics within the hospital wards is one of the major causes of increased amr. there have been published studies that positively correlate the high consumption of antibiotics to that of the increased amr (miliani et al., 2011). the high incidence of amr further increases the antibiotic consumption as the multi-drug resistant (mdr) microbes are the basic cause of the healthcare associated infections (hai) or the nosocomial infections that not only renders the patients to the toxicological impact of the antibiotics but also elevates the overall cost of treatment (khan et al., 2017). the statistics of the antimicrobial consumption within the hospital wards worldwide highlight the trend that 30% to 50% antibiotic prescriptions do not match with antimicrobial spectrum of the antibiotics (livermore et al., 2013). the overuse of antibiotics within the hospital wards increase the total cost of treatment which is a matter of concern for both the patients and the healthcare providers (chandy et al., 2014). in order to overcome the overutilization of the antibiotics and to minimize the cost of antimicrobial treatment for the in-patients various hospitals worldwide have adopted a strategy which is the set of interventions commonly known as antimicrobial stewardship (ams) program (knox & wiemiller, 2017). the improvement of the prescribing patterns of the antibiotics is one of the most important goals of the ams program so as to ensure a rational use of antibiotics (chen et al., 2014). the ams interventions being implemented during an in-patient ams program are classified as persuasive, restrictive and structural interventions (teixeira rodrigues et al., 2019). the persuasive ams interventions are comprised of education of the prescribers, development of the ams guidelines along with the modification of the clinical pathways (neo et al., 2020). the formulary restriction (pre-authorization) of the prescriptions mainly from an infectious disease (id) physician, microbiologist or clinical pharmacist, stop order (de-escalation) practice for some highly consumed antibiotics along with the practice of antibiotic cycling or switch over between various antibiotic groups are some of the restrictive interventions being implemented as part of the hospitals’ ams programs (dutcher et al., 2020). the structural interventions for the ams are mainly the prospective (post-prescription) audit and the introduction of the computerized decision support system (cdss) that automatically guides the prescribers regarding the ams guidelines for a particular antibiotic (huh et al., 2016). introduction of the computerized prescribing and recording of the patient data within the electronic software of a hospital for traceability is an important structural intervention practiced worldwide so as to implement the ams efficiently (kauppinen et al., 2017). the effective and successful implementation of an ams program requires a team of multi-disciplinary healthcare professionals holding the responsibility to ensure a prudent antimicrobial practice within the hospital wards dealing with the antibiotics (apisarnthanarak et al., 2018). the infectious diseases society of america (idsa) and the society for healthcare epidemiology of america (shea) in the policy guidelines pertaining to the ams describe the structure of the ams team and the responsibilities of its members regarding the enforcement and implementation of the ams policy (barlam et al., 2016). according to the ams policy guidelines an ams team should contain an id physician and a clinical pharmacist (with a specialised training regarding the infectious diseases) being the core members (waters, 2015). additionally, the inclusion of a clinical microbiologist, an information technology (it) technician and a hospital epidemiologist is highly recommended for the efficient performance of the ams team (murri et al., 2018). additional to the above-mentioned members of the ams team in the healthcare settings of england a physician expert for acute care, a surgeon, a member from the pharmacy department team of management, a paediatrician, a senior nurse and an anaesthetist is also included as core members of the ams team (ashiruoredope et al., 2016). considering the assessment of the ams impact on the targeted outcomes various study designs are followed that categorically describe and evaluate the impact of the ams interventions on the study population. the most commonly implied study designs for page 18 the said purpose are controlled before after (cba), randomized controlled trial (rct), controlled clinical trial (cct) and interrupted time series (its). methodology in order to collect the data pertaining to the impact of ams programs within the in-patient settings worldwide the databases such as pubmed, google scholar, science direct, ovid (medline) and scopus were searched systematically. the searching terms used for data exploration were antimicrobial stewardship programs, impact of antimicrobial stewardship program on antibiotic utilization, cost and resistance for in-patients etc. the studies published in english and covering the scope of the hospitals’ ams programs for the in-patients describing at least one ams intervention with the resultant impact on the reduction of the antibiotic utilization, cost of treatment along with the control of healthcare associated infections (hai) and amr were included. the studies which were focused on the ams programs of the primary care settings and pediatric care wards and did not clearly described the ams interventions and their impact on the antibiotic utilization, cost and resistance patterns for the in-patients were excluded for this review. results the primary search of the relevant articles for this review from the said databases consisted of 2767 articles from 2014 2021 out of which 130 articles were short listed. finally, 49 studies were included in this review and evaluation of these articles was performed to describe the impact of the ams interventions on antibiotic utilization, cost and resistance patterns of the antibiotics used for the hospitalized patients. 24 of the sorted studies (table 1) primarily reported the ams impact on antibiotic utilization whereas 12 studies (table 2) focused on the economic impact of the ams. 13 studies elaborating the ams impact on the infection control and the control of the amr are presented in table 3. out of 49 studies included for this review 20 studies used cba study design to assess the impact of hospitals’ ams programs, 7 studies followed the rct study design, 6 studies used the cct study design whereas 16 studies used its study design to assess the impact of the ams interventions on antibiotics utilization, cost and resistance patterns for the in-patients. studies describing the ams impact on antibiotic utilization patterns the table 1. describes the impact of ams on the antibiotic utilization patterns. most of the studies reported the defined daily doses (ddd) and days of therapy (dot) of the antibiotics in order to describe the impact of the ams interventions on utilization patterns. 4 studies described the educative interventions that were implied by the ams team, id physicians or by the clinical pharmacists to enhance the awareness of the prescribers and antibiotic handlers. 11 studies described that the prospective audit of the antibiotic prescription orders by the ams team and id specialists as an intervention to control the over utilization of certain antibiotics within the hospitalized patients. 2 studies described that the over utilization of the ams included antibiotics was controlled by improving the clinical pathways. 5 studies mentioned that the formulary restrictive (pre-authorization) interventions were implemented to control the irrational use of antibiotics within the in-patient settings. 2 included studies elaborated the guideline development and the steps taken by the ams team to ensure the adherence to such ams guidelines as an ams intervention to ensure a judicious use of antibiotics. studies describing the ams impact on cost patterns the table 2. describes the impact of ams on the antibiotics cost patterns. 4 studies described the educative interventions being implemented to elucidate the prescribers about the importance of ams to control the overall cost of treatment with the antibiotics. 3 studies described the prospective audit done by the ams team, id physician or the clinical pharmacists as an intervention to improve the cost patterns of the antimicrobial treatment. 1 study performed in the hospital of usa described that to control the cost of treatment with antibiotics the improvement of the clinical pathways was done as an ams intervention. a study performed in a german hospital mentioned the formulary restrictive intervention as a tool to control the high cost of treatment with 3rd generation cephalosporins. 1 study conducted in the in-patient setting of spain elaborated that adherence to the ams guidelines resulted in the control of the cost of treatment with meropenem. a study of a malawian hospital claimed that the cdss based ams interventions were found an effective tool to control the antibiotics cost of treatment for the in-patients. studies describing the ams impact on resistance patterns the table 3. describes the impact of ams on the infection control and amr patterns. 6 studies described the formulary restrictive interventions that were implemented within the hospital wards helped to control the amr. the improvement of the clinical pathway during the ams program of a taiwanese hospital helped in infection control for the in-patients. 3 studies described that a decline of the incidence of the amr and certain nosocomial infections namely clostridium difficile infection (cdi) was observed as a result of continuous medical education (cme) of the hospital staff. page 19 figure 1: study selection flowchart according to prisma checklist page 20 table 1: studies describing the outcomes ams impact on utilization author/country/ reference study design intervention/activity ams intervention category results (garcell et al., 2017), qatar rct education of the prescribers for judicious prescribing of antibiotics after appendectomies. educative post-ams fall of the antibiotics defined daily dosses (ddd) by 18.9% (okumura et al., 2015), brazil cct pharmacist led bundled ams program educative post-ams fall of antibiotic use by 140.2 ddd/1000 bed days (bd) (murri et al., 2018), italy cct enhanced involvement of microbiologists to onset definitive therapy quickly clinical pathway development significant (p<0.002) fall of the number of days of therapy (dot) (ruiz et al., 2018), spain rct regular review of the prescriptions by ams team and relevant feedback for prescribers prospective audit significant (p<0.015) fall of the number of ddd/100 stays (pitiriga et al., 2018), greece cct mandatory order form introduced for broadspectrum antibiotics prescribing formulary restriction significant (p<0.05) fall of the number of ddd (tang et al., 2018), united states of america (usa) cba continuous medical education of ward staff by clinical pharmacist by twice weekly ward rounds educative significant (p=0.01) fall of the number of dot (palmay et al., 2014), canada rct audit & feedback-based review of the prescriptions in icu by the ams staff. prospective audit significant (p=0.004) post-ams fall of the number of ddd by 21% (didiodato et al., 2016), canada its prescription review by ams staff for the patients with community acquired pneumonia (cap) prospective audit post-ams decline of the dot by 29%. (khdour et al., 2018), palestine cba regular prescription review by the ams team for the intensive care unit (icu) patients prospective audit significant (p<0.001) post-ams fall of the use by 21.2 ddd/100 bd (lesprit et al., 2015), france rct regular post prescription review by the infectious disease (id) physician prospective audit significant (p=0.003) post-ams fall of dot by 3 days (nilholm et al., 2015), sweden cba id specialist led twice weekly audit of antibiotic prescriptions prospective audit significant (p<0.001) post-ams fall of the number of dot (boyles et al., 2017), south africa cba review of antibiotic prescriptions during ward rounds by ams team and feedback prospective audit post-ams fall of antibiotic use by 178 ddd/1000 bd (trupka et al., 2017), usa cct de-escalation of antibiotics for non-responsive ventilator patients formulary restriction fall of the number of ddd (nonsignificant) (seah et al., 2017), singapore cct dose optimization of carbapenems after prescription review by ams team formulary restriction significant (p<0.001) post-ams fall of the number of ddd/1000 bd of carbapenems (tartof et al., 2020), usa cba targeted de-escalation of ams included antibiotics implemented by the ams team formulary restriction post-ams fall of ddd by 6.1% & dot by 4.3% (garcía-rodríguez et al., 2021), spain cba post prescription audit of carbapenem prescriptions prospective audit significant (p<0.05) fall of the number of ddd of carbapenems page 21 table 1 (cont.): studies describing the outcomes ams impact on utilization author/country/ reference study design intervention/activity ams intervention category results (dutcher et al., 2020), usa cba pharmacist led education for prescribers to encourage implementation of ams prescribing guidelines for antibiotics educative significant (p<0.001) fall of the number of ddd of co-trimoxazole (surat et al., 2021), germany cba introduction of ams guidelines for postoperative treatment with antibiotics guideline development significant (p<0.035) fall of the number of ddd/100 bd (paulson et al., 2020), usa cba introduction of time out alerts for antibiotics used for 72 hours for the icu patients formulary restriction significant (p<0.014) decline of the number of dot (shively et al., 2020), usa its post prescription review of antibiotic prescriptions by id physician prospective audit significant (p<0.001) fall of dot by 24.4% (du et al., 2020), china its pharmacist led antibiotics prescription audit prospective audit significant (p<0.01) decline of ddd (pineda et al., 2020), usa its mrsa screening ams introduction for rapid onset of definitive therapy clinical pathway development decline of use by 2.1 dot/1000 bd (faraone et al., 2020), italy its adherence to the multimodal ams guidelines for carbapenem use guideline development fall of carbapenem use by 3.6 ddd/100 bd (knight et al., 2020), usa cba post prescription review of broad-spectrum antibiotics by id physician prospective audit post-ams fall of the dot/1000 bd by 4.6% page 22 table 2: studies describing the ams impact on cost of treatment with antibiotics author/country/ reference study design intervention/activity ams intervention category results (lee et al., 2014), canada cba introduction of electronic check list for antibiotics to perform twice weekly audit prospective audit fall of cost of treatment with antibiotics by $69424 (box et al., 2015), usa its rapid diagnosis of gram +ve bacteremia clinical pathway development fall of cost of treatment with antibiotics by $7240 (cisneros et al., 2014), spain rct counselling sessions for prescribers by ams team educative fall of post-ams antibiotics treatment cost by 42% (so et al., 2018), canada cct academic detailing to treat leukemia patients in oncology unit educative significant (p=0.03) decline of cost of treatment with antibiotics (chandrasekhar & pokkavayalil, 2019), india cba continuous medical education (cme) courses by ams personnel educative significant (p<0.05) fall of cost of treatment with antibiotics by 19.5% (libertin et al., 2017), usa cba counselling sessions for prescribers by ams team as part of continuous medical education (cme) educative decline of antibiotics treatment cost by 50% with savings of $280000/year (garcía-rodríguez et al., 2019), spain cba formal guidelines issued to meropenem prescribing physicians by the id physician guideline development significant (p<0.05) reduction of post-ams cost of treatment with meropenem (seah et al., 2014), singapore its post prescription review by ams team prospective audit significant (p=0.01) post-ams fall of antibiotics treatment cost by $149/patient (cisneros et al., 2014), spain rct counselling sessions for prescribers by ams team educative post-ams fall of antibiotics cost by 42% (borde et al., 2014), germany its replacement of 3rd generation cephalosporins with penicillins and fluoroquinolones formulary restriction significant (p<0.05) reduction of treatment cost with 3rd generation cephalosporins (day et al., 2015), usa rct hiring of an id physician to check accurate susceptibility of microbes with the prescribed antibiotics prospective audit fall of cost of treatment with antibiotics by 42% (lester et al., 2020), malawi cba introduction of software for the selection of empiric therapy of antibiotics to reduce high 3rd generation cephalosporin consumption cdss post-ams savings by $15000 for antibiotics treatment cost page 23 table 3: impact of the ams on resistance patterns author/country/ reference study design intervention/activity ams intervention category results (wenisch et al., 2014), austria cba preauthorization declared compulsory for moxifloxacin supply to patients formulary restriction significant (p<0.005) decline of the incidence of clostridium difficile infection (cdi) by 46% (wang et al., 2014), taiwan its rapid onset of blood culture guided definitive therapy clinical pathway development gradual post-ams decline of infections (libertin et al., 2017), usa cba cme for prescribers by ams team educative decline of the occurrence of cdi from 3.35 to 1.35 cases/1000 bd (percival et al., 2015), usa cba cme done for prescribers dealing with the urinary tract infections (uti) educative significant (p<0.05) decline of the occurrence of amr by 5% after ams (hecker et al., 2019), usa its syndrome specific use for fluoroquinolones formulary restriction significant (p<0.05) control of pseudomonas aeruginosa infection (tedeschi et al., 2017), italy its protocol revision for prophylactic therapy with antibiotics formulary restriction significant (p<0.001) fall of pseudomonas aeruginosa infections (horikoshi et al., 2017), japan its investigation of gram-negative bacteria (gnb) resistance against carbapenems prospective audit significant (p<0.01) fall of pseudomonas aeruginosa resistance for carbapenems (lawes et al., 2015), united kingdom its health screening termed mandatory for the penicillin, cephalosporins and fluoroquinolone use formulary restriction significant (p=0.006) fall of the occurrence of methicillin resistant staphylococcus aureus (mrsa) by 50% (peragine et al., 2020), canada its regular ward rounds by ams team for audit and feedback of antibiotic prescriptions prospective audit post-ams decline of antibiotic resistant organisms by 9% (yusef et al., 2021), jordan cba preauthorization policy introduced for carbapenems formulary restriction significant (p<0.024) fall of carbapenem resistant acinetobacter baumannii infections (mardani et al., 2020), iran its regular audit and feedback of antibiotic prescriptions by ams team prospective audit significant (p<0.05) reduction of the incidence of cdi (strazzulla et al., 2020), france cba training of prescribers and nurses by ams team in urological ward educative significant (p<0.004) post-ams fall of ofloxacin resistance by 16% (al-omari et al., 2020), saudi arabia its restrictive antibiotic usage policy for carbapenems and fluoroquinolones formulary restriction significant (p<0.024) fall of cdi & significant (p=0.001) fall of ventilator associated pneumonia (vap) page 24 discussion this review mainly focused on the impact of the ams programs implemented within the hospital wards that resulted in the control of the overuse of antibiotics, the reduction of the antimicrobial cost of treatment and the control of the amr. impact of ams programs on antibiotic use the ams programs that primarily aimed to rationalize the antimicrobial use indirectly contribute to minimize various healthcare associated infections (hai) (deptuła et al., 2015). the occurrence of nosocomial infections within the in-patient populations contribute to the extra usage of broad-spectrum antibiotics which not only impart the adverse effects on patients’ health but also increase the cost of treatment (so et al., 2018). the reviewed studies describing an ams impact on the antibiotic utilization showed that ddd and dot were the main matrices that were recorded to determine the ams impact on antibiotic utilization. impact of ams on ddd was estimated in 14 studies while 10 studies described the ams impact of dot of the antibiotics. the implementation of prospective audit intervention to reduce the antibiotic utilization was the major ams intervention being implemented during the ams programs. formulary restrictive and educative interventions were the other ams interventions mainly used to curb the unjustified antibiotic utilization. it is suggested that an estimation of the impact of ams on prescribed daily doses (pdd) other than ddd and dot could outline the prescribing frequency of the antibiotics. but it is a common trend that most of the studies that focus on antibiotic utilization mainly use ddd and dot as the pdd is a more efficient matrix to evaluate the drug (antibiotic) utilization for a particular clinical condition or infection (gagliotti, et al., 2014). among the antibiotic groups carbapenems were the antibiotics that were targeted by the ams interventions in order to minimize their unjustified utilization within the hospital wards. since carbapenems are the broad-spectrum antibiotics and must be protected by the ams interventions as they have the ability to cure various deadly infections. due to this reason most of the ams programs have aimed to minimize carbapenem use for empiric therapy. impact of ams programs on antibiotic cost the high antimicrobial treatment cost is the matter of concern both for the patients and the healthcare providers (chandrasekhar & pokkavayalil, 2019). due to such concerns the health governing bodies of usa such as the cdc and shea since 2014, have recommended a mandatory ams program for every in-patient setting that deals with the antibiotics and infectious diseases (lee et al., 2014). out of the 12 studies in which the post-ams reduction of the cost of treatment with antibiotics was observed 5 studies were found to report a significant (p<0.05) fall of the cost of treatment. the educative interventions being implemented during the ams programs were found highly effective to minimize the antimicrobial cost of treatment. similar to the educative interventions the prospective audit proved an efficient intervention in controlling the cost of treatment with the antibiotics where the post-prescription audit by the id specialist and the ams team effectively improved the unjustified inclusion of the antibiotics which indirectly minimized the antimicrobial treatment cost. it is suggested that the implementation of antibiotic rotation or antibiotic cycling (replacement of broad-spectrum expensive antibiotics with cost-effective antibiotics) interventions in the hospitals could be highly lucrative pertaining to the reduction of the in-patients’ cost of treatment with antibiotics (bruno-murthaet al.,2005). impact of ams programs on antimicrobial resistance the ams interventions performed for the inpatients targeting the control of various nosocomial infections mainly depicted a fall in the incidence rate of clostridium difficile, pseudomonas aeruginosa, acinetobacter baumannii, enterobacteriaceae and mrsa infections which due to their multi-drug resistant capacity contribute to high utilization of the broad-spectrum antibiotics and sequentially a higher cost of antimicrobial treatment (thampi et al., 2019). out of the 13 studies describing the ams interventions on the control of the amr, 10 studies described a significant (p<0.05) fall of the amr and certain nosocomial infections. formulary restriction was found the most common intervention being implemented during the ams programs that retarded the incidence of amr and the nosocomial infections. the control of cdi was the main outcome being observed in majority of the ams programs designed to control the amr and nosocomial infection outbreaks. it is suggested that the hospitals that primarily deal with the infectious diseases and wide range of antimicrobials must hire an id specialist physician to monitor the ams activities targeting the infection control. as in developing countries the financial constraints hinder the availability of infectious disease physicians thus, to overcome such constraints the training of the physicians and pharmacists in the perspective of infection control could be a highly effective ams intervention aimed to minimize amr and nosocomial infections’ outbreaks. such moves would not only help the developing countries but also globally minimize the spread of amr along with the preservation of the existing antibiotics’ efficacies against various infections as the spread of antibiotic resistant microbes from one part of the world to other is much easier nowadays due to high tourism and efficient transportation (dancer, 2013). page 25 an ams program is the set of interventions that requires a coordinated support from the health governing bodies to enforce the ams guidelines and sequentially, the adherence of the prescribers (physicians and surgeons) to such policy guidelines is of utmost importance for the success of the ams (barlam et al., 2016). the id physicians, clinical pharmacists and microbiologists are the leading healthcare personnel responsible to implement the ams interventions and to monitor the degree of adherence of the antibiotic prescribers to the hospital’s ams policy (waters, 2015). most of the studies are performed over a limited period of time with brief follow up pertaining to the impact of ams programs. the successful implementation of the ams programs is highly dependent on the coordination of the various services and units of the hospital under the supervision of an ams team led by the id specialist (garcell et al., 2017). the availability of the ams experts and the monetary support ensured by the hospital directors is of prime importance as it has been observed that in lowand middle-income countries (lmic) as compared to the developed countries the commitment of hospital management in terms of fund allocation for the ams is much lower (boyles et al., 2017). therefore, a more systematic formative evaluation of the hospital’s antibiotic related problems is of prime importance before finalizing the hospital’s ams policy as the intensity of the infection and antibiotic related problems vary country wise and region wise (knight et al., 2020). such formative evaluation prior to the onset of an ams program must include the identification of the infections related problem, designing of a suitable intervention for the identified problem needing the ams, successful implementation of the ams intervention by id specialists and finally the evaluation the outcomes of the ams program is considered highly important (adhikari et al., 2018). the coordination of pharmacy department, medical wards, medical record department, information technology department (itd), microbiology department under the supervision of the id specialists being the members of the hospital’s ams team is mandatory for a successful ams program (tartof et al., 2020). the ams related data of the individual hospitals could be compiled into national database which enables the health policy makers to evaluate the ams progress in national perspective (aldeyab et al., 2012). ultimately, a data sharing among various countries could contribute to the achievement of the united nation’s goal number 3 being designed for the global healthcare that also addresses the common threat of the onset of a post antibiotic era worldwide (sadiq et al., 2018). conclusion the ams programs outlined in this review mainly implemented the prospective audit, formulary restrictive, educative, clinical pathway development and guideline development related interventions that controlled the misuse of antibiotics with the aim of the reduction of the antibiotic use related resistance and ultimately the higher cost of treatment. most of the included studies focused the in-patient settings in relation to the ams impact on antibiotic utilization, cost and infection control which invites the researchers to further explore the regionwide ams related data so as to adopt a common global ams policy that will also guide the lmic lacking the strategy and resources for the ams implementation. the findings of this study elucidate the effectiveness of ams programs in controlling the irrational use of antibiotics within the in-patient clinical settings that is considered the first step towards the infection control and to minimize the cost of treatment with antibiotics. acknowledgements this research project was funded by the fundamental research grant scheme (frgs) of the ministry of education (moe), malaysia for the research acculturation of early career researchers (racer) having the project identification number mentioned as racer19-025-0025 with the funder code of racer/1/2019/skk09/uiam/1. conflict of interest the authors declare that there is no conflict of interest. references adhikari, s., piza, 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(2023) journal of pharmacy, 3(2), 140-153 page 140 silver nanoparticle biogenically synthesised by psychotria malayana jack: physicochemical, cytotoxic and antimicrobial characterisations nur afifah mohd zulkafly1, deny susanti2, tengku karmila tengku mohd. kamil3 and muhammad taher1* abstract introduction: silver nanoparticles are targeted for antimicrobial and cytotoxic properties to combat antimicrobial resistance and chemoresistance. green synthesis of silver nanoparticle method is widely used because it is environmental-friendly using biological substances as reducing and stabilising agents. psychotria malayana jack is rich with a wide range of phytochemicals that able to synthesise silver nanoparticle. methods: the leaves of p. malayana jack was extracted with ethanol-water solvent via ultrasound assisted extraction and the extract was analysed using liquid chromatographymass spectrometry (lc-ms). the extract was then added to silver nitrate solution for 24 hours. the formation of agnps-pm was analysed using uv-visible spectrophotometry, scanning electron microscopy, zeta particle size and zeta potential analysis. the synthesised agnps-pm were tested for their cytotoxicity on human colorectal adenocarcinoma cells (caco-2) and human epithelial breast adenocarcinoma cells (mcf-7) using 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (mtt) colourimetric assay. for antibacterial activity, the nanoparticles were tested on gram-negative escherichia coli and pseudomonas aeruginosa and gram-positive bacillus subtilis and staphylococcus aureus using disc diffusion method. results: agnps-pm were successfully synthesised using p. malayana jack extract. lc-ms analysis showed the presence of flavonoids, amino acids and heterocyclic compounds . an attempt in cytotoxic activity test showed that at concentrations between 12.5 µg/ml to 400 µg/ml of agnps-pm, no cytotoxic activity was observed. whereas, in antibacterial assay, 2 mg/ml agnps-pm tested on the bacterial strains showed weak inhibition on their growth. conclusion: agnps-pm has been successfully synthesised and characterised. however, the agnps-pm possess low bioactivities of cytotoxic and antibacterial activities. article history: received: 3 july 2023 accepted: 25 july 2023 published: 31 july 2023 keywords: silver nanoparticles, p. malayana jack, flavonoids, cytotoxic, antimicrobial how to cite this article: mohd zulkafly n. a., susanti d., tengku mohd kamil t. k. & taher m. (2023). silver nanoparticle biogenically synthesised by psychotria malayana jack: physicochemical characterisations, cytotoxic and antimicrobial activities. journal of pharmacy, 3(2)., 140-153 doi: 10.31436/jop.v3i2.244 authors’ affiliation: 1 department of pharmaceutical technology, faculty of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, bandar indera mahkota, pahang, malaysia. 2 department of chemistry, faculty of science, international islamic university malaysia, jalan sultan ahmad shah, bandar indera mahkota, pahang, malaysia 3department of pharmacy practice, faculty of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, bandar indera mahkota, pahang, malaysia. *corresponding author: email address: mtaher@iium.edu.my mohd zulkafly et al. (2023) journal of pharmacy, 3(2), 140-152 page 141 introduction in the last few decades, research on nanotechnology is being widely adapted worldwide. nanoparticles refer to particles within the size range of 1 nm to 100 nm and they have many potential applications in various areas such as biotechnology, medicine, pharmaceutics, industries, biology and agriculture (song & kim, 2009; susanti, haris, taher, & khotib, 2022). in medical study, nanoparticles assist in more rapid diagnosing of diseases and more efficient disease treatments because functional molecules can be attached selectively to the metallic nanoparticles such as silver, platinum and gold, which allow the transportation of the molecules to the target site under the influence of magnetic field (ahmed et al., 2022; muhamad, ab.rahim, wan omar, & nik mohamed kamal, 2022). in other field such as nanotechnology, metallic nanoparticles are being widely utilised due to their high reactivity and high surface area to volume ration (muhamad et al., 2022; susanti et al., 2022). silver nanoparticles (agnps) are gaining attention among other metallic nanoparticles as they have remarkable biological and physicochemical properties due to their distinct surface plasmon resonance (muhamad et al., 2022). this surface plasmon resonance refers to electronic oscillations of the conduction electrons on nanoparticle’s surfaces due to the interaction with the electromagnetic radiation (eze, tola, nwabor, & jayeoye, 2019). in synthesising the metallic nanoparticles such as agnps, a few methods have been introduced: physical, chemical and biological (susanti et al., 2022). physical method renders a few disadvantages which include small number of agnps yield, a long completion period of the whole process and the utilisation of high energy which can cause a release of excessive heat to the surroundings. with chemical method, the production yield may be high, but the production of the toxic by-products is a big downturn of this process (muhamad et al., 2022). due to disadvantages presented via physical and chemical methods, the synthesis of nanoparticles using biological method such as bacteria, fungi and plant mediated synthesis is now being widely adapted (susanti et al., 2022). from the economical factor, green synthesis is cost-effective as the biological component of the biological agents which include bacteria, fungi, yeast, plant, viruses and by-products of these agents can act as the reducing agents (alarjani, huessien, rasheed, & kalaiyarasi, 2022; susanti et al., 2022). they also function as capping agents which are crucial for the stability and biocompatibility of nanoparticles (susanti et al., 2022). figure 1: the leaves and fruit bunch of p. malayana jack. the mature plant possesses dark-green leaves with a fruit bunch (figure 1). the phytochemicals presence in the leaves extract of p. malayana jack, or also known as “meroyan sakat” or “salung” in malaysia can be utilised as the reducing and stabilising agents for the synthesising process of agnps in this study. plants from genus psychotria are rich with alkaloids as their major compounds such as indole, quinoline, isoquinoline, monoterpene indole, flavonoids, cyclic peptides, terpenoids and coumarins (calixto et al., 2016). among these phytochemicals, flavonoids are well known for its role as reducing and capping agent, as a replacement for the use of toxic chemical products (ahmad et al., 2022). in this study, the leaves extract of p. malayana jack was utilised to produce agnps. other advantages of using green synthesis method include environmentally friendly as toxic chemicals are not being used with no application of high pressure, temperature, and energy. it is also stated that the use of plant mediated synthesis in nanoparticles production is simple as it can be produced in a single step at a room temperature, easy to manage commercial-scale processes and highly stable in storage (alarjani et al., 2022; susanti et al., 2022). mohd zulkafly et al. (2023) journal of pharmacy, 3(2), 140-152 page 142 studies reported that the emergence of multidrugresistant bacterial strains due to misuse of antibiotics has become a major concern to the human health all over the world (murray et al., 2022; wang, hu, & shao, 2017). a predictive statistical models stated that the deaths associated with antimicrobial resistance in the year 2019 was estimated to be 4.95 million deaths (murray et al., 2022). it was proven in many previous literatures that silver ions and silver-modified inorganic materials such as agnps exhibit multiple antimicrobial activity. this includes their ability to interfere with the bacterial dna activity and generation of free radicals which induce the reactive oxygen species leading to bacterial cell death (ahmad et al., 2022). these mechanisms are different to most antibiotics which commonly work by targeting the cell wall synthesis, translation machinery and dna replication machinery. these unique properties of agnps make them a comparable choice to antibiotic in the treatment of microbial infections as antibiotic’s resistance mechanisms are not applicable to them (ahmad et al., 2022; susanti et al., 2022). aside from that, reports state that agnps also demonstrate antitumourigenic activity on tumour models (muhamad et al., 2022). based on the research, agnps which is concentration dependent can induce apoptosis or the programmed cell death in in vitro studies. furthermore, agnps can also induce the alterations in the cell morphology, reduce cell viability and its metabolic activity and causing an increase in oxidative stress which lead to mitochondrial damage. this then leads to significant dna damage and cell death. thus, there is a potential usage of the agnps in cancer treatment (zhang, ma, gu, huang, & zhang, 2020). therefore, this study was done to investigate the antimicrobial activity and cytotoxicity of agnps-pm for their potential application in human use. methodology 1. plant sample collection and extraction fresh leaves of p. malayana jack, voucher specimen (piium 0008-1) was collected at kuantan, pahang, malaysia. the leaves were air dried at a controlled temperature drier (40 °c) for three days. the leaves were ground mechanically into fine powder using a mechanical grinder. 50g of the leaves powder were mixed with 500 ml ethanol-water (ethanol 95%, gene chemicals) (80:20) in a 500 ml erlenmeyer flask. the extraction was done via ultrasound-assisted extraction method at a temperature of 48 °c for 40 minutes using a probe sonicator (qsonica ultrasonic sonicator converter model cl-334). the leaves extract was then filtered using filter papers (nice qualitative 102) and stored in a refrigerator at 4 °c (bimakr, ganjloo, zarringhalami, & ansarian, 2017). 2. liquid chromatography mass spectrometry quadrupole of flight (lc-ms-qtof) analysis sample preparation: p. malayana jack leaves extract was dried via rotary evaporator (ika hb 10 basic) at a speed of 130 rpm and a temperature of 50°c. the dried extract was reconstituted with methanol to a final concentration of 10 mg/ml, then it was diluted to a concentration of 1 mg/ml with methanol. prior to analysis via lc-ms-qtof (6520 agilent techologies, sa, usa), the extract was filtered using a 0.22 µm pore of pvdf membrane size syringe filter. lc-ms method: the chromatographic separation was operated using agilent zorbax eclipse plus c18 rapid resolution ht (2.1 x 100 mm) 1.8 µm with (a) 0.1% formic acid in distilled water and (b) 0.1% formic acid in acetonitrile for positive mode. the gradient elution programme was 0.00 – 18.00 min, 5 – 95% (b); 18 – 23 minutes; 95% (b); 23.0 minutes; 5% (b). it was run for 30 minutes. prior to new injection, re-equilibration of lc condition was conducted for 2 minutes. the sample injection volume and the flow rate of mobile phase was set at 2 µl and 0.25 ml/min, respectively. the mass spectrometer was operated in positive electrospray ionisation (esi) mode with optimum gas temperature at 325°c, gas flow at 11 l/min and nebuliser at 35 psi. data analysis: the chromatographic profiles were analysed using agilent mass hunter qualitative analysis b.05.00 software (agilent technologies, santa clara, ca, usa) based on the accurate mass data identified and the predicted compounds were annotated using metlin database (al-abd et al., 2015). 3. preparation of silver nitrate (𝑨𝑨𝑨𝑨𝑵𝑵𝑵𝑵𝟑𝟑) solution 1 mm and 5 mm of agno3 solutions were prepared by dissolving 0.0153g and 0.0764g of agno3 powder (emsure, macedonia) into 90 ml deionised water, respectively. 4. synthesis of silver nitrate (𝑨𝑨𝑨𝑨𝑵𝑵𝑵𝑵𝟑𝟑) solution 10 ml of p. malayana jack leaves extract was added slowly into a 90 ml agno3 solution of two different concentrations (1 mm and 5 mm) under continuous stirring (100 rpm) using a magnetic stirrer to ensure the 1:9 ratio of the plant extract to agno3 solution. the synthesis process was continued for 24 hours. after 24 hours, the synthesised solution was centrifuged at 7500 rpm for 15 minutes at 4 °c to purify it (supra 22k, korea). the resulting pellet was resuspended with a small amount of deionised water and was dried in an oven at 40 °c. the resulting powder of agnps-pm was left at room temperature for future use. mohd zulkafly et al. (2023) journal of pharmacy, 3(2), 140-152 page 143 5. characterization of silver nanoparticles (agnps-pm) the characterisation of the synthesised agnps-pm was carried out by ultraviolet and visible spectrophotometer (uv-vis), scanning electron microscopy (sem), zetasizer and zeta potential analysis. uv-vis spectroscopy analysis: the green synthesised agnps-pm were sampled at 1, 2, 4, 8 and 24 hours for analysis via uv-visible double beam spectrophotometer (shimadzu uv-1800, japan). deionised water was used as a blank and reference solution. the spectrum was then recorded in the scanning range of 350 nm to 800 nm. morphological analysis: the image of the biosynthesised agnps-pm, the nanoparticles were analysed under zeiss evo-50x scanning electron microscopy (sem) instrument. then, on a non-conduction carbon tape that functions as the stabiliser, agnps-pm powder was prepared by simply sprinkling 2 mg of the powder sample on the tape on the sample holder. after being fixed, sem analysis was performed and the agnps-pm powder was analysed at room temperature. zetasizer and zeta potential analysis: the analysis sample was an aqueous solution of biosynthesised agnps-pm that was placed inside a malvern zetasizer instrument. the instrument then identified the electrical potential of ions surrounding a particle at its border as well as ions adsorbed in the diffuse layer at 25oc that was run 12 times. 6. cytotoxic assay cell culture: human colorectal adenocarcinoma cells (caco-2) and human epithelial breast adenocarcinoma cells (mcf-7) were cultured at 37℃ in a 5% co2 incubator (thermo scientific heraeus bb15) in eagle’s minimal essential medium, emem (atcc 30-2003, manassas, va) supplemented with 10% fetal bovine serum, fbs (gibco, brazil) and 1% (v/v) penicillinstreptomycin (nacalai tesque, inc., kyoto, japan). after the cells reached confluency at 80%, trypsinisation process was applied to detach and subculture the cells. the cells were then seeded into a 96-well plate at a density of 15,000 cells per well. cell viability assay: the evaluation of the cytotoxic activity of the synthesised agnps was done via 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (mtt) colourimetric assay. the seeded cells were treated with various concentrations (400 µg/ml, 200 µg/ml, 100 µg/ml, 50 µg/ml, 25 µ/ml and 12.5 µg/ml) of agnps-pm and an anticancer drug tamoxifen as positive control. the cells were incubated for 24 hours. after 24 hours, the cells were treated with 20 µg of mtt (5 mg/ml) and re-incubated for 30 minutes at 37℃. the formed crystals of formazan were dissolved using 200 µl of dimethyl sulfoxide, dmso (emplura, usa) and reincubated for another 30 minutes at 37℃. using a microplate reader, the difference in colour intensities (absorbance) was recorded at 630 nm. 7. antimicrobial assay two gram positive bacteria, bacillus subtilis and staphylococcus aureus and two gram negative bacteria, escherichia coli and pseudomonas aeruginosa were cultured in nutrient broth medium. then, they were placed in the incubator for 18 hours at 37 ± 1℃. disc diffusion assay was employed to screen for antimicrobial activity of agnps-pm. the grown microbes were sub-cultured on petri dishes and the discs were treated with 10 µl of 2 mg/ml agnps-pm, 10% p. malayana plant extract, 2 mg/ml agno3 solutions and deionised water (negative control). amoxicillin discs were used as the positive control. then, the petri dishes were put in a co2 incubator (binder) at 37oc for 24 hours. the zone of inhibition around the discs was measured in mm and was compared with the negative control. 8. statistical study statistical analysis was done via statistical package for social sciences (spss). the tests were conducted in sets of three and the data were reported as mean ± standard deviation (sd) using one-way anova test. the individual correlations were obtained via duncan’s technique. if p < 0.05, the value will be considered as significantly different (anbumani et al., 2022). results and discussion 1. lc-ms-qtof analysis of p. malayana jack leaves extract the preliminary compound identification was performed using lc-ms-qtof analysis by comparing the m/z spectra belonging to each compound to the mass spectra database of metlin (figure 2). the analysis provided that there was a total of 80 compounds detected. information on the chemical composition of the 10 major compounds from the analysis such as name, molecular formula, m/z, mass and classification of each compound were listed in table 1. among the compounds analysed were categorised as phospholipid, steroid, ketone, amino acids and flavonoids (flavans, falavanols and leucoanthocyanidin) (table 1). although, there were some unknown compounds as the data of the compounds are not available in the metlin database. the available literatures studying genus psychotria reported that among chemical compounds found were indole alkaloids, cyclic peptides or cyclotides, quinoline and isoquinoline alkaloids, flavonoids, terpenoids, coumarins and tannins (calixto et al., 2016). this is in line with the findings from lc-ms analysis of p. malayana jack (figure 2 and table 1). many literatures also reported that among the phytochemicals that were responsible for synthesis of agnps were flavonoids, amino acids, tannins, polyphenols, sterols, heterocyclic compounds, triterpenoids, terpenoids, alkaloid, etc. due their ability to mohd zulkafly et al. (2023) journal of pharmacy, 3(2), 140-152 page 144 act as the reducing, capping and stabilising agents (ahmad et al., 2022; alarjani et al., 2022; nadaf et al., 2022; patil & kim, 2017). thus, attributable to the biological activity of p. malayana jack, it can be utilised in the synthesis of agnps-pm. 2. green synthesis of silver nanoparticles, agnps-pm for the synthesis, 1 mm and 5 mm agno3 solutions were added to the plant extract and changes were observed every 1, 2, 6, 8 and 24-hours (figure 3a-3b). the changes in colour intensity when synthesising nanoparticles with 1 mm concentration were minimal, but darkest after 24 hours incubation. when the process was done with 5 mm, the colour of the solutions turned from green to dark brown after 1 hour incubation. as cited in many literatures, the formation of dark brown solution which is due to the excitation of agnps’ surface plasmon resonance confirms the successful synthesis of agnps (ahmad et al., 2022). the yield of the agnps-pm was weighed after high-speed centrifugation process. the findings provided that 1 mm yielded the lowest and 5 mm the most (figure 3c). the development of agnps-pm synthesised using 5 mm agno3 solution was further observed using other characterisation methods. 2.1 proposed mechanism for synthesis of agnps a general mechanism for the formation of agnps include three main phases which are activation phase, growth phase and termination phase (makarov et al., 2014). during the activation phase, there will be reduction of metal ions and nucleation of the reduced metal atoms. phytochemicals such as flavonoids in its keto form will convert into enol form. this will in turn release reactive hydrogen. however, due to the presence of two hydroxyl groups on the same carbon, the enol form is deemed as unstable, and thus, will convert back to its keto form. at this stage, the liberated reactive hydrogen causes the reduction of metal ions to metal atom, specifically ag+ to ag0, which then combine with each other forming small agnps. in addition to flavonoids, tannins also act as reducing agents (ahmad et al., 2022). in the growth phase, the small adjacent agnps coalesce spontaneously into larger particles. this process continues until the particles assume a stable shape and size. finally, in the termination phase, agnps will acquire the most favourable conformation due to the influence of the phytochemicals that function as stabilising agents (makarov et al., 2014). figure 2: lc-ms analysis of p. malayana jack. the sample was reconstituted with methanol to the final concentration of 10 mg/ml, then diluted to the concentration of 1 mg/ml with methanol. the sample was then filtered using a 0.22 μm pore size syringe filter before analysis. chromatographic separation was performed at 40°c using agilent zorbax eclipse plus c18 rapid resolution ht (2.1 x 100 mm) 1.8 μm with (a) 0.1% formic acid in dh20 and (b) 0.1% formic acid in acetonitrile for positive mode. mass spectrometer was operated in positive electrospray ionisation (esi) mode with optimum gas temperature at 325°c, gas flow at 11 l/min and nebuliser at 35 psi, respectively. mohd zulkafly et al. (2023) journal of pharmacy, 3(2), 140-152 page 145 table 1: 10 major compounds of p. malayana jack analysed by lc-ms-qtof analysis no compound name molecular formula m/z mass (g/mol) classification 1 lysope(0:0/18:4(6z,9z,12z,15z)) c23h40no7p 491.286 473.252 lipid (phospholipid) 2 eplerenone c24h30o6 415.2116 414.2043 lipid (steroid) 3 4-(o-carboxybenzamido) glutaramic acids c13h14n2o6 295.0926 294.085 4 n-cyclohexanecarbonyloentadecyl amine c22h43no 338.3421 337.3347 ketone 5 2-amino-3-methyl-1-butanol c5h13no 104.1069 103.0997 amino acids 6 unknown 533.3315 532.3242 7 oritin-4beta-ol c15h14o6 291.0862 290.079 flavonoids (flavans, flavanols and leucoanthocyanidin) 8 unknown 393.2862 375.2525 9 unknown 568.4265 567.4192 10 purine c5h4n4 121.0509 120.0473 heterocyclic aromatic organic compound figure 3: agnps-pm incubated at 1, 2, 6, 8 and 24hours incubation, respectively using (a) 1 mm agno3 solution (b) 5 mm agno3 solution (c) yield of agnps-pm obtained from two different molarity of agno3 mohd zulkafly et al. (2023) journal of pharmacy, 3(2), 140-152 page 146 3. characterisation of silver nanoparticles, agnps-pm 3.1 uv-visible spectrophotometer analysis the formation of the synthesised agnps-pm was confirmed via uv-visible spectroscopy analysis by measurement of surface plasmon resonance (patra & baek, 2014). uv-vis absorption spectra of p. malayana jack leaves extract showed the hypsochromic and hyperchromic shift of the uv-visible spectra of the leaves extract, and agnps-pm at different incubation time (figure 4). in this study, the collected absorption spectra within 350 nm to 800 nm showed maximum absorption at 412.0 nm (1 hr), 416.1 nm (2 hr), 422.2 nm (6 hr), 437.2 nm (8 hr) and 449.0 nm (24 hr). it was also observed that the absorption and intensity of the peak increased as incubation time increased. the broad peak in the range of 550 nm to 650 nm were attributed to the presence of flavonoids with aromatic benzene conjugated at c-2 and c-3. another broad band that was centred at 520 nm suggested a conjugated benzene with electron donating group such as nh and oh, also suggesting flavonoids (ahmad et al., 2022). the data from lc-ms-qtof analysis provided that one of the major phytoconstituents of p. malayana jack extract was flavonoids. the observations on this analysis provided that flavonoids were the major constituent of p. malayana jack that was responsible for the reduction and stabilisation of agnpspm (patil & kim, 2017). the observation on the agnpspm bands from this analysis provided that the hypochromic shift moved towards lower wavelength. these new maximum absorption peaks of agnps range from 410 nm to 450 nm represent the surface plasmon resonance of agnps-pm. . figure 4: uv-vis absorption spectra of (a) the green synthesized agnps-pm after incubation at 1, 2, 6, 8 and 24 hours (b) p. malayana jack leaves extract measured between 350-800 nm. an arrow indicated a specific band for silver nanoparticles. mohd zulkafly et al. (2023) journal of pharmacy, 3(2), 140-152 page 147 3.2. scanning electron microscopy (sem) analysis the morphological characteristics of the synthesised agnps-pm such as size and shape were directly viewed using sem via electron scanning. these characteristics were associated with the toxicity, drug and tissue targeting, drug release and the biological fate of agnpspm (patra & baek, 2014). the sem image indicated that the agnps-pm formed were mostly aggregated, which is attributed to the function of the phytochemicals of p. malayana jack leaves extract (figure 5). the formed agnps-pm were having a hexagonal cluster and the particle sizes of agnps-pm analysed ranged from 75 nm to 145 nm, which was magnified under 4000x. some factors could have an influence in the formation of the size and shape of agnps-pm such as temperature of the reaction medium, time of the synthesising reaction, exposure to light and the storage conditions (patra & baek, 2014; ye et al., 2022). aggregation of nanoparticles may occur during the storage, in which they might shrink or grow, thus affecting their potential activities. however, the information provided by sem about the size distribution and the true population average is limited (patra & baek, 2014). figure 5: sem analysis of agnps-pm. characteristic morphology of silver nanoparticles produced in this study. 3.3 zeta particle size and zeta potential analysis dynamic light scattering (dls) analysis provided information on the particle hydrodynamic size, zeta potential and polydispersity index (pdi) of agnps-pm (suriyakala et al., 2022). in this study, the average size distribution of the synthesised agnps-pm as analysed via zetasizer nanomachine was 110.1 ± 64.66 nm (table 2). particle size and their morphology are the most important parameters in determining their properties. it had been proven in many studies that nanoparticles are more efficient in delivering drugs than microparticles due to nanoparticles having larger surface areas, thus more drug interactions can be observed (patra & baek, 2014). many literatures accepted the descriptive size of nanoparticles to be between 1 nm to 100 nm (susanti et al., 2022). in pharmaceutical field, those particles having a diameter of 10 nm to 1000 nm are also regarded as nanoparticles (mazayen, ghoneim, elbatanony, basalious, & bendas, 2022). zeta potential measurement can provide predictive data on the surface charge, which affects the storage stability of the colloidal dispersion (patil & kim, 2017; suriyakala et al., 2022). the maximum zeta potential value of the formed agnps-pm (table 2) was approximately -117 ± 15.2 mv. this high negative value indicated that there were electrostatic repulsion between the synthesised agnps-pm, making them stable. the negative value also indicated that there were presence of negatively charged functional groups from the p. malayana jack leaves extract (suriyakala et al., 2022). mohd zulkafly et al. (2023) journal of pharmacy, 3(2), 140-152 page 148 in addition, zeta potential at ± 35 mv suggested a formation of stable particles (buszewski et al., 2018). previous findings also stated that in order to avoid particle aggregation and ensuring its stability, either a high positive or a high negative value of zeta potential must be achieved (patra & baek, 2014). table 2. average size distribution and zeta potential of agnps-pm analysed using zetasizer instrument. particle size analysis particle size average (d.nm) 110.1 ± 64.66 pdi 0.287 zeta potential analysis zeta potential (mv) -117 ± 15.2 zeta deviation 18.2 conductivity (ms/cm) 0.551 4. cytotoxic assay cancer is one of the most leading death-causing diseases and 1 out of 3 people has the possibility to get cancer (alyami, alyami, & almeer, 2022; zhang et al., 2020). different types of cancer therapies being offered include chemotherapy, surgery, radiation, hormonal, targeted and immunotherapy. however, the therapies are challenging due to induction of enormous side effects, which include neuropathies, alopecia and gastrointestinal and skin disorders. in addition, high rate of recurrence and multi-drug resistance against common chemotherapeutic drugs are other factors that limit the therapeutic efficacy (alyami et al., 2022; gavas, quazi, & karpiński, 2021). thus, to avoid the systemic side effects and drug resistance, many researchers are focusing on the development of nanomaterials as an alternative formulation that can specifically target tumour cells (zhang et al., 2020). cytotoxic assay was used to measure the ability of tested compounds in killing cell lines (shelembe, mahlangeni, & moodley, 2022). in this study, the cytotoxic effect of agnps-pm at different concentrations of 400 µg/ml, 200 µg/ml, 100 µg/ml, 50 µg/ml, 25 µg/ml and 12.5 µg/ml was investigated against caco-2 and mcf-7 cell lines using mtt cell viability assay. the formation of purple formazan crystals is dependent on nadph and oxidoreductase enzymes of the cancer cells. thus, the intensity of purple colour is directly proportional to the cell viability (shelembe et al., 2022). the absorbance of the dissolved formazan crystals was measured via microplate reader at 570 nm. the number of viable cells is proportional to the absorbance and percentage viability (%) was calculated using the following formula: % cell viability = asample auntreated x 100 (eq. 1) many studies from in vitro assays reported that agnps possess cytotoxic activity on several human cell lines, which include human peripheral blood mononuclear cells, human bronchial epithelial cells, red blood cells, liver cells (hepg2), human colorectal cells (caco-2) and human epithelial breast cells (mcf-7) (liao, li, & tjong, 2019; van der zande et al., 2016). in this study, the percentage viability of caco-2 and mcf7 cell lines was reduced after interaction against various concentrations of agnps-pm (figure 6). in caco-2 cells, after incubation with 12.5 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml and 400 µg/ml, the cell viability calculated was 89.2%, 84.6%, 82.2%, 88.6%, 80.18% and 81.675, respectively. however, previous study reported that at a concentration of 5 µg/ml of agnps, 50% of cell will be inhibited after exposure for 48 hours (zein, alghoraibi, soukkarieh, salman, & alahmad, 2020). in mcf-7 cells, the cell viability calculated against the increasing concentration of agnps-pm was 97.5%, 96.7%, 90.2%, 95.14%, 90.97% and 89.7%, respectively. whereas the ic50 of agnps against mcf-7 in a study conducted by fard, tafvizi, and torbati (2018) was found to be 9.85 µg/ml. mohd zulkafly et al. (2023) journal of pharmacy, 3(2), 140-152 page 149 figure 6: percentage viability of agnps-pm on caco-2 and mcf-7 against various concentrations ranging from 12.5 µg/ml to 400 µg/ml of agnps-pm however, the findings from this study showed that the cytotoxicity of agnps-pm on caco-2 cells and mcf-7 were both insignificant. it was also expected that the percentage viability of the two cell lines to decrease with increasing concentration of agnps-pm as agnps exhibit concentration-dependent cell death (alyami et al., 2022; zhang et al., 2020). however, in this study, the trend fluctuated. in addition to that, the standard errors of this assay were high, indicating that the results were not reliable, thus, the percent viability might not reflect a true value of the overall cytotoxic assay. this less reliable result might be due to variable density of cell in each well, unsuitable incubation time of cells in mtt, the wavelength at which the optical density was measured, and the type of culture media used (ghasemi, turnbull, sebastian, & kempson, 2021). in this assay, it was also observed that the percentage inhibition of agnps-pm against caco-2 cells were more prominent than mcf-7 cells. this finding might be attributed to the higher sensitivity of caco-2 cells to agnps-pm than mcf-7 cells (van der zande et al., 2016). 5. antibacterial assay many pathogenic bacteria are showing resistance to various antibiotics. to address this issue, new antibiotics are necessary (ahmad et al., 2022). agnps were proven to possess antimicrobial properties, making them suitable alternatives to antibiotics (nguyen et al., 2021). this study investigated the activity of agnpspm, agno3 solutions and p. malayana jack leaves extract on four bacterial strains via disc diffusion method. the tested bacteria were b. subtilis, e. coli, p. aeruginosa and s. aureus. in this study, the negative control discs were treated with deionised water, and the positive control discs were treated with antibiotic amoxicillin. for the sample discs, they were treated with 10% p. malayana plant extract, 2 mg/ml agnps-pm and 2 mg/ml agno3 solutions 2 mg/ml agnps-pm and 2 mg/ml agno3 solutions both showed antimicrobial activity on the microbial growth against all the tested bacterial strains (figure 7). at a same concentration of 2 mg/ml, agnps-pm exhibited good inhibition on the growth of p. aeruginosa, followed by s. aureus, b. subtilis and e. coli at 3.00 ± 0.17 mm, 2.00 ± 0.28 mm, 1.75 ± 0.14 mm and 1.25 ± 0.26 mm, respectively. the activity of 2 mg/ml agno3 solutions were almost comparable to agnpspm, but with a larger zone of inhibition than agnpspm. the zone of inhibition caused by agno3 solutions was 2.00 ± 0.35 mm, 2.50 ± 0.41 mm, 4.00 ± 0.00 mm and 3.50 ± 0.22 mm on p. aeruginosa, followed by s. aureus, b. subtilis and e. coli, respectively. this is in line with the previous publications stating that silver ions and silver-modified materials like agnps possess multiple antimicrobial activity such as induction of reactive oxygen species (ahmad et al., 2022). since the activity of agnps-pm was both greatest on p. aeruginosa and least on e. coli, it is unknown 0 20 40 60 80 100 12.5 25 50 100 200 400 pe rc en ta ge v ia bi lit y (% ) concentration of agnps (ug/ caco2 mcf7 mohd zulkafly et al. (2023) journal of pharmacy, 3(2), 140-152 page 150 whether agnps work better on gram-negative bacteria or gram-positive bacteria. in addition, the true mechanism of how agnps exhibit antimicrobial activity is still unclear (ahmad et al., 2022). the proposed mechanisms are grouped into three main actions which are induction of oxidative stress, release of metal ion and non-oxidative mechanism. these actions can either act independently or simultaneously. this can lead to denaturation of the proteins and leaking of the cell contents (goyal, verma, kharewal, gahlaut, & hooda, 2022). however, when the bacterial strains were tested against the 10% p. malayana plant extract, no zone of inhibition was seen and measured, indicating that the extract does not possess antimicrobial activity. from this assay, it can be concluded that the antimicrobial activity of agnps-pm was caused by the synthesised agnps solely (nguyen et al., 2021). there is no significant difference of the antibacterial activity between the samples (agnps-pm and agno3 solutions) and amoxicillin against b. subtilis and e. coli. however, testing on p. aeruginosa and s. aureus showed that there is a large significant difference observed between the samples (agnps-pm and agno3 solutions) and amoxicillin. . figure 7: zone of inhibition (mm) of four bacterial strains against agnps-pm, agno3, p. malayana jack extract. the test was conducted by disc diffusion method using amoxicillin disc as a positive control and deionised water as negative control. conclusion the study was conducted by synthesising agnps-pm from p. malayana leaves extract via biological route which is known as green synthesis method. the major phytochemicals of p. malayana jack as analysed by lcms-qtof, namely flavonoids, amino acids and heterocyclic aromatic organic compound which were possible for the formation of agnps-pm as they act as the reducing and stabilising agents for the process. characterisations were done on the synthesised agnpspm to study their characteristics as the nature of the agnps play an important role in their application. the investigations proposed that the formed agnps-pm were having hexagonal cluster with the size of around 75 nm to 145 nm as viewed under electron microscope. another analysis also confirmed that the size distribution of agnps-pm was 110.1 ± 66.64 nm with a zeta potential of approximately -117 ± 15.2 mv, indicating that stable agnps-pm were successfully synthesised using green synthesis method. their cytotoxicity was also determined via mtt colourimetric assay and it was found out that at concentrations of 12.5 µg/ml, 25 µg/ml, 50 µg/ml¸ 100 µg/ml, 200 µg/ml and 400 µg/ml of agnps-pm, the cell viability of both caco-2 and mcf-7 cell lines were over 80%. this finding indicated that the percentage inhibition was low and insignificant. however, in this study, a few confounding factors may have an effect on the result, thus, making it unreliable. agnps-pm were also studied for antimicrobial activity against b. subtilis, e. coli, p. aeruginosa and s. aureus using disc diffusion method, and 0 2 4 6 8 10 12 14 b. subtilis e. coli p. aeruginosa s. aureus zo ne o f i nh ib iti on (m m ) bacterial strains agnps-pm agno3 solution p. malayana jack extract amoxicillin deionised water mohd zulkafly et al. (2023) journal of pharmacy, 3(2), 140-152 page 151 it was observed that agnps-pm inhibited the growth of the four bacterial strains, with the highest inhibition on p. aeruginosa and the lowest inhibited was e. coli. thus, the biogenic synthesis of agnps-pm might be a promising process for the production of other metallic nanoparticles which could have potential applications in various fields. however, the improvement on this study for cytotoxic and antimicrobial assays can be made by adjusting the size and formulation of nanoparticles. author contributions mt and ds design the study. mt, ds, tk supervise the works. na conduct the research and collect the data. na wrote the manuscript. mt review the manuscript. all authors have read the manuscript. acknowledgements the research was supported by department of pharmaceutical technology, kulliyyah of pharmacy, international islamic university malaysia. conflict of interest the authors declare that there is no conflict of interest in the writing of this manuscript. references ahmad, n., fozia, jabeen, m., haq, z. u., ahmad, i., wahab, a., … khan, m. y. 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(2020, october 29). green synthesis of metallic nanoparticles and their potential applications to treat cancer. frontiers in chemistry, vol. 8. frontiers media s.a. https://doi.org/10.3389/fchem.2020.00799 introduction in the last few decades, research on nanotechnology is being widely adapted worldwide. nanoparticles refer to particles within the size range of 1 nm to 100 nm and they have many potential applications in various areas such as biotechnology, medicine,... silver nanoparticles (agnps) are gaining attention among other metallic nanoparticles as they have remarkable biological and physicochemical properties due to their distinct surface plasmon resonance (muhamad et al., 2022). this surface plasmon resona... due to disadvantages presented via physical and chemical methods, the synthesis of nanoparticles using biological method such as bacteria, fungi and plant mediated synthesis is now being widely adapted (susanti et al., 2022). from the economical facto... figure 1: the leaves and fruit bunch of p. malayana jack. the mature plant possesses dark-green leaves with a fruit bunch (figure 1). the phytochemicals presence in the leaves extract of p. malayana jack, or also known as “meroyan sakat” or “salung” in malaysia can be utilised as the reducing and stabilising... studies reported that the emergence of multidrug-resistant bacterial strains due to misuse of antibiotics has become a major concern to the human health all over the world (murray et al., 2022; wang, hu, & shao, 2017). a predictive statistical models ... aside from that, reports state that agnps also demonstrate antitumourigenic activity on tumour models (muhamad et al., 2022). based on the research, agnps which is concentration dependent can induce apoptosis or the programmed cell death in in vitro s... methodology 1. plant sample collection and extraction fresh leaves of p. malayana jack, voucher specimen (piium 0008-1) was collected at kuantan, pahang, malaysia. the leaves were air dried at a controlled temperature drier (40 c) for three days. the leaves were ground mechanically into fine powder usi... 2. liquid chromatography mass spectrometry quadrupole of flight (lc-ms-qtof) analysis sample preparation: p. malayana jack leaves extract was dried via rotary evaporator (ika hb 10 basic) at a speed of 130 rpm and a temperature of 50 c. the dried extract was reconstituted with methanol to a final concentration of 10 mg/ml, then it was ... 3. preparation of silver nitrate (𝑨𝒈,𝑵𝑶-𝟑.) solution 1 mm and 5 mm of ag,no-3. solutions were prepared by dissolving 0.0153g and 0.0764g of ag,no-3. powder (emsure, macedonia) into 90 ml deionised water, respectively. 4. synthesis of silver nitrate (𝑨𝒈,𝑵𝑶-𝟑.) solution 10 ml of p. malayana jack leaves extract was added slowly into a 90 ml ag,no-3. solution of two different concentrations (1 mm and 5 mm) under continuous stirring (100 rpm) using a magnetic stirrer to ensure the 1:9 ratio of the plant extract to ag,no... 5. characterization of silver nanoparticles (agnps-pm) the characterisation of the synthesised agnps-pm was carried out by ultraviolet and visible spectrophotometer (uv-vis), scanning electron microscopy (sem), zetasizer and zeta potential analysis. uv-vis spectroscopy analysis: the green synthesised agnps-pm were sampled at 1, 2, 4, 8 and 24 hours for analysis via uv-visible double beam spectrophotometer (shimadzu uv-1800, japan). deionised water was used as a blank and reference solution. the s... morphological analysis: the image of the biosynthesised agnps-pm, the nanoparticles were analysed under zeiss evo-50x scanning electron microscopy (sem) instrument. then, on a non-conduction carbon tape that functions as the stabiliser, agnps-pm powde... zetasizer and zeta potential analysis: the analysis sample was an aqueous solution of biosynthesised agnps-pm that was placed inside a malvern zetasizer instrument. the instrument then identified the electrical potential of ions surrounding a particle... 6. cytotoxic assay cell culture: human colorectal adenocarcinoma cells (caco-2) and human epithelial breast adenocarcinoma cells (mcf-7) were cultured at 37℃ in a 5% ,co-2. incubator (thermo scientific heraeus bb15) in eagle’s minimal essential medium, emem (atcc 30-200... 7. antimicrobial assay two gram positive bacteria, bacillus subtilis and staphylococcus aureus and two gram negative bacteria, escherichia coli and pseudomonas aeruginosa were cultured in nutrient broth medium. then, they were placed in the incubator for 18 hours at 37 ± 1℃... 8. statistical study statistical analysis was done via statistical package for social sciences (spss). the tests were conducted in sets of three and the data were reported as mean ± standard deviation (sd) using one-way anova test. the individual correlations were obtaine... results and discussion 1. lc-ms-qtof analysis of p. malayana jack leaves extract the preliminary compound identification was performed using lc-ms-qtof analysis by comparing the m/z spectra belonging to each compound to the mass spectra database of metlin (figure 2). the analysis provided that there was a total of 80 compounds det... the available literatures studying genus psychotria reported that among chemical compounds found were indole alkaloids, cyclic peptides or cyclotides, quinoline and isoquinoline alkaloids, flavonoids, terpenoids, coumarins and tannins (calixto et al.,... 2. green synthesis of silver nanoparticles, agnps-pm for the synthesis, 1 mm and 5 mm ag,no-3. solutions were added to the plant extract and changes were observed every 1, 2, 6, 8 and 24-hours (figure 3a-3b). the changes in colour intensity when synthesising nanoparticles with 1 mm concentration were mi... 2.1 proposed mechanism for synthesis of agnps a general mechanism for the formation of agnps include three main phases which are activation phase, growth phase and termination phase (makarov et al., 2014). during the activation phase, there will be reduction of metal ions and nucleation of the re... figure 2: lc-ms analysis of p. malayana jack. the sample was reconstituted with methanol to the final concentration of 10 mg/ml, then diluted to the concentration of 1 mg/ml with methanol. the sample was then filtered using a 0.22 μm pore size syringe... table 1: 10 major compounds of p. malayana jack analysed by lc-ms-qtof analysis figure 3: agnps-pm incubated at 1, 2, 6, 8 and 24hours incubation, respectively using (a) 1 mm agno3 solution (b) 5 mm agno3 solution (c) yield of agnps-pm obtained from two different molarity of agno3 3. characterisation of silver nanoparticles, agnps-pm 3.1 uv-visible spectrophotometer analysis the formation of the synthesised agnps-pm was confirmed via uv-visible spectroscopy analysis by measurement of surface plasmon resonance (patra & baek, 2014). uv-vis absorption spectra of p. malayana jack leaves extract showed the hypsochromic and hyp... . figure 4: uv-vis absorption spectra of (a) the green synthesized agnps-pm after incubation at 1, 2, 6, 8 and 24 hours (b) p. malayana jack leaves extract measured between 350-800 nm. an arrow indicated a specific band for silver nanoparticles. 3.2. scanning electron microscopy (sem) analysis the morphological characteristics of the synthesised agnps-pm such as size and shape were directly viewed using sem via electron scanning. these characteristics were associated with the toxicity, drug and tissue targeting, drug release and the biologi... 3.3 zeta particle size and zeta potential analysis dynamic light scattering (dls) analysis provided information on the particle hydrodynamic size, zeta potential and polydispersity index (pdi) of agnps-pm (suriyakala et al., 2022). in this study, the average size distribution of the synthesised agnps-... zeta potential measurement can provide predictive data on the surface charge, which affects the storage stability of the colloidal dispersion (patil & kim, 2017; suriyakala et al., 2022). the maximum zeta potential value of the formed agnps-pm (table ... table 2. average size distribution and zeta potential of agnps-pm analysed using zetasizer instrument. many studies from in vitro assays reported that agnps possess cytotoxic activity on several human cell lines, which include human peripheral blood mononuclear cells, human bronchial epithelial cells, red blood cells, liver cells (hepg2), human colorec... however, the findings from this study showed that the cytotoxicity of agnps-pm on caco-2 cells and mcf-7 were both insignificant. it was also expected that the percentage viability of the two cell lines to decrease with increasing concentration of agn... 5. antibacterial assay many pathogenic bacteria are showing resistance to various antibiotics. to address this issue, new antibiotics are necessary (ahmad et al., 2022). agnps were proven to possess antimicrobial properties, making them suitable alternatives to antibiotics ... 2 mg/ml agnps-pm and 2 mg/ml ag,no-3. solutions both showed antimicrobial activity on the microbial growth against all the tested bacterial strains (figure 7). at a same concentration of 2 mg/ml, agnps-pm exhibited good inhibition on the growth of p. ... since the activity of agnps-pm was both greatest on p. aeruginosa and least on e. coli, it is unknown whether agnps work better on gram-negative bacteria or gram-positive bacteria. in addition, the true mechanism of how agnps exhibit antimicrobial act... . figure 7: zone of inhibition (mm) of four bacterial strains against agnps-pm, agno3, p. malayana jack extract. the test was conducted by disc diffusion method using amoxicillin disc as a positive control and deionised water as negative control. conclusion the study was conducted by synthesising agnps-pm from p. malayana leaves extract via biological route which is known as green synthesis method. the major phytochemicals of p. malayana jack as analysed by lc-ms-qtof, namely flavonoids, amino acids and ... author contributions mt and ds design the study. mt, ds, tk supervise the works. na conduct the research and collect the data. na wrote the manuscript. mt review the manuscript. all authors have read the manuscript. acknowledgements the research was supported by department of pharmaceutical technology, kulliyyah of pharmacy, international islamic university malaysia. conflict of interest the authors declare that there is no conflict of interest in the writing of this manuscript. references barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework taher et al. (2021) journal of pharmacy, 1(1), 34-44. page 34 potential nanospray inhalation of remdesivir and hydroxychloroquine using poly (lactic-co-glycolic) acid as fast delivery for covid-19 treatment. muhammad taher1*, siti syazwani shaari1 and deny susanti2 abstract introduction: the oral medication of remdesivir and hydroxychloroquine face several limitations in covid-19 therapy. despite having the first-pass metabolism, it also has a limitation in the patient who has hospitalised with a severe covid-19 infection. it is especially for a drug that is targeting the angiotensin-converting enzyme ii (ace2) receptor where the receptors are found abundantly in the lung, kidney, heart, and gastrointestinal tract. therefore, an alternative delivery such as nanospray inhalation would provide a great benefit to those patients. methods: scientific sources from scopus, pubmed, google scholar, ebsco, sciencedirect, and elsevier were accessed for publication of this review article regarding the nanospray inhalation for covid-19. results: since the main organ infected by sars-cov-2 is the trachea and lung, inhalation may be the best route to deliver the drug to the site of action. it is proposed that poly (lactic-co-glycolic) acid to be used in the formulation. conclusion: poly (lactic-co-glycolic) acid (plga) is considered a suitable polymer since it is biocompatible and noncytotoxic, it is the most widely applied in drug delivery either as carrier or excipient for the optimal formulation and distribution of the drugs. dry powder inhalation of remdesivir and hydroxychloroquine may be an alternative way to deliver the drug against covid-19. article history: received: 30 august 2020 accepted: 17 december 2020 published: 8 january 2021 keywords: covid-19, nanospray, hydroxychloroquine, remdesivir, inhalation, antiviral. how to cite this article: taher, m., shaari, s. s. & susanti, d. (2021). potential nanospray inhalation of remdesivir and hydroxychloroquine using poly (lactic-co-glycolic) acid as fast delivery for covid19 treatment. journal of pharmacy, 1(1), 34-44. authors’ affiliation: 1 department of pharmaceutical technology, kulliyyah of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia. 2 department of chemistry, kulliyyah of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, 25200 kuantan, pahang, malaysia. *corresponding author: email address: mtaher@iium.edu.my tel:+609 570 4842 fax: +609 571 6775 tel:+609 taher et al. (2021) journal of pharmacy, 1(1), 34-44. page 35 introduction in december 2019, the world has been threatened with a recent coronavirus outbreak caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which is believed to be originated from a seafood market in hubei province, wuhan in central china (al-tawfiq et al., 2020; morse et al., 2020; she et al., 2020). however, the seafood market is not the only source of the new coronavirus as 13 out of 41 cases in china were not linked to the market (she et al., 2020). this new coronavirus has caused the pandemic of pneumonia in humans (she et al., 2020; wang et al., 2020) and causes severe respiratory failure requiring icu admission (bouadma et al., 2020). the disease, known as coronavirus disease 2019 (covid19), had been declared by the world health organization (who) as a pandemic in just 45 days from the onset of the new coronavirus emergence, indicating significant public health issues around the world (panati & narala, 2020). the virus not only affected global public health but also local economies (liu et al., 2020). on january 12, 2020, the world health organization (who) had named the new coronavirus as the 2019-novel coronavirus (2019-ncov). disease caused by this new coronavirus was named by the who as covid-19 on 11 february 2020, the same day of the coronavirus study group (csg) of the international committee proposed to name the new coronavirus as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (guo et al., 2020). as of december 31, 2019, 104 cases were diagnosed and the covid-19 infection was extended to all the 34 districts of china including macau, hong kong, and taiwan in just 30 days. by february, the confirmed case increased rapidly to 2,175,567 with 2239 deaths reported in china, and 1151 cases were confirmed with 8 losses reported in 26 countries ( zhou et al., 2020a). recently, the death rate of 2019-ncov worldwide is 6.93% with 265,084 deaths reported (covid-19 visualiser) compared to 9.6 % of sars-cov and 34.4% of middle east respiratory syndrome coronavirus (mers) (she et al., 2020). as of may 12, 2020, the number of deaths worldwide is 303, 371 (covidvisualizer.com). it is estimated that the basic reproductive figure for covid-19 infection lies between 2.2 and 4.8 (bouadma et al., 2020; g. zhou et al., 2020). thus it is believed that the increase in the widespread of sars-cov-2 infection is due to genetic variation and regular recombinant of the viral genomes as well as increased human and animal activities (she et al., 2020). generally, coronaviruses (members of the family coronaviridae) are rna viruses with features of enveloped, single-stranded and positive-sense, (guo et al., 2020; she et al., 2020), which able to spread to birds and all mammals including humans, thus affecting the body systems such as respiratory, enteric, hepatic, and neurologic (she et al., 2020). it was reported that the genome sequence of sars-cov-2, sars-cov, merscov is 96.2%, 75-80%, and 40% similar to the betacoronaviruses detected in bats respectively (guo et al., 2020; she et al., 2020; wang et al., 2020). the sars-cov and mers-cov are both fatal coronaviruses that occurred in different areas in 2002 and 2012 respectively (she et al., 2020). meanwhile, sars-cov-2 shares 82% similarity of the sequence identity with the sars-cov (morse et al., 2020; wang et al., 2020). because of the comparable genomic sequence, the bat was allegedly to be the natural host of the virus originated in which the virus is spread by an unknown intermediate host from bats to humans (guo et al., 2020). in the early of the pandemic, remdesivir and hydroxychloroquine have been suggested as possible prevention or treatment of covid-19 based on evidence of in vitro inhibition of sar-cov-2. the current available formulation of remdesivir and hydroxychloroquine are not suitable for critically infected patients, therefore, we would like to provide a brief insight regarding an alternative preparation for critically ill patients. methods information on the nanospray inhalation for covid-19 was obtained via a literature search conducted for publications using various electronic databases, such as pubmed, google scholar, international islamic university malaysia iium ebsco, sciencedirect, and elsevier. the publications selected for this review is ranging from 2000 to 2020 to make sure the information gathered is validated and updated. the keyword ‘sars-cov-2’ and ‘covid-19’ were used as primary searches, while terms such as nanospray, inhalation, and antiviral were applied as secondary searches. as there was a limited source on invivo and in-vitro studies related to nanospray inhalation for covid-19 at the time of writing this article, the only scientific theory regarding nanospray inhalation from the selected literature search was included to highlight the therapeutic use of nanospray inhalation for covid-19. literature search on potential medications to be delivered as nanospray inhalation such as remdesivir and hydroxychloroquine were also included. therefore, this review article is not exhaustive to medications used for covid-19 that cannot be delivered through nanospray inhalation technology. transmission and clinical presentation as mentioned before, covid-19 is a disease that is spread from an unidentified animal source to a human and it is transmissible from one person to another (bouadma et al., 2020). sars-cov-2 is transmitted primarily through respiratory droplets generated after coughing, sneezing, and speaking (itani et al., 2020) as well as through direct contact with an infected person for a low infective dose taher et al. (2021) journal of pharmacy, 1(1), 34-44. page 36 (guo et al., 2020). the symptomatic or asymptomatic infected person may transmit the sars-cov-2 to a healthy person who is in close contact, or when he or she touches an infected surface and subsequently touches his or her face especially at the area of eyes, nose, or mouth (itani et al., 2020). family members, such as relatives and friends having in direct contact with patients or incubation carriers, are at high risk of infection transmission (guo et al., 2020). although the sars-cov-2 droplets can move less than 2 meters and do not stay in the air, a study found that the sars-cov-2 can remain sustainable in the aerosols for up to 3 hours, and on copper, cardboard as well as on plastic or stainless steel for 4 hours, 24 hours and, 2 to 3 days respectively (itani et al., 2020). the incubation period is mainly between 3 to 7 days (within 14 days) (guo et al., 2020; itani et al., 2020; she et al., 2020) and it is contagious during the latency period (guo et al., 2020). there are no specific symptoms but the most frequent ones are an increase in body temperature, cough, exhaustion, sputum production, breathlessness, sore throat, and headache (guo et al., 2020; she et al., 2020). some patients may experience gastrointestinal symptoms (diarrhea and vomiting) (guo et al., 2020). if the disease is uncontrolled, it can develop severe complications including acute respiratory distress syndrome (asds), septic shock, multiple organs failure, or even death (guo et al., 2020; itani et al., 2020; she et al., 2020). the most susceptible people to severe diseases are older patients (>65 years old) and people with comorbidities such as hypertension, chronic obstructive pulmonary disease, diabetes, and cardiovascular disease (guo et al., 2020; itani et al., 2020). site of action it was found that the sars-cov-2 multiplies better in epithelial cells of the primary human airway than in normal tissue-culture cells as compared to the sars-cov and mers-cov (she et al., 2020) and it utilizes angiotensin-converting enzyme2 (ace-2) receptor (figure 1), just like sars-cov, to infect human being (guo et al., 2020; she et al., 2020; g. zhou et al., 2020). ace2 can be found in type ii alveolar cells of the lung and other epithelial cells located at the heart, kidney, ileum, esophagus, and bladder (zhou et al., 2020). the receptors are abundant in lung alveolar epithelial cells and enterocytes of small intestines (guo et al., 2020). the coronaviruses use their spike protein to attach to a host cell receptor for entry (morse et al., 2020). structural analysis showed that the spike of sars-cov-2 glycoprotein has a higher binding affinity towards the ace-2 receptor (morse et al., 2020; g. zhou et al., 2020). the s-glycoprotein of the sars-cov-2 has 2 subunits; s1 and s2. s1 is responsible for evaluating the number of virus-hosts and cell tropism with receptor-binding domain (rbd), while s2 is responsible for the fusion of virus into cell membrane through a pair of domains, heptad repeats 1 (hr1) and (hr2) (guo et al., 2020). besides that, the sars-cov-2 also requires two proteins which are coronavirus main proteinase (3clpro) and the papain-like protease (plpro) to mediate the formation of new virions through the proteolysis process and one protein which is replicate or rna-dependent rna polymerase (rdrp) to replicate once it enters the human epithelial cells (morse et al., 2020). figure 1. typical binding of sars-cov-2 to ace2 receptor. ace2 receptor is an entry point by the virus to propogate inside the human cells. treatment approaches currently, there is no definite treatment or vaccine available in the market to kill the sars-cov-2 (bouadma et al., 2020; panati & narala, 2020; she et al., 2020; wang et al., 2020). in fact, in clinical practices, supportive therapy, oxygen therapy, or mechanical ventilation are used to prevent complications or reduce symptoms of the covid-19 disease associated with respiratory disorders (she et al., 2020). it was reported that the sars-cov-2 invades the human body through its entry via ace-2 receptors which are expressed on the epithelial cells of the lung, intestines, kidney, and blood vessels (panati & narala, 2020). targeting directly the ace-2 receptors using ace inhibitors or angiotensin-receptor antagonists had led to high morbidity in a patient with diabetes and hypertension as the drugs promote overexpression of the ace-2 receptors, making them more favorable situation for the virus to invade the body system (gaurav & ramarao, 2020; panati & narala, 2020). due to that, several drugs and treatment options were undergoing clinical trials and being tested for their efficacy against covid 9 disease. among them, remdesivir (an experimental drug) and hydroxychloroquine are the most frequent drugs that have been tested extensively (gaurav taher et al. (2021) journal of pharmacy, 1(1), 34-44. page 37 & ramarao, 2020) due to their efficient action in impeding sars-cov-2 infection in-vitro (morse et al., 2020). remdesivir, a novel antiviral drug, and chloroquine, an anti-malarial drug, were found to be effective in preventing the current novel coronavirus at an effective concentration (ec) of 1.1 µm (colson et al., 2020). remdesivir is a prodrug and an analogue of a nucleoside. it was produced by gilead sciences located in the united state of america (usa) (liu et al., 2020). in the earliest case report, the first covid-19 patient from the usa had shown an improvement in his clinical condition after being treated with intravenous remdesivir (guo et al., 2020; holshue et al., 2020; liu et al., 2020; she et al., 2020). to block the covid-19 infection, remdesivir may inhibit the viral receptor-binding domain (rbd)-ace-2 interaction by using peptides and their cocktails derive from rbd and ace-2 (morse et al., 2020). meanwhile, chloroquine was commonly used to treat malarial infection and autoimmune disease, but nowadays, it has been reported that this drug has a wide-spectrum activity against viral infection. it can prevent virus infection by raising the endosomal ph necessary for virus and cell fusion, as well as obstructing the glycosylation of cellular ace-2 receptors by sars-cov (wang et al., 2020). since ace-2 receptors are abundant in lung epithelial cells and its associated with respiratory problems among infected patients with sars-cov-2, this article review focuses to target directly the lungs through localised therapy by using nanospray inhalation. this formulation can produce an ideal particle size of a drug (about 1-5 µm) which can efficiently reach the deep lung, allowing its penetration and deposition in the alveolar (arpagaus et al., 2018; karathanasis et al., 2005). this formulation is also able to avoid the first-pass metabolism and allow for more rapid onset of therapeutic action (bartolucci, 2017). thus, the lung which is infected by sars-cov-2 would be more reachable with pulmonary delivery compared to oral and intravenous injection. besides that, poly(lactic-co-glycolic acid) (plga) will be used as a polymer since it is biocompatible, noncytotoxic, and widely used as carrier or excipient in drug delivery to achieve the best formulation and delivery of the drugs (arpagaus, 2019a). properties and clinical trials of remdesivir and hydroxychloroquine remdesivir remdesivir (gs-5734), is an investigational nucleoside analog prodrug that has not yet approved or licensed anywhere including by the food and drug administration (fda) of the united states (us) (altawfiq et al., 2020; guo et al., 2020; ko et al., 2020). it is a small molecule with a molecular weight of 602.6 g/mol and its chemical formula is c27h35n6o8p (ko et al., 2020). it was manufactured by gilead sciences in 2017 to treat the ebola virus that was occurred in 2016 (al-tawfiq et al., 2020; eastman et al., 2020; gordon et al., 2020). besides, it was reported that remdesivir can treat various rna viruses including coronaviruses like sars-cov and mers-cov which were discovered in 2017. currently, it has been investigated as one of the promising medications for sars-cov-2 infection (eastman et al., 2020). this drug demonstrated wide antiviral activities such as inhibit human and zoonotic coronavirus (including sars-cov-2 and ebola virus) in vitro and produce preventive and therapeutic effects in animal models of mers-cov and sars-cov infections ( zhou et al., 2020). inside the cell, the prodrug remdesivir (gs-5734) is metabolised into adenosine nucleotide analogue (gs441524). since the nucleotide analogue, gs-441524 is not highly cell-permeable, it requires diand triphosphorylation to generate active nucleoside triphosphate (ntp) (eastman et al., 2020; ko et al., 2020; sheahan et al., 2017). this is because the initial phosphorylation of nucleotide analogue into monophosphate form, is quite polar, so it could not diffuse back through the cell membrane and being trapped in the cells (eastman et al., 2020). the ntp then will be misintegrated into the rna viral via the viral rna-dependent rna polymerases (rdrp) during genome replication thus suppressing viral rna synthesis (eastman et al., 2020; gordon et al., 2020; ko et al., 2020). the ntp resembles adenosine triphosphate (atp) (gordon et al., 2020). the ntp confuses the viral rdrp by working as an incorporation competitor with adenosine triphosphate (atp) to enter into the nascent chain of viral rna and results in immature cessation (wang et al., 2020). a case report associated with the earlier confirmed case of covid-19 in the united states (us) has proven the efficacy of remdesivir to combat sars-cov-2. intravenous remdesivir was given on the 7th day to an infected covid-19 patient following reports on the development of severe pneumonia. surprisingly, the patient’s medical status showed a positive outcome on the following day with no observed adverse effects. this finding has encouraged other researchers to conduct several clinical studies for the use of remdesivir in dealing with covid-19 disease (holshue et al., 2020). on the other hand, three out of twelve positive covid-19 patients had been given with remdesivir treatment for 4-10 days, in which 200 mg iv was administered on the first day, and 100 mg for each subsequent day. it was noted that all the treated patients encountered “transient gastrointestinal symptoms, such as nausea, throwing up, gastroparesis, or rectal bleeding” after the initial dose. however, the therapy proceeded until there was an amelioration in the respiratory symptoms. nevertheless, this finding is omitted from clinical effectiveness or health review due to the limited number of sample study involvement and lack of controlled randomisation (eastman et al., 2020). taher et al. (2021) journal of pharmacy, 1(1), 34-44. page 38 in china, a phase 3 randomized, quadruple-blind, placebo-controlled clinical trial was enrolled at capital medical university, to determine the safety and efficacy of remdesivir in 308 hospitalised and infected adult patients with manifestations of mild to moderate symptoms of covid-19 infection (nct04252664). on the next day, another phase 3 clinical trial (nct04257656) was enrolled at the same university to measure the efficacy and safety of remdesivir in 452 hospitalised patients with severe sars-cov-2 respiratory disease. all the participants involved in both trials were randomly administered with either placebo or remdesivir 200 mg loading dose on the 1st day followed by 100 mg intravenous once-daily as maintenance doses for the following 9 days. after 28 days of both treatments, the patients’ conditions such as fever, oxygen saturation, and respiratory rate became normal and there was also alleviation of cough for about 72 hours (altawfiq et al., 2020; eastman et al., 2020). to further evaluate the safety and efficacy of remdesivir, several phases 3 clinical studies were launched on infected covid-19 patients around the world. among the studies are nct04292899, nct04292730, nct04280705, nct04323761, isrctn83971151, nct04315948, 2020-001052-18, nct04321616, nct04314817, nct04302766 (eastman et al., 2020) and nct04252664 (itani et al., 2020). however, since remdesivir is an experimental drug, it would be insufficient to be used for dealing with a huge number of patients promptly. thus, another choice of potential drugs for large-scale use is chloroquine as it is largely available with proven safety record as well as relatively low cost (liu et al., 2020). chloroquine (cq) and hydroxychloroquine (hcq) chloroquine (4-aminoquinoline) is known as a drug used to treat malarial infection and autoimmune disease, and now, has been reported as a possible antiviral drug with broad-spectrum activity (o’neill et al., 1998; wang et al., 2020). it was synthesised by hans andersag, in 1934, under german chemical and pharmaceutical company bayer ag (thomé et al., 2013). it is a safe and cheap drug that has been used for more than 70 years (gao et al., 2020; wang et al., 2020). cq with the chemical structure c18h26cin3 is a small molecule and has a molecular weight of 319.872 g/mol (o’neill et al., 1998). in 1946, the first derivate of cq, hydroxychloroquine (hcq) sulfate was synthesised by inserting a hydroxyl group into chloroquine and exhibited lesser (~40%) toxic than chloroquine in animals (liu et al., 2020; o’neill et al., 1998). both drugs, cq, and hcq are widely available as anti-inflammatory agents for treating rheumatoid arthritis and lupus erythematosus (gao et al., 2020; liu et al., 2020; moore, 2020). these drugs are weak base drugs with similar chemical structures (liu et al., 2020) and mechanisms of action as antiviral, anti-inflammatory, and immunomodulator. thus, chloroquine and hydroxychloroquine may effective in treating patients with covid-19 pneumonia (gao et al., 2020; wang et al., 2020). both cq and hcq interfere with the fusion of sarscov with the ace-2 receptors on cells via de-acidification of lysosomes, thereby preventing cathepsins that require acidic conditions for optimal sars-cov spike protein cleavage (gao et al., 2020; liu et al., 2020; singh et al., 2020). originally, chloroquine (cq) is lipophilic and in unprotonated form. it diffuses passively through cell membranes and into endosomes, lysosomes, and golgi vesicles; where it becomes di-protonated (doubly positively charged), trapping the chloroquine in the organelle and elevating the surrounding ph. the resultant increased in the ph value of the endosomes prevents viral replication including fusion and uncoating (colson et al., 2020; thomé et al., 2013; warhurst et al., 2003). cq and hcq do not affect the level of ace-2 expression on cell surfaces, but instead, they prevent the viral entry by inhibiting terminal glycosylation of ace-2 receptor (target receptor for sars-cov and sars-cov-2 to enter the body), making it less efficient to interact with the sars-cov-2 spike protein (thomé et al., 2013). a group of researchers from china had previously conducted an in-vitro study to compare the antiviral effect of hcq and cq on veroe6 cells infected with sarscov-2 infection. the outcome showed that both drugs can efficiently inhibit covid-19 infection (by reducing viral replication) with conventional dosing because of its high distribution in tissues as well as in the lung. they inhibited the entry, transport, and post-entry of sars-cov-2 into the cells. it was also reported that hcq is less potent and less toxic than cq in combating sars-cov-2 (liu et al., 2020; singh et al., 2020). this is because the hydroxyl group in the hcq that makes it less permeable to the blood-retinal barrier thus lessen the risk of retinal toxicity (singh et al., 2020). however, hcq has a low selectivity index (si) as compared to cq thus, careful designing and safe management of the sars-cov-2 infection is required (liu et al., 2020). besides, in vivo evaluation using infected sars-cov2 veroe6 cells had been conducted to assess the effects of seven potential drugs including remdesivir and cq on the cytotoxicity, virus production, and the rate of virus infection. at low-micromolar concentration, both remdesivir (ec50: 0.77 µm, ec90: 1.76 µm) and cq (ec50: 1.13 µm, ec90: 6.9 µm) effectively hindered the sarscov-2 infection. it was known that remdesivir blocked at post-stage virus entry meanwhile chloroquine blocked both entry and following-entry of the sars-cov-2 infection in the veroe6 cells. the antiviral activity of cq is also enhanced with the synergy effect of its immunemodulating activity (wang et al., 2020). interestingly, in taher et al. (2021) journal of pharmacy, 1(1), 34-44. page 39 another study, it has been reported that the serum level of hcq sulfate in humans is between 1.4-1.5 µm when given at doses of 6-6.5 mg/kg/day. this suggests that high distribution and concentration of hcq sulfate in the liver, spleen, kidney, and lung could be achieved to inhibit sars-cov-2 infection (liu et al., 2020). to evaluate the efficacy and safety of cq and hcq, several ongoing clinical trials were currently conducted. the clinical trials can be tracked by the following numbers: nct04303299, nct04303507, nct04304053, nct04304053, nct04286503, nct04307693, nct04261517, and nct04308668 (singh et al., 2020). nanospray inhalation the coronaviruses use their spike protein on its surface to recognise and bind to the angiotensin-converting enzyme (ace)-2 receptor (abundant in the lung epithelial cells) of the host cell causing respiratory tract infection (yang & wang, 2020). this gives the idea that local delivery of inhaled drugs which is usually used to treat lung diseases such as asthma, chronic obstructive pulmonary disease (copd), and cystic fibrosis (ungaro et al., 2012) may be necessary to treat sars-cov-2 infection. lungs offer a unique route for localised therapy because of the large surface area of the alveolar region, the equivalence of inhalation to arterial injection, reasonably low proteolysis activity in the alveolar space, and lack of first-pass hepatic metabolism (karathanasis et al., 2005; ungaro et al., 2012). inhalation of powder particles with a size below 5 µm can be directly and efficiently delivered into the lower part of the respiratory tract (ahmad et al., 2015; beck-broichsitter et al., 2012; karathanasis et al., 2005; ungaro et al., 2012). this local type of treatment provides fewer systemic effects than oral therapy (ungaro et al., 2012). spray-dried powder is a suitable dosage form for the delivery of drugs through inhalation. it can be produced by a spray drying technique which has been an important technique for the pulmonary delivery of pharmaceutical proteins (haggag & faheem, 2015). spray drying is onestep, rapid, reproducible, and scalable solvent evaporation system that is suited to convert a solution, suspension, or emulsion into controlled size and shape of dried powder particles (arpagaus, 2012, 2018, 2019b) (figure 2). it is widely used in the pharmaceutical sectors for processing excipient, microencapsulation, or granulation (arpagaus, 2018). there are two types of spray dryers available in the market nowadays; standard spray dryer and nanospray dryer. a standard spray dryer uses a pneumatic nozzle system while a nanospray dryer uses a piezoelectric system to produce liquid feed droplets by spraying the starting liquid material containing the drug into a hot drying medium (patrizia et al., 2014). a standard spray-drying procedure comprises of four steps which are 1) atomisation of the liquid feed, 2) evaporating of the liquid feed through the drying gas, 3) formation of a particle, and lastly 4) separation and collection of the formed particle (arpagaus, 2019b; schafroth et al., 2012). interestingly, the innovative nano spray dryer has become a more promising technology to produce powder particles for inhalation as compared to standard spray dryers because of its ability in controlling and reducing powder particles (patrizia et al., 2014). nanospray dryer could produce liquid droplets using a piezoelectric system that functions at a certain ultrasonic frequency (60 khz) and thus forming a mist of droplets that has a remarkable ultra-fine particle size. the final dried particles eventually have an electrostatic charge which enables them to be deposited on the surface of the collecting electrode (arpagaus, 2012; schafroth et al., 2012). the uniqueness about this new spray dryer is that it utilises a gentle laminar airflow to minimise sample loss. besides, it has a spray head device consists of a stainless-steel membrane. this membrane has several specific micron-sized holes (spray meshes with hole sizes of 4.0, 5.5, or 7.0 µm) to generate small particles (size ranging from 3 to 15 µm) in a very narrow size distribution. last but not least, the novel spray dryer has an electrode collector that collects particles electrostatically charged to produce high yields and thus reduce particle wastages (arpagaus, 2012; patrizia et al., 2014; schafroth et al., 2012). figure 2. principle operation of nanospray drier (adapted from arpagaus, 2012) taher et al. (2021) journal of pharmacy, 1(1), 34-44. page 40 the advantages of nanospray drying are 1) particle size and shape optimization for pulmonary drug delivery, 2) high bioavailability of the drug in the lungs as compared to oral administration, 3) reduction of drug doses, 4) minimizing systemic side effects, and 5) increases patient adherence (arpagaus, 2012, 2019b; arpagaus et al., 2018). besides that, nano spray drying is suited for heat-sensitive drugs and various excipients (haggag & faheem, 2015). it also has been used to encapsulate both hydrophobic or hydrophilic drugs, peptides, and proteins into particles for controlled drug release (swider et al., 2018). a reasonable size range (approximately 0.5-3.3 um) can also be achieved especially for locally acting inhaled drug particles (arpagaus & meuri, 2010). an amount of powder about 30 mg can be collected from the electrostatic particles collector thus allowing economical uses of expensive pharmaceuticals ingredients. small sample quantities of about 5 to 10 ml are also possible to be spray dried with the help of a nanospray drying technique (arpagaus, 2012, 2019b). proposed strategies there is a high expectation of using remdesivir and hydroxychloroquine as an effective treatment against sars-cov-2 infection due to its promising anti-sarscov-2 activity; antiviral, anti-inflammatory, and immunomodulator. a targeted administration of nasal hcq has been reported to reduce histamine and leukotriene release in one of the animal studies with no significant acute toxicity (barrett et al., 2008), and in cell cultures and animal model, remdesivir has shown the inhibition of replication of coronavirus. besides, its proven safety profile and low price in the market make it a drug of choice to be produced on a large-scale amid the sars-cov-2 pandemic outbreak. nanospray inhalation of remdesivir and hydroxychloroquine might be the best option to deliver the drug quickly to the lung epithelial cells particularly, the alveolar regions, where ace-2 receptors are mostly located. the ability of a drug to deposit and retain on the lung epithelial cells, and to overcome extracellular and cellular pulmonary barriers are essential factors to be considered in the design of inhaled powder particles (ungaro et al., 2012). the capability of the powder particles to deposit on the lung relies on its aerodynamic diameter (daer) while rapid macrophage uptake and clearance of noxious particles (including solid drug particles) depend on the dgeo. to get to the deep lung, powder particles should have a daer and dgeo below 5 µm. however, the 5 µm of geometric diameter (dgeo) of the particles can promote aggressive pulmonary macrophage activity, thus resulting in a brief period of delivery for an inhaled drug (karathanasis et al., 2005). in contrast, nanoparticles with a size <1 µm will have difficulty to deposit at the pulmonary airways as they are mostly will be exhaled after inhalation (beck-broichsitter et al., 2012). to overcome this disadvantage, it is highly recommended to utilise polymeric drugs that are easy to control and target their delivery to a specific region especially the lungs. a polymer such as biodegradable poly(lactic-co-glycolic-acid) (plga) is the most frequently used as a carrier-mediated lung targeting for the delivery of inhalation particles (ungaro et al., 2012). plga co-polymer consists of polylactic acid (pla) and polyglycolic acid (pga). it can be easily hydrolysed by body fluids into non-toxic metabolites monomers, lactic acid, and glycolic acid causing minimal systemic toxicity (arpagaus, 2019b; bartolucci, 2017; danhier et al., 2012; essa et al., 2020; pandey & jain, 2015; swider et al., 2018). it is available for commercial use and licensed by the united states food and drug administration (us fda) and the european medicine agency (ema) at different molecular weights and lactide/glycolide ratios (bartolucci, 2017; danhier et al., 2012; hirenkumar & steven, 2012; pandey & jain, 2015). interestingly, plga copolymer can be stored in powder form for a lengthy period (swider et al., 2018). several studies found that nanospray dried plga particles could produce from approximately 2 µm to below 200 nm spherical powder particles (arpagaus, 2019a) which is within an ideal nanoparticle size for use in dry powder inhaler (dpi) (bartolucci, 2017; beck-broichsitter et al., 2012). by applying the nanospray drying technique, any hydrophobic or hydrophilic active ingredients can be encapsulated in polymeric wall materials to improve the drug formulation and to protect and deliver the drug to the correct site and time in the body (arpagaus, 2018). generally, encapsulated nanoparticles are described as solid particles (size less than 1 µm) suspended in a liquid medium. the smaller the size of the nanoparticles, the larger their surface area. subsequently, the dissolution and absorption rate of the nanoparticles is improved, and thus, lead to the high bioavailability of the encapsulated drug (arpagaus, 2019b). the common method for plgabased nanoparticles formulation that is suitable for hydrophobic drugs is the emulsification-solvent evaporation technique (danhier et al., 2012; hirenkumar & steven, 2012). in this technique, an organic solvent like dichloromethane will be used to dissolve both polymer and the desired drug. after that, the polymer solution will be mixed with water and a surfactant such as polysorbate-80 or poloxamer-188 to form an oil in water (o/w) emulsion (danhier et al., 2012). the mixture then is atomised into hot gas to evaporate the solvent. the resulting dried particles that contain the drug will be dispersed in an amorphous polymer matrix with a geometric mean diameter of the particles of about 1-2.7 µm and a mean mass diameter of <4 µm, (using 4 µm spray mesh size) (arpagaus, 2018). the size range is ideal for the delivery of the drug to the lower respiratory region. taher et al. (2021) journal of pharmacy, 1(1), 34-44. page 41 several formulation parameters need to be considered to prepare an ideal plga-based nanoparticle. the plga molecular weight and the ratio of poly-lactic acid to polyglycolic acid can be manipulated to obtain the desired degradation rate and mechanical strength (arpagaus, 2019b; danhier et al., 2012; pandey & jain, 2015). the higher molecular weight of plga possesses more structural stability, therefore it degrades slowly in in-vivo (arpagaus, 2019b; essa et al., 2020). to modify the degradation rate of the polymer, different end-capped functional groups also can be used. it has been shown that ester end-capped polymers degrade slower than acid-end capped polymers, thus, useful for slower release applications (essa et al., 2020). pla is rigid, and hydrophobic because of methyl groups' existence. meanwhile, pga is more flexible and less hydrophobic (blasi, 2019; essa et al., 2020). therefore, plga copolymers that contain a high proportion of lactic acid are hydrophobic, making them absorb water slightly, and degrade more gradually than plga that contains a high amount of glycolic acid (blasi, 2019; pandey & jain, 2015). plga copolymers that made up of d,l-pla, and d,lpga are amorphous and allow the embedded active ingredients to disperse more homogenously than in the semi-crystalline form of copolymers which comprised of l-pla and l-pga) (blasi, 2019; hirenkumar & steven, 2012; pandey & jain, 2015). for this amorphous state of plga, the important physical-chemical property is the glass transition temperature (tg). plga polymer at a rubbery state is vulnerable to chemical and physical changes which in turn could influence the release mechanisms and kinetics of the embedded active ingredients. the tg decreases in conditions such as 1) low lactic acid content, 2) low plga molecular weight, and 3) addition of compounds such as excipients or active ingredients (blasi, 2019). an amorphous form of plga co-polymers that consists of 70% lactic acid content is believed to be suitable for drug delivery application (essa et al., 2020). the surface of plga nanocarriers can be modulated to prevent it from being recognised and phagocytosed by the pulmonary macrophages. polyethylene glycol (peg) and chitosan (natural polymer) are commonly used copolymer coated onto the surface of the plga carrier to shields it from being taken up by opsonins (an antibody that makes foreign particles susceptible to phagocytosis) and promotes stronger cellular interaction and retention (danhier et al., 2012). the addition of surfactant in the polymeric solution (polyvinyl alcohol (pva), polysorbate 80, 60, and 20, poloxamer or poloxamine) is suggested to prevent particle aggregation and optimise the colloidal stability by increasing miscibility and dispersion of drugs in the polymeric solution (beck-broichsitter et al., 2012; essa et al., 2020). besides, a combination of plga-based nanoparticles with ligands that specifically bind to receptors on the cell of interest, enables the plga carrier to enter the cell by receptor-mediated endocytosis (essa et al., 2020). this strategy could enhance the targeted delivery systems of the plga nanoparticles to the targeted site of action. for the spray parameters, the preferred organic solvents used in the nanospray drying of plga polymers are dichloromethane (dcm), acetone, acetonitrile, ethyl acetate, and mixtures of dcm/methanol. the boiling point of dcm and acetone which is 40°c and 56°c respectively has allowed a low drying temperature which results in a fast-drying process and hampers particles from binding to or agglomerating on the walls. the ideal inlet drying gas temperature for plga dissolved in dcm lies between 29° and 32°c (arpagaus, 2018, 2019b; schafroth et al., 2012), meanwhile, the outlet temperature is between 21°c and 35° c, which is below the glass transition temperature of most plga polymers (about between 37° c and 54°c) (arpagaus, 2019a; hirenkumar & steven, 2012). the outlet temperature must not exceed the plga biopolymer glass transition temperature to avoid softening and yield reduction (arpagaus, 2019b). therefore, heat-sensitive pharmaceuticals are the most suitable materials to be used in nanospray drying (amsalem et al., 2017; arpagaus, 2019a). the slow and gentle drying produces an almost smooth and spherical surface of compact carrier particles (ahmad et al., 2015; arpagaus & meuri, 2010). however, the smooth surface of the carrier particles may result in the generation of auto-adhesive layers surrounding the carrier particles. consequently, the micronised drug particles could agglomerate thus hinders their detachment during inhalation. to improve the aggregation and disaggregation of the micronised powder particles, chitosan may be used as a co-polymer as it gives a rougher surface (groove space) onto the carrier particle which is a good space to bind a drug and help it spread in the oral cavities (better aerosolisation properties) (ahmad et al., 2015). a diluted polymer solution with solids concentration ranging from 0.1 to 1% (w/v) could produce spray-dried particles down to a size of 100 nm (arpagaus, 2018; arpagaus et al., 2018). feed rates depend on the spray cap membrane’s size, type of formulation, temperature inlet and spray rate. the feed rate must be adjusted to within a range of 10ml/h to 16ml/h when using a 4.0 um spray cap (arpagaus, 2012). typically, inert gases such as nitrogen and carbon dioxide are used as drying gases to avoid a blast of explosives from occurring. the concentration is adjusted to be less than 4% and the inert gases are recirculated entirely within the circuit (arpagaus et al., 2018). the nozzle spray pretreated with a surfactant solution (leucine) could generate a free-flowing powder with a satisfactory aerosol functioning. this could be taher et al. (2021) journal of pharmacy, 1(1), 34-44. page 42 beneficial for the nanospray dryer to produce efficient powder for inhalation (patrizia et al., 2014). the encapsulated product must be kept under a controlled condition and a stabilizer may be added into the feed formulation to maintain its activity after nanospray drying. because most nanosprays dried powders are amorphous after a brief drying time, it is important to store the powders under dry conditions to prevent recrystallization (arpagaus, 2019b; arpagaus et al., 2018; arpagaus & meuri, 2010). conclusion nano spray drying technology is economically simple, easy to use, and very efficient in the formulation of nanospray inhalation. plga has several advantages to be used in nanospray inhalation according to its properties such as biodegradable, biosafety, biocompatibility, versatility in formulation and functionalization. the inhaled drugs provide a rapid and direct local effect on the respiratory regions as compared to oral drugs, suggesting its beneficial uses in diseases associated with respiratory disorders. several formulation parameters (hydrophilicity/hydrophobicity of drug, polymer molecular weight, polymer composition, and degradation rate) and spraying parameters (spray mesh diameter, spray rate, sample concentration, sample flow rate, drying gas temperature, solvent types, selection of excipients, stabilizers, or surfactants) are important to take into considerations as they have a great impact on the particle size, production yield, encapsulation efficiency, solid-state solubility, and controlled release profiles of the engineered nanoparticles. the demand for large scale production of powder particles may increase especially during the pandemic of the severe acute respiratory syndrome (sars) caused by coronaviruses. future research should, therefore, focus on a pilotand industrial-scale commercialization of this technology to promote the use of nanospray inhalation for the treatment of sars coronaviruses infection. acknowledgements the authors are thankful to the international islamic university malaysia for funding this work via grant no. p-rigs18-028-0028. conflict of interest the authors declare no conflict of interest. references ahmad, m. i., ungphaiboon, s., & srichana, t. 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(2020). coronavirus disease 2019 (covid-19): a clinical update. frontiers of medicine, 2019. https://doi.org/10.1007/s11684-0200767-8 microsoft word 180-article review galley proof.docx page 86 *corresponding author: email address: amiraraudhah@usim.edu.my synergistic interaction between combination of existing therapy with polyphenols in several human diseases: a review amira raudhah abdullah1*, hermizi hapidin2, nor munirah zakaria2 abstract introduction: the complicated pathology of current diseases requires an intricate treatment. today, current application of individual single-target drugs or therapeutic approaches is inadequate to target these diseases not to mentioned perceived shortcomings and presented with numerous adverse effects. the extensive and successful documented findings in natural product researches urges the need to make use of these knowledge in the development of new generation of medicine. polyphenols are compounds naturally derived from plants and have been describe in many research to have tremendous medical benefit. therefore, a synergistic combination of readily available drugs or other therapeutic approaches is a favourable approach to enhance efficacy, overcome toxicity and optimize safety. the objective of this review is to describe the synergistic effects between the combination of a variety of polyphenols with synthetic drugs or other therapeutic approaches which can help to improve therapeutic efficacy subsequently minimize the adverse effects of a substance targeted in various diseases focusing mainly on cancer, diabetic, microbial infections and tissue regeneration along with their underlying mechanism. article history: received: 13 july 2022 accepted: 13 february 2023 published: 31 july 2023 keywords: polyphenols, synergistic effects, anti-cancer, anti-microbial, antidiabetic how to cite this article: abdullah, a. r., hapidin, h., & zakaria, n. m. (2023). synergistic interaction between combination of existing therapy with polyphenols in several human diseases: a review. journal of pharmacy, 3(2), 86-102 doi: 10.31436/jop.v3i2.180 authors’ affiliation: page 87 introduction natural products from plants have been widely applied by humans for the past centuries for treatment of various diseases (firenzuoli & gori, 2007). the use of natural products for medicinal purpose were being practiced by about 75% to 80% of the world population mainly because of its cultural acceptability as well as superiorly compatibility with human body (set et al., 2010). contrary to synthetic drugs which typically are chemically isolated compound, natural products consist of multicomponent of phytoconstituents (ginsburg & deharo, 2011). there are numerous elements that influence the efficacy mechanism of natural products which includes the geographical origin, plant parts (leaves, stem, root, and fruit), storage, processing, extraction as well as types of solvents used (doughari, 2012; s.-y. pan et al., 2013). over the recent years, researchers are currently focusing on natural products for drug discovery targeted for various diseases. in spite of the facts that the process of drug discovery is now moving forward resulting in advancement of various technology platforms, drug development remains a considerable long process with a low rate of success and large investment. it takes up to years for a newly discovered chemically synthetic compound to become a successful marketable therapeutic agent (barden & weaver, 2010). not to mention, readily available synthetic drug usually perceived shortcomings and presented with numerous adverse effects (pöch, 2012). today, thanks to scientists over the world, approximately 80% of antimicrobial, cardiovascular, immunosuppressive, and anticancer drugs are from plant origin or developed from a natural compound (krief et al., 2004). polyphenols are one of the natural products that has been extensively researched upon and has been described successfully either in vitro (curti et al., 2017; h. sun et al., 2018); in vivo (kujawska & jodynis-liebert, 2018; wang et al., 2017) or in clinical trial (borges et al., 2016; wauquier et al., 2019) to effectively target various disease documented with low toxicity and minimum adverse effects to human (cory et al., 2018; edwards et al., 2012; nash et al., 2018). throughout recent years, more attention have been given by researcher to study the potential interaction between synthetic drugs with natural product targeting diverse type of diseases (lehár et al., 2009). thus, with the presence of recent technological advancement and abundant discovery on the effects of natural product and its health benefits; the development of a new generation medicine can be expedited by combining natural products with synthetic drugs or other therapeutic approaches (pan et al., 2013). the potential benefits that can be gained from interaction of natural product with synthetic drug or other recent therapeutic approaches includes increased efficiency, reduction of undesirable effects increase in bioavailability of the free agents as well as gaining sufficient therapeutic effect with relatively small doses in comparison with a synthetic medication (chanda & rakholiya, 2011). the focal point of this review is on the discovery of synergistic effect of combination of naturally derived polyphenols with synthetic drugs or other therapeutic approaches on various diseases and medical discoveries. the medicinal role of polyphenols polyphenols represent a group of common phytochemicals that are structurally characterized by the presence of one or more phenol units which includes hydroxybenzoic acids, hydroxycinnamic acids, anthocyanins, proanthocyanidins, flavonols, flavones, flavanols, flavanones, isoflavones, stilbenes, and lignans (gupta et al., 2008) . flavonoids for example can be further subdivided into flavones, flavonols, flavanones, isoflavones, anthocyanidins, chalcones, and catechins predominantly found in fruits, vegetables, legumes, red wine, and green tea and have a potential effect on radical scavenging activity and inflammatory reactions (xiao et al., 2011). additionally, stilbenes or resveratrol are polyphenols found in product of grapes, red wine, and peanuts. meanwhile, phenolic acids found in coffee, tea, cinnamon, blueberries, kiwis, plums, apples, and cherries; all has been reported to elicit tremendous health benefits (hasan et al., 2013; sales & resurreccion, 2014). it has been documented that different groups of polyphenols have shown to have different biochemical mechanism and acts differently in response to various modes of diseases. numerous studies confirm that treatment by using variety of polyphenolic compounds either single compounds or in groups as well as dietary intake of natural sources rich in polyphenols, reduced incidence of chronic diseases. polyphenols mainly acts by interacting with reactive oxygen species (ros). ros are typically formed within human body in a controlled amount and are vital compounds that are related in the regulation of processes in maintaining cell homeostasis and functions such as signal transduction, gene expression, and activation of various signaling receptors (kumar & pandey, 2015). the involvement of polyphenols on ros has suggested to be associated with the etiological effects on prevention of different disease pathologies (lima et al, 2014). tremendous researches have shown the therapeutic potential of polyphenols (figure 1)(ganesan & xu, 2017) such as anti-diabetic (cao et al., 2017), anticancer (devi et al., 2017), anti-inflammatory (yahfouf et al., 2018), cardioprotective (arbeláez et al., 2018), osteoprotective (brzóska et al., 2016), neuroprotective, antiasthmatic, antihypertensive, antiageing, antiseptic, hepatoprotective, antifungal, page 88 figure 1: role of polyphenols in humans (ganesan & xu, 2017) antibacterial and antiviral properties (ganesan & xu, 2017; gorzynik-debicka et al., 2018; gupta et al., 2008). synergistic mechanism development of combined pharmaceuticals or therapeutics substances primarily aimed to achieve synergism in order to remain clinically significant. researcher has denoted how latest molecular biological methods and new genomic technologies allow us to unwinds the various synergistic mechanisms underlying these effects (david et al, 2015). however, recognition of successful drug combination is complicated and often remains a setback due to lack of standardization in the aspect of terminology, experimental protocols as well as drug modelling. synergistic effects are produced when compounds interact with one another of the same constituents or in combination with other compounds such as synthetic drugs, biomaterials and other therapeutics approaches. the mechanism of synergy will effect different targets in order to improve the solubility and by that means enhance the bioavailability of one or several substances of a compounds (pöch, 2012). the general standardized method used to measure synergisms and antagonisms is the isobologram method in which concludes the iso-dose effect of two or more substance acting together (berenbaum, 1989). moreover, the construction of a dose response curve also allows investigation of combined effects of investigated compounds (chou, 2006). additionally, to achieved a successful combined therapy synergism it is important to also consider numerous factors affecting drug-drug interactions or compounds interactions (natural products) such as compound target site, affected pathway of compound, process or pathogenesis the compounds acts on and most importantly patients’ ability to absorb, metabolize and excretes a substance or compounds (pemovska et al., 2018; x. sun et al., 2013). methodology the relevant articles were searched through pubmed, google scholar and sciencedirect database. the following keywords and search terms were used: “synergistic combination of polyphenols therapy” in “anti-cancer”, “anti-microbial”, “anti-diabetic,” “cardio-protective” and “regenerative effect”. the articles were screened and the articles that are related to the search keywords dated from the year from 2000 to 2020 were included in the review. results synergistic interaction between combination of existing therapy with polyphenols on various diseases over the recent years, consistent efforts have been made to translate the benefits offered by natural product into clinically relevant substances or therapy in many diseases setting. in this review, we listed the successful work of the combination treatment; by employing existing standardized therapy such as synthetic drugs and biomaterials commonly used in clinical setting with polyphenols (table 1) and its mechanism. in most cases, transforming experimental research for clinical based trial might be hard, however, in combination therapy; through the use of existing standardize regimes and protocol; polyphenols can simply be introduced safely in clinical trials for example through diet or in the form of page 89 table 1: therapeutic combination of existing therapy with polyphenol on various diseases target. drugs or therapeutics approach polyphenols targets experimental models experimental findings ref anticancer tamoxifen green tea extract breast cancer in vitro mcf-7, zr75, t47d breast cancer cell line and in vivo breast cancer induced mice model i. in vitro green tea extract increased the inhibitory effect of tamoxifen on the proliferation of estrogen receptor (er) -positive mcf-7, zr75, t47d human breast cancer cells. ii. in vivo mice treated with both green tea and tamoxifen showed highest apoptosis in tumor tissue compared with either agent administered alone. (sartippour et al., 2006) trastuzumab oleuropein aglycone from extra virgin olive oil extract breast cancer in vitro skbr3 breast cancer cell line increased efficacy of trastuzumab in the presence of oleuropein aglycone via significant reduction of her2 gene; that are associated with unfavorable breast cancer prognosis includes high fatality and relapse rate. (menendez et al., 2007) docetaxel curcumin breast cancer phase i clinical trial on advanced metastatic breast cancer patients fourteen patients were accrued in the trial. findings showed combination dose of curcumin for seven consecutive days every 3 weeks with a standard dose of docetaxel improved biological response and clinical presentation in most patients indicated encouraging efficacy results. (bayet-robert et al., 2010) doxorubicin (dox) quercetin breast cancer in vivo mice model transplanted with 4t1 breast cancer cells. combination of dietary quercetin with intratumoral dox injection synergistically induced potent rejection of 4t1 breast cancer, induced t-cell tumor specific response that results in long-term, tumor-free survival in mice. (du et al., 2010) doxorubicin (dox) silymarin (slm) from seeds of silybum marianum breast cancer in vitro 4ti breast cancer cell line combination of slm-dox exerts synergistic growth inhibitory effects on 4ti breast cancer cell line. (gheybi et al., 2019) page 90 table 1 (cont.): therapeutic combination of existing therapy with polyphenol on various diseases target. drugs or therapeutics approach polyphenols targets experimental models experimental findings ref doxorubicin (dox) epicatechin gallate (ecg) and epigallocatechin gallate (egcg) from green tea extract liver cancer in vitro human hepatocellular carcinoma (hcc) cell line; bel-7404 and bel-7404/dox and in vivo mice model transplanted with bel7404/dox hcc cells. ecg and egcg increased chemosensitivity to dox and increase dox cytotoxicity in bel7404/dox cells by inhibiting p-gp pump function that contribute to the reversal of multidrug resistance (mdr) in vitro and in vivo. (liang et al., 2010) sahasuberoylanilidine hydroxamic acid zolinza (vorinostat) epigallocatechin3-gallate (egcg) from green tea extracts melanoma in vitro a-375, hs-294t and g-361, human melanoma cell line. combination treatment egcg with varinostat resulted in significantly higher inhibition of cell proliferation, increased apoptosis via modulation of the cyclin-cdk-cki network, bcl2 family proteins and nf-κb activity. (niha et al., 2010) cisplatin theaflavin (tf) and epigallocatechin3-gallate (egcg) encapsulated in biodegradable nanoparticulate (tf/egcg-nps) lung cancer, acute monocytic leukemia (aml) and cervical cancer in vitro a549 human lung adenocarcinoma cell line, thp-1 human acute monocytic leukemia cell, hela human epithelial cervical cancer and in vivo ascites carcinoma induced mice. combination of tf/egcg-nps with cisplatin inhibited nf-κb activation and suppressed cyclin d1 activation, matrix metalloproteinase9, and vascular endothelial growth factor (vegf), involved in cell proliferation, metastasis, and angiogenesis in vitro. in vivo findings showed that combination treatment increased the life span of mice model with apparent regression of tumor volume compared to either agent alone. (singh et al., 2015) leptomycin b (lmb) epigallocatechin3-gallate (egcg) lung cancer in vitro a549 human lung adenocarcinoma cell line combination treatment of egcg enhanced lmb cytotoxicity through enhanced ros production and modulation of drug metabolism via p21/survivin pathways. (cromie & gao, 2015) doxorubicin (dox) and etoposide quercetin, apigenin, emodin, rhein and cis-stilbene (commercial compounds) lymphoid and myeloid leukemia in vitro tib-152 peripheral blood t cell leukemia, ccrf-cem acute lymphoblastic leukemia, thp-1 acute monocytic leukemia and kg-1a acute myelogenous leukemia dox alone combined with quercetin, apigenin, emodin, and cis-stilbene synergistically leads to the downregulation of glutathione and increased apoptosis via caspase 8 and 9 activation in myeloid leukemia. (mahbub et al., 2015) page 91 table 1 (cont.).: therapeutic combination of existing therapy with polyphenol on various diseases target. drugs or therapeutics approach polyphenols targets experimental models experimental findings ref bleomycin (blm) tea polyphenols (tpp) antioxidant based therapy for cervical cancer in vitro siha cervical cancer cell line the tpp-blm treatment synergistically induced apoptosis through caspase-3, caspase8 and caspase-9 activation, bcl-2 upregulation and p53 overexpression as well as increased the percentage of apoptotic nuclei in nuclear staining. (alshatw et al., 2016) tamoxifen (tam) genistein (soybased extracts) hepatocellular carcinoma in vitro hepe2 human hepatocellular carcinoma cell line genistein and tam significantly inhibit proliferation and induce apoptosis in hepg 2 cell line. (sanaei et al., 2017) dacarbazine and evorolimus oleuropein braf mutated melanoma in vitro a375 human melanoma cell line induced with braf mutation oleuropein successfully increased the cytotoxic effect of dacarbazine and significantly enhanced everolimus effects on braf melanoma cells, via inhibition of the pakt/ps6 pathway. (ruzzolini et al., 2018) cisplatin theaflvineflavin3,3’-digallate (tf3) ovarian cancer in vitro a2789, cp70 and ovcar3 ovarian cancer cell line tf3 and cisplatin synergistically induced apoptosis and g1/s cell cycle arrest in ovarian cancer cells as well as downregulated akt phosphorylation in ovarian cancer cells. (h. pan et al., 2018) 2-methoxyestradiol (2-me) oleuropein osteosarcoma in vitro 143b human osteosarcoma (os) cell line oleuropein significantly enhanced anti-cancer effects of 2-me on highly metastatic 143b os cells. (przychodzen et al., 2019) antimicrobial amphotericin b epigallocatechin3-gallate (egcg) antimycoticsusceptible and -resistant candida albicans in vitro microbial culture combined treatment between egcg and amphotericin b enhances the antifungal effect of amphotericin b by inhibiting growth of antimycotic-susceptible and -resistant c. albicans by 98.5%–99.7%. as well as allows the use of lower doses of antimycotics. (hirasawa & takada, 2004) norfloxacin (nor), ampicillin (amp), oxacillin (oxa), ciprofloxacin (cip) curcumin methicillinresistant staphylococcus aureus (mrsa) in vitro microbial culture curcumin in combination with all the four antibiotics effectively inhibit s.aureus growth via reduction in minimal inhibitory concentration (mic) against mrsa. (mun et al., 2013) page 92 table 1 (cont.): therapeutic combination of existing therapy with polyphenol on various diseases target. drugs or therapeutics approach polyphenols targets experimental models experimental findings ref ciprofloxacin phenolic-rich maple syrup extract (prmse); active components catechol gram-negative clinical strains of escherichia coli, proteus mirabilis, and pseudomonas aeruginosa in vitro microbial culture prmse with ciprofloxacin exhibit synergistic interaction by targeting bacterial biofilm which helps reduced biofilm formation and increased the susceptibility of bacterial biofilms to antibiotics. (maisuria et al., 2015) fluconazole and amphotericin b catechins from assam and himachal pradesh green tea candida albicans and candida glabrata in vitro microbial culture and in vitro vero cell line purified catechins showed synergistic activity with fluconazole and amphotericin b against candida species with twice mic compared to any agent alone. cytotoxicity analysis of the combined treatment depicted high percentage viability from 91.4% to 100% of vero cell line; suggesting non-cytotoxic activity of proposed composition on healthy cells. (anand & rai, 2017) rimfampin (r) and isonic acid (inh) polyphenols from punica stranatum or pomegranate extracts multidrug resistance turbeculosis (mdr-tb) in vitro microbial culture synergistic effects were observed between r and inh with punica stranatum extracts against mdr-tb strains. however, combination therapy of r was more effective than inh. combination of r with punica stranatum extracts at 15% inhibited 100% (mic 200%) against mdr-tb strains. (almatar et al., 2019) anti-diabetic insulin curcumin diabetes in vitro c2c12 mouse myoblast cell line treatment of insulin and curcumin synergistically and strongly induced glucose uptake and the phosphorylation of ampactivated protein kinase in vitro with increased insulin sensitivity in muscle cells. (kang & kim, 2010) oral hypoglycemic drugs (ohd), namely, thiazolidinedione (thz) and metformin, ferulic acid, pcoumaric acid, eugenol, chlorogenic acid, and caffeic acid from dietary polyphenol diabetes in vitro 3t3-li adipocytes cell line cinnamic acid, ferulic acid, p-coumaric acid, eugenol, chlorogenic acid, and caffeic acid in combination thiazolidinedione (thz) and metformin, increases glucose metabolism via increased uptake of 2-deoxyglucose (2dg) by 3t3-l1 adipocytes cells. (prabhakar & doble, 2011) page 93 table 1 (cont.): therapeutic combination of existing therapy with polyphenol on various diseases target. drugs or therapeutics approach polyphenols targets experimental models experimental findings ref pioglitazone ellagic acid type ii diabetes in vivo diabetes induced rat diabetic rats received combination of 10 mg of ellagic acid/kg with 10 mg of pioglitazone/kg showed improvements in all biochemical parameters in comparison to single treatment along with increased the expression levels of glut4, ppar-γ and adiponectin in skeletal muscle. (nankar & doble, 2017) cardio-protective effects simvastatin chokeberry flavonoid extract (anthocyans, polymeric procyanidines and phenolic acids) myocardial infraction (mi) clinical trial in patients that survived mi and have received statin therapy at least 6 months forty-four patients with mean age 66 years undergone double-blind, placebo-controlled trial. together with simvastatin, flavonoids from chokeberry extract reduce the severity of inflammation by significantly decreased serum 8-isoprostans and ox-ldl levels, as well as hscrp and mcp-1 levels with the reduction in systolic and diastolic blood pressure. (naruszewi et al., 2007) simvastatin or atorvastatin flavonoid-enriched chocolate contained short-term flavan-3-ol and isoflavone cardiovascular disease risk randomized, doubleblind, placebocontrolled clinical trial in postmenopausal women with type ii diabetes receiving mg simvastatin or atorvastatin 118 participants aged less or equal to 75 years old undergone the clinical trial. the chocolate enriched flavonoid intervention with existing therapy (simvastatin or atorvastatin) improved pulse pressure variability equated to a 10% cardiovascular disease risk reduction with larger reductions in diastolic blood pressure and mean arterial pressure indicating clinically relevant improvements in arterial stiffness. (curtis et al., 2013) atorvastatin calcium curcumin atherosclerosis in vitro human aortic endothelial cells and in vivo apoe knockout (apoe-/-) mice synergistic suppression of adhesion molecules (e-selectin and icam-1) and plasma lipid along with secretion of inflammatory factors (il-6 and mcp-1) on combined atorvastatin calcium and curcumin delivery in vitro. reduced atoinducible cytotoxicity in vivo observed in combined treatment. both in vitro and in vivo results demonstrated drastically reduces atherosclerotic lesions. (li et al., 2019) page 94 table 1 (cont.): therapeutic combination of existing therapy with polyphenol on various diseases target. drugs or therapeutics approach polyphenols targets experimental models experimental findings ref atorvastatins resveratrol percutaneous coronary intervention (pci) for coronary artery disease in vivo rabbits with induced abdominal aorta injury followed by drug eluting stents (dess) implantation and in vitro bone marrow stem cells (bmscs) the area of proliferation and migration of vascular smooth muscle cells in the tunica intima and mean thickness were greater in the combined atorvastatin and resveratrol treatment in vivo. biochemical assays on in vitro bmscs resulted in significantly upregulated akt, p -akt, enos, p enos , and cxcr4 expression. both findings exhibited improved re-reendothelialization. (chen et al., 2020) regenerative effects bone marrow stromal cells (bmscs) green tea polyphenols (gtps) blood-spinal cord barrier (bscb) after spinal cord injury in vivo rat model with spinal cord injury combination of bmscs and gtps shown to decrease bscb permeability that helps to improve spinal cord compression, improve motor function, up-regulated expression of tight junction associated proteins claudin-5, occludin and zo-1 in rat model with spinal cord injury. (yu et al., 2015) collagen scaffold curcumin based chitosan nanoparticle wound healing in vivo rat model synergistic combination of curcumin based chitosan nanoparticle and collagen scaffold indicated faster contracted wound in wound closure analysis and complete epithelialization with thick granulation tissue formation in vivo. (karri et al., 2016) pamidronate quercus infectoria extracts bone regeneration in vitro hfob 1.19 human osteoblast cell line increased rate of proliferation and significant elevation of runx2 and osx expression; biochemical markers for bone tissue regeneration in cells treated with combination of quercus infectoria extract and pamidronate. (raudhah et al., 2018) collagen scaffold p-coumaric acid mandible tissue regeneration in vivo rat model with critical size mandible defect. impregnation of collagen scaffold loaded with pcoumaric acid and cartilage oligomatrix protein (comp) enhanced formation of new bone and showed up-regulation of osteogenesis related biochemical markers; osx, ocn and opn as well as angiogenesis markers; fibroblast growth factor2 and vegf in rat model with critical size mandible defects. (bhattarai et al., 2019) page 95 capsulation. the main aims are primarily to increase the efficacy and effectiveness of the readily available therapy; determining the required doses; thus, allowing synergistic interaction. mechanism of interaction of combined existing therapy with polyphenols the capability of polyphenols to regulate the activity of various enzymes and thus to interfere in signaling mechanisms in various cellular processes may be ascribed in part to the physiochemical properties of these compounds that allow them to participate in different metabolic cellular oxidation–reduction reactions. most documented mechanism of action of polyphenols are via savaging ros. as mentioned, ros are produced by human body in a controlled quantity and are capable of unrestricted oxidation of various cellular components that controlled different signal transduction in cells; it can also lead to the oxidative destruction of the cells (mittler, 2002). commonly, oxidatively modified forms of proteins accumulate during aging, oxidative stress, and in most pathological conditions of diseases; these had scientist to focused their attention on the modification of biological molecules by various kinds of ros. collectively, these ros can lead to oxidation of amino acid residue side chains, formation of protein-protein cross-linkages, and oxidation of the protein backbone and deoxyribonucleic acid (dna) modification that may help in development of targeted therapy for management of various disease (balaban et al., 2005; buttke & sandstrom, 1994; finkel & holbrook, 2000). as previously mentioned, development of newly discovered drugs or synthetic compounds could take up to years involving tremendous experimental research and multiple clinal trial in order for it to be marketable and confirmed the safety of its use (barden & weaver, 2010). hence, taking into consideration the immense benefits offered by polyphenolic compounds; as listed in table 1; this review uncovers successful studies on the combination of polyphenols with readily available drugs or therapeutic approaches targeted on various diseases and its ability to increase drugs along with therapeutic efficacy. as a consequence, may fast tract the process of new drug-natural product discovery. combination of chemotherapeutic agents with polyphenols despite many advances in the treatment for most forms of cancer, the mortality and relapse rate remain high. since there is no definitive treatment for cancer, many efforts have been made in order to find a breakthrough in cancer. we have compiled a list of successful combination treatment in cancer therapy employing the use of standard chemotherapeutics drugs with polyphenols targeted for different types of cancer. an ideal treatment combination should achieve synergistic effects via measurement of various biochemical markers. studied on the successful in vitro assessment of combined treatments between chemotherapeutics agents and polyphenols on targeted cancer cell lines exhibited improved or increased inhibition of cancer cells via direct anti-proliferative and pro-apoptotic effects (alshatwi et al., 2016; cromie & gao, 2015; gheybi et al., 2019; liang et al., 2010; mahbub et al., 2015; menendez et al., 2007; nihal et al., 2010; przychodzen et al., 2019; ruzzolini et al., 2018; sanaei et al., 2017; sartippour et al., 2006; singh et al., 2015). additionally, some of the studies has shown that the presence of polyphenols increased the efficacy of chemotherapeutic agents used against the studied cancer cell line compared to any of the agents alone (cromie & gao, 2015; liang et al., 2010; mahbub et al., 2015; menendez et al., 2007; ruzzolini et al., 2018; sartippour et al., 2006). the biological activity of combined treatment between chemotherapeutic drugs and polyphenols has been extensively studied in the preclinical assays. some combined treatments are more effective in overcoming cancer chemotherapeutic resistance by modulating cancer cells with multiple drug resistance (mdr) overexpression phenotype (liang et al., 2010; sartippour et al., 2006). a part from that, some of the studies has shown that synergy combination between chemotherapeutic drugs and polyphenols, exert an important role in apoptosis induction, cell cycle arrest and oxidative stress in vitro (alshatwi et al., 2016; kiemlian kwee, 2016; pan et al., 2018). moreover, in vivo study demonstrated suppression of angiogenesis is translated to larger areas of necrosis and lower blood vessel density in the treated xenografts (sartippour et al., 2006). in vivo experimental results also showed promising potential of long-term tumor free survival in the experimental model (du et al., 2010; singh et al., 2015). regardless of the successful preclinical findings documented, clinical trial has yet to be truly investigated for combined treatment between chemotherapeutic drugs and polyphenols. bayetrobert et al, however; demonstrated significant improvement in clinical presentation and biological response in a phase i clinical trial conducted among advanced metastatic breast cancer patients receiving doxetacel (chemotherapeutics drugs) and curcumin (polyphenols) (bayet-robert et al., 2010). hence, this has shown the significant possibility of successful clinical trial of the combined treatment. combination of antimicrobial drugs with polyphenols antimicrobial drugs act by interfering with the life cycle of an organism in various ways by binding to a cellular target that results in the alteration of the normal function of the microorganism, leading to either inhibition of growth or cell death. a part from that, in order for the antimicrobial agent to reach its target site, the drug must also possess sufficient affinity for its receptor (neely & page 96 jelliffe, 2017). these pharmacological characteristics are the primary determinants of antimicrobial activity. some of these drugs is notorious for developing rapid resistance to antibiotics, caused primarily by antibiotic selection and the horizontal transfer of resistance genes. based on the previous findings, it has been reported that polyphenols also serve a beneficial role as potent antimicrobial agents. therefore, this study was investigated to assess whether polyphenols in combination with antibiotics has the potential qualities of alternative therapeutic agents to overcome the antibiotic resistance of various microbial strains. studies from various documented literature has shown synergistic interaction between combination treatment of antimicrobial drugs and polyphenols causing increased in drug’s efficacy against various mdr strains includes candida albicans (anand & rai, 2017; hirasawa & takada, 2004), mrsa (mun et al., 2013) and mdrtb (almatar et al., 2019) in in vitro microbial culture. moreover, research by maisuria et al, indicated the successful combined treatment between prmse and ciprofloxacin on targeting bacterial biofilm and reducing its formation in clinical strains of escherichia coli, proteus mirabilis, and pseudomonas aeruginosa (maisuria et al., 2015). based on the successful synergistic combination recorded, there’s the need for further in vivo testing for development of an efficient and safer combinational drug against various microbial agent. combination of antidiabetic agents with polyphenols diabetes is a group of heterogeneous disorders that are commonly presented with hyperglycemia and glucose intolerance, due to insulin deficiency, impaired insulin action or sometimes could be both. according to the world health organization (who), diabetes mellitus (dm) is the most common endocrine disorder that are now affecting at least 171 million people worldwide (wild et al., 2004). hence, there is a necessity for new antidiabetic agents with a better therapeutic efficacy and less adverse effects. previous study has demonstrated significant roles of polyphenols in glucose metabolism through scavenging of free radicals, and its role on oxidative stress-linked cell signaling which are the key towards uncovering the therapeutic intervention of dm (kamalakkannan & prince, 2006; veerapur et al., 2017). hence, in this literature, we present the successful increase in efficacy of antidiabetic drugs when combined together with polyphenols in vitro (kang & kim, 2010; prabhakar & doble, 2009; prabhakar et al., 2011) and in vivo (nankar & doble, 2017) experimental model. the ability of polyphenols; curcumin as a potent antioxidant in glucose metabolism was shown through ampk/acc pathway activation that results in enhanced insulin sensitivity in vitro (kang & kim, 2010). meanwhile, dietary polyphenols from cinnamic acid, ferulic acid, pcoumaric acid, eugenol, chlorogenic acid, and caffeic acid in combination thiazolidinedione (thz) and metformin also demonstrated to help increases glucose metabolism (prabhakar & doble, 2011). the dietary intake from plant source and their ingredients could be a more effective strategy for the management of dm because of the likelihood of high compliance not to mention; these dietary polyphenols are free from side effects, have better effectiveness, act on multiple target sites, and relatively cost effective. hence, these studies has shown the potential reduced therapeutic concentration of anti-diabetic drugs when combined with polyphenols, that in returns will cause the side effects to be decreased to a large extent (prabhakar & doble, 2009) in addition, understanding the metabolism and bioavailability of the interaction between these two compounds are vital. a part from that, these findings support a potential clinical application of combination treatment between polyphenols and antidiabetic agents in measurement of dm and its complications. cardioprotective effects in combined polyphenols treatment with synthetic drugs the antioxidant properties of many polyphenols alone could have been proclaim to exhibit vasodilator, antithrombotic, anti-inflammatory, antiapoptotic, hypolipemic or antiatherogenic effects (williams et al., 2004) that have been associated with decreased cardiovascular risk. studies have shown that polyphenols contribute to vasodilator effects and can help to improve lipid profiles as well as mediates the oxidation of lowdensity lipoproteins. in addition, these polyphenols can also attenuate apoptotic process in vascular endothelium and contributes to anti-inflammatory effects. hence, polyphenols can be considered good candidates for the prevention and treatment of cardiovascular diseases (quinones, et al., 2013). based on table 1, combination treatment between selected cardiovascular drug and polyphenols causes reduced atherosclerosis lesion and improved vascular re-reendothelialization in vivo (chen et al., 2020; li et al., 2019). these findings were supported by successful clinical trial incorporating combined polyphenols intake with cardiovascular drug therapy. results of the clinical trial indicated that the action of combined compounds causes decrease of endothelial inflammation and mediation of vascular wall repair (naruszewicz et al., 2007) moreover, it was uncovered that the combination treatment also causes significant clinical improvement that includes decreased in pulse pressure variability, diastolic blood pressure and mean arterial pressure indicating relevant improvements in arterial stiffness (curtis et al., 2013). generally, the evidence listed provide researchers and clinicians with an outline to consider these combined approached as a readily available solution to helps reduced cardiovascular disease risk among high-risk patients. page 97 regenerative effects of combined therapeutic approaches and synthetic drugs with polyphenols regenerative medicine has been extensively explored to as an ideal solution for numerous existing diseases. the key factor in regenerative medicine is to replace or "regenerating" human cells, tissues or organs to restore or establish normal human function. the use of natural product and drug either by itself of synergistically combined, has been published in many researches as a drive component in regenerative medicine (douglas et al., 2018). current approaches in regenerative medicine is either via targeted drugs, cellular material, biomaterials as well as other therapeutic approaches that aims to mediate cellular regeneration and growth. hence, in this review, the potential used of polyphenols combined with some of readily applied therapeutic approaches or drugs on cell regeneration to promotes healing were demonstrated. yu et al, demonstrated the successful combination between bone marrow stromal cells (bmscs), and green tea polyphenols on improving the condition of spinal cord injury in a rat model in vivo (yu et al., 2015). moreover, raudhah et al, indicated successful combination between osteoporotic drug and polyphenols in bone regeneration in vitro (raudhah et al., 2018). additionally, the incorporation of material such as collagen scaffold combined with polyphenols was found to be successful for improving wound healing (karri et al., 2016) and bone regeneration (bhattarai et al., 2019) in vivo. thus, these findings indicated various potential that can be explored to mitigate readily available drugs, biomaterial and synthetic material in order to promotes cell regeneration. conclusion in summary, synergistic effects are achieved when two compounds increase each other’s effectiveness by more than the sum of their single agent responses. we conclude that the use of polyphenols in combination of synthetic drugs or other therapeutic approaches against various diseases has significant preclinical effects; however, the evidence on clinical trial is still lacking and need to be uncover. the advantage of designing treatment regimens of synergistic combination in a particular disease setting provide the opportunity to lower the dosage of an individual agent, thereby reducing toxicity while maintaining the wanted effect on the target cells (jia et al., 2009). additionally, synergistic combination on the use of biomaterials was shown to enhanced its acceptance. thus, provided numerous successful researches reported in this literature; it is strongly recommended that prioritizing the pharmacodynamics and pharmacokinetics concept in designing treatment combination should be considered in the later stages of clinical testing for optimization of successful clinically utilizable therapies. acknowledgements the authors thank assoc. prof. dr. hermizi hapidin, universiti sains malaysia (usm) for her valuable insight and comments to help refine the review in the earlier stage of the manuscripts writing. funding the author(s) received no financial support for the research, authorship, and/ or publication of this article. availability of data and materials all materials analyzed for this review are included in this article. authors’ contribution ara, hh and nmz drafted the manuscript. all authors read and approved the final manuscript. first author information ara (ph.d) is a lecturer in the department of biomedical science 1, faculty of medicine and health science, universiti sains islam malaysia. conflict of interest the authors declare that they have no competing interests. references almatar, m., var, i., kayar, b., eker, e., kafkas, e., zarifikhosroshahi, m., & köksal, f. 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(2021) journal of pharmacy, 1(1), 1-7. page 1 experiential education as a strategy to preserve maqasid al-shariah by identifying and addressing stigmatic views held by pharmacy students of patients with substance use disorders. nor hidayah mohd taufek1,2*, syafiqah nadiah halimi1, norny syafinaz ab rahman1,2, che suraya zin1,2, che anuar che mohamad3 and christopher john turner4 abstract introduction: stigmatising people with a history of substance use disorders (phsuds) is discriminatory, causes harm by hindering access to health services and promotes relapse. it jeopardises the preservation of maqasid al-shariah, particularly the protection of life and well-being. experiential education (ee) is a potential strategy to overcome stigma through direct experience with phsuds. this study aimed to identify stigmatised views of phsud's held by pharmacy students and, in accord with maqasid al-shariah, the effectiveness of ee in changing those views. materials and methods: interviews were conducted with seven undergraduate pharmacy students who provided care to phsuds through an ee programme. a semi-structured interview guide was used to explore students' perceptions on stigma. each interview was audio-taped, transcribed verbatim and translated into english. data were manually sorted and coded using microsoft excel 2016 and subjected to thematic analysis. results: the following themes related to stigma were identified: 1) individuals are to blame 2) moral versus biological views of addiction 3) stereotypes of unpredictability and dangerousness 4) lack of didactic education/training for pharmacy students regarding phsuds and 5) lack of face-to-face experiential education with phsuds. less stigma and greater empathy towards phsuds were reported post-intervention attributable to increased knowledge about substance abuse and face-toface interactions with phsuds. conclusion: pharmacy students have pre-conceived stigmatic views regarding phsuds modifiable through experiential education. contact by pharmacy students with stigmatised patients promotes health care without discrimination as advocated in the teaching of islam. future interventions to reduce stigma are required to preserve maqasid al-shariah. article history: received: 17 july 2020 accepted: 8 october 2020 published: 8 january 2021 keywords: stigma, experiential education, substance use disorders, protection of life, maqasid alshariah. how to cite this article: mohd taufek, n. h., halimi, s. n., ab rahman, n. s., zin, c. s., che mohamad, c. a. & turner, c. j. (2021). experiential education as a strategy to preserve maqasid alshariah by identifying and addressing stigmatic views held by pharmacy students for patients with substance use disorders. journal of pharmacy, 1(1), 1-7. authors’ affiliations: 1 department of pharmacy practice, kulliyyah of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, 25200 kuantan, pahang. 2 substance use disorders research group, kulliyyah of pharmacy, international islamic university malaysia, kuantan, malaysia. 3 department of basic medical sciences, kulliyyah of pharmacy, international islamic university malaysia, jalan sultan ahmad shah, 25200 kuantan, pahang. 4 retired but formerly with skaggs school of pharmacy and pharmaceutical sciences, university of colorado anschutz medical campus, aurora, united states. *corresponding author: email address: hidayahtaufek@iium.edu.my tel:+609 571 6400, fax: +609 571 6775 mohd taufek et al. (2021) journal of pharmacy, 1(1), 1-7. page 2 introduction stigma complex is defined as "the set of interrelated, heterogeneous system structures, from the individual to the society, and processes, from the molecular to the geographic and historical, that constructs, labels, and translates difference into marks" (pescosolido & martin, 2015). negative marks associated with substance use disorders (suds) include health, social and behavioural dispositions at odds with societal expectations (e.g. unemployment and crime) that result in social rejection (committee on the science of changing behavioral health social norms et al., 2016). manifestations of stigma affect health outcomes negatively. they hinder health-seeking behaviour, limit allocation of resources for treatment/intervention and dissuade health care professionals from providing services (yang et al., 2017). accordingly, it is important to identify and address negative views held by current and future healthcare professionals towards people with a history of substance use disorders (phsuds). that is especially important for muslim healthcare professionals because holding stigmatised views is inconsistent with the islamic principles of maqasid alshariah (raysuni, 2005). maqasid al-shariah refers to the higher objectives of the islamic law that emphasise the protection/preservation of basic human rights including five fundamental elements: faith/religion, life, lineage/offspring, intellect and wealth/property (raysuni, 2005). preservation of the elements can be achieved through efforts related to establishing and strengthening them and by removing potential threats such as poor health (ibn ashur, 2006). stigma towards phsuds has been associated with poor health outcomes (crapanzano et al., 2018). didactic and experiential education are effective strategies in changing stigmatised attitudes towards suds (committee on the science of changing behavioral health social norms et al., 2016). experiential education in pharmacy education involves direct interaction between students and patients in clinical and public healthcare settings and is an important strategy to improve the competency of future pharmacists (legal, 2019). however, research is lacking in addressing stigma associated with suds among pharmacy students. this study sought 1) to identify stigmatised views held by pharmacy students towards phsuds and the influence of experiential education on those views and 2) to discuss the value to students of providing healthcare for phsuds with respect to the islamic principles of maqasid al-shariah. methodology this study received ethical approval from the international islamic university malaysia (iium) research ethics committee (id no. irec 2019-026). eligibility criteria for participants were students who completed the iium drug abuse course phm 3282, volunteered to be trained in providing face-to-face health screening services to phsuds and consented to be interviewed. phsuds were people who completed drug rehabilitation treatment and were monitored by the malaysian government's national anti-drug agency (nada) in kuantan, malaysia. the activities undertaken by the students during the programme were screening of blood pressure, glucose, lipid and carbon monoxide levels. a qualitative study that involved semi-structured face-to-face interviews was conducted with all (7) students who met the eligibility requirements, at the faculty of pharmacy, iium. it was conducted from january until may 2019 at mutually convenient times arranged by the interviewer and each student. students were contacted by the researchers who explained the purpose and nature of the study. one-on-one interviews were conducted in the malay language by one interviewer using a general interview guide designed by the researchers to allow students to comment on their didactic knowledge, perceptions, experience, patient interactions and reflections pertinent to the study. questions addressing barriers, problems, challenges during the experiential learning as well as positive and negative feelings specific to phsuds were emphasised to obtain detailed elaboration on stigma, importance of health services to phsuds from educational, health and islamic perspectives. when deemed necessary, questions were repeated and/or rephrased to confirm the students' understanding of the questions asked and to confirm the interviewer's understanding of the students' answers. the interviews were audiotaped, transcribed verbatim and translated into english. the names of participants were kept anonymous for privacy protection. a thematic analysis of the transcripts was undertaken to identify, analyse, and report recurring themes relevant to the study (braun & clarke, 2006). data (relevant phrases, sentences, etc.) were copied from each transcript to a microsoft excel spreadsheet and manually sorted to facilitate the identification and coding of recurring themes. that process was conducted independently by two researchers and differences in the identification and coding of themes were resolved through discussion. there is no established ideal sample size when using thematic analysis. most qualitative studies use the concept of saturation, the point at which no new information or themes are observed in the data (guest, bunce & johnson, 2006; willig, 2013; fugard & potss, 2015). mohd taufek et al. (2021) journal of pharmacy, 1(1), 1-7. page 3 table 1: views held by pharmacy students towards suds and the influence of experiential education on those views. themes quotes individuals are to blame "we tend to blame the drug addicts that they don't want to try to go back to the right path…after they go for rehab, they relapse" (p6) "i had this stigma on why we should spend a large amount of money in helping them because it is their own problem. why did they involve in drug abuse and difficult to be cured?" (p2) "mostly come from low education status... rural area, they involved with drugs influence by their friends" (p4) "drug abusers as those who are from disorganised family institution" (p5) "i felt uncomfortable because people who involved with drugs may have hiv...they were also not really friendly" (p7) moral versus biological views of addiction "i learned how drugs affect the health of drug users, addiction can affect their mental health, physiological, quality of life and attitude…i can see that it is a disease, not only because of themselves" (p2) "i thought that people who took drugs just wanted to have fun, they didn't want to quit drugs…we know that withdrawal syndromes involve physiology of their body…same with other chronic diseases…so it is partly not their will" (p7) stereotypes of unpredictability and dangerousness "at first i was quite scared because may be some of them could become aggressive...cheating…we don't know if they have withdrawal symptoms" (p1) "from afar, i saw that they were quite rough in terms of personality, so i was a bit scared to have a chat with them" (p4) "concern about their behaviour...those who have difficulty to cooperate" (p5) "how do we communicate with these people...because drug (addiction) is a sensitive issue" (p6) lack of education/training among students "we only learned theory previously, we know better now from experience" (p1) "experiential learning is important. when we understand about how drugs affect them in reality…it can change our thought on them" (p2). "those who did not take the course (iium drug abuse course phm 3282), they lack preparation…we need to have more hands-on experiences so that students are confident to do it...no more fear of doing such activities. we can also learn from psychological aspect...or behavioural knowledge…about the best method to approach them" (p7) mohd taufek et al. (2021) journal of pharmacy, 1(1), 1-7. page 4 themes quotes lack of contact with phsuds "there should be more exposure to ex-drug addicts in terms of teaching and learning" (p3) "it was a totally new experience, i got to know their behaviour...some of my friends did not want to involve when they knew that this program involved ex-drug users" (p4) "he told us about how many cigarettes he smoked before...this population is a bit unique...we need to know their behaviour...because different people require different methods" (p5) "this programme was an opportunity for me to meet and observe them...positive experience... i could interact with them and they shared their stories how they got involved with drugs" (p6) reduced stigma and increased empathy following direct contact "before the programme, i felt a bit scared and nervous, and some negative thought. after that i felt like they were similar to other patients...they told stories...we got some insights how hard it was for them to quit drugs…when they went to the clinic to seek treatment, the public had sceptical opinion towards them…sometimes even the hospital staff were being sarcastic and teasing them…they felt inferior, they did not want to go anymore" (p1) "it changed my stigma about them...i wanted to help them" (p2) "they said that they did not usually do health screening…so when we do it, they seemed to appreciate the services…we should get to know their community better" (p3) "in islam, smoking cigarette is forbidden...i observed the bad things that happened to the patients from smoking based on their stories...so it strengthens the fact that smoking should be avoided completely...if we do not care about them properly they could relapse" (p5) "improved my perspective on drug addict, not all of them wanted to involve with drugs...a patient told us that he took morphine to relieve the pain due to an illness...then he got addicted" (p6) "this programme gave us more understanding and empathy towards this population" (p7) mohd taufek et al. (2021) journal of pharmacy, 1(1), 1-7. page 5 saturation was deemed to have occurred in this study after five students were interviewed. data triangulation process related to stigma and substance use disorders, as well as islamic concept of both components in relation to maqasid al-shariah were compared with the literature and audit trail conducted during the interview for differences and similarities. results the seven participants (age 23-25 years) were female, malay, single, final year undergraduate pharmacy students. the total duration of the interviews was 204 minutes and ranged from 21 minutes to 37 minutes and average length of 29 minutes. the variation in length was due to different student responses to questions and the requirement to ask for clarification regarding some responses. six themes are identified and are described in table 1 using italicised quotes from the transcripts. the participants were identified using the participant number (e.g. p6). individuals are to blame stigma was manifested by blaming individuals for 1) becoming drug addicts, 2) relapsing after treatment, 3) succumbing to peer pressure, 4) unwillingness to quit drugs or to be treated, 5) the large amount of resources required to treat addiction and its co-morbidities including human immunodeficiency virus (hiv) infections and 6) their backgrounds they described phsuds as individuals with low education and coming from a dysfunctional family in rural areas. they implied that phsuds were unfriendly. moral versus biological views of addiction students expressed both moral and biological views and could link both components. the moral views were described in terms of addiction habit, reluctance to seek professional help, negative attitudes and desire to have short-term pleasure. the biological view was implied by knowledge regarding suds in terms of biological changes in the body and viewing drug addiction as a chronic disease associated with withdrawal syndromes and relapse. the biological view of addiction was linked with mental health, quality of life and attitude. stereotypes of unpredictability and dangerousness students reported that they had negative thoughts on the behaviour and personality of phsuds. they described them as aggressive, cheating, uncooperative, anxious and rough as well as the possibility that they might exhibit withdrawal symptoms. they were concerned as to the best way to communicate with phsuds given the sensitivity of some issues. lack of education/training among students the students reported that their education and training in suds was lacking and that theory and experiential education needs to be balanced. they reported on the need to focus on the best ways to gain the trust of phsuds such that they are willing to engage with healthcare practitioners. they expressed concern that their peers who did not participate in the course and programme will not have the knowledge and skills to care for phsuds. lack of contact with phsuds students reported that the programme was their first encounter phsud patients. they described it as a positive experience where interaction with phsuds improved their understanding of suds. they perceived that phsuds possessed unique behaviours compared with the general public and were concerned that some of their peers refused to participate in the programme because the patients were phsuds. the face-to-face interactions enabled the students to gain patients' trust and to discuss and address each patient's individual needs. reduced stigma and increased empathy following direct contact the students reported that their experience improved their empathy towards phsuds and, with a changed perception, now saw phsud patients in the same way they saw other patients. the exchange of information and stories during the programme provided the students with insights into the reality of their patients' struggles to quit drugs and the circumstances surrounding the development of their addiction. they expressed empathy in terms of the challenges to accessing healthcare due to stigma. they were motivated to help this population with their overall health care and to prevent relapse. islamic values were reflected regarding smoking tobacco in that smoking may lead to other forms of addiction. discussion the study provides evidence that pharmacy students hold stigmatised views of phsuds likely driven by preconceived ideas regarding suds common in the general population. in addition, the results provide evidence that experiential education involving face-to-face contact with phsuds can improve pharmacy students' views of phsuds and promote their interest in experiential education and didactic education regarding suds. the finding that pharmacy students hold stigmatised views of phsuds is important because stigma limits phsuds access to health care (merril & monti, 2015). avoidance of negative labels has been reported as an important factor in encouraging individuals with suds to seek help from healthcare providers (ciftci, jones & corrigan, 2012) and, accordingly, it is important to identify and address negative views held by healthcare professionals regarding suds. it has been reported that lack of education, training and support for health professionals working with stigmatised patients creates barriers, reduces engagement and mohd taufek et al. (2021) journal of pharmacy, 1(1), 1-7. page 6 diminishes empathy (van boekel et al., 2013). the finding that face-to-face interactions reduced stigma and generated empathy towards phsuds is important because it provides evidence that experiential education can change malaysian students' pre-conceived stigmatised views of phsuds. the finding that students felt their experience should become a core component of the curriculum (e.g. involve all pharmacy students) and that more didactic education regarding suds should be included in the curriculum is important in demonstrating that experiential education can motivate and improve student learning. early and continuous experiential education has been identified in an evidence-based literature review as an important component of the pharmacy curriculum (speedie et al., 2012). relevant to this study, though not reported directly in the published manuscript, even first-year pharmacy students have had interactions with phsuds in community pharmacy-based methadone clinics (winn & turner, 2016). malaysia is a muslim country and healthcare practitioners need to be culturally competent with respect to the maqasid al-shariah principles of basic human rights including life, health and well-being (attum et al., 2020). islamic teaching encourages understanding and empathy and discourages assumption and speculation. in that light, holding stigmatised views can be considered as sinful as demonstrated in the following quranic verses: "o you who have believed, let not a people ridicule [another] people; perhaps they may be better than them; nor let women ridicule [other] women; perhaps they may be better than them. and do not insult one another and do not call each other by [offensive] nicknames. wretched is the name of disobedience after [one's] faith. and whoever does not repent then it is those who are the wrongdoers" (49:11). "o you who have believed, avoid much [negative] assumption. indeed, some assumption is sin…." (49:12). these verses establish one important rule which is to avoid negative suspicion as this act is regarded as sinful. this would leave the heart to be clean together with more positive thoughts towards others (qutb, 2002). the verses also indicate that it is forbidden for one group to deride other groups as we do not know for certain which groups enjoy a better status in the eyes of god. phsuds may possess values known only to god despite being socially disadvantaged. with proper care and treatment, those values could be identified and nurtured as part of the recovery process. the verses also imply the right of a person not to be treated disrespectfully which occurs commonly in stigmatised populations. islam teaches that despite their history or previous sins, people should not call others by labels that inflict pain. islamic teaching goes even further in establishing a noble society with high standard of justice and fairness as reflected in the following quranic verse: "and do not let your dislike of a people lead you to be unjust (5:8). in our context, this verse teaches us as healthcare professionals to maintain our standard of care to all patients regardless of their appearance or background. islamic teachings are against harmful practices such as substance abuse that intoxicates the mind leading to loss of self-control and prevents remembrance of allah. however, those teachings acknowledge human limitations and require that individuals should be given opportunities to repent and correct their mistakes. people should be helped to return to normal life instead of being stigmatised and discriminated against in ways that can limit access to health care and lead to relapse. healthcare professionals including pharmacists are responsible for delivering fair and equal services to all patients. they should care for phsuds with compassion and empathy to develop mutual trust and promote compliance as required by maqasid al-shariah. our study suggests that higher education models should explore the potential strategies of using experiential educational intervention to eliminate stigma among current and future health professionals. we propose the islamic guideline of good manners e.g. avoid negative assumptions, being judgmental and degrading others, not to allow hatred to cause unequal services and injustice. these components need to be incorporated into the pharmacy curriculum. failure to overcome stigma will eventually compromise the accessibility to basic healthcare services in marginalised groups of patients resulting in poor health outcomes. the limitation of this study included a lack of variability in the student participants due to the small sample size (other themes or sub-themes may have emerged with a larger sample size). the findings of this study cannot be generalised to other population with different characteristics. future research should include studies with larger sample sizes and with study subjects more characteristics of the general population. conclusion pharmacy students had pre-conceived stigmatic views toward patients with a history of substance use disorders. experiential educational was a potential strategy in promoting empathy for stigmatised patients. experiential education upholds the principle of maqasid al-shariah that achievement of optimal health promotes the preservation of life. acknowledgements the authors wish to thank the drug rehabilitation committee of national anti-drug agency kuantan district for sponsoring the health programme for phsuds and the pharmacists at the faculty of pharmacy iium (syed mohd syahmi syd mohmad faudzi, wan zuhaira amirah wan amran) who provided assistance and training for students and during programme. mohd taufek et al. 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