Preformulation and Formulation Studies of the Alcoholic Extract of Punica Granatum L., (Lythraceae) Exocarp As Antimicrobial Ointment
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JAASP http://www.aaspjournal.org Journal of Asian Association of Schools of Pharmacy 2019; 8: 50–57 © 2019 The Asian Association of Schools of Pharmacy Research Paper Preformulation and formulation studies of the alcoholic extract of Punica granatum L., (Lythraceae) exocarp as antimicrobial ointment Anna Muriel T. Jacinto*, Jose Rennel M. Talavera and Catherine B. Galvez College of Pharmacy, University of Perpetual Help System DALTA, Las Piñas City, Metro Manila, Philippines Received May 30, 2019 ABSTRACT Revised August 13, 2019 Accepted September 19, 2019 Punica granatum L., (Lythraceae) is commonly known as pomegranate and contains various pharmacological property among which is its antimicrobial property. The * Corresponding author objective of the study was to formulate an antimicrobial ointment. Methods used include College of Pharmacy, University of Perpetual physicochemical evaluation, microbial assay, compatibility of excipients, formulation, Help System DALTA, Las Piñas City, Alabang- Zapote Road, Pamplona 3, Las Piñas City, quality control and stability test of the ointment. Results revealed that the extract was a Metro Manila, 1740 Philippines dark brown colored powder with like bark odor, disagreeable taste, acidic (pH 3), lighter E-mail: [email protected] than water (< 1.0 density), melts at 117.10ºC and was soluble in the following solvents: 95% ethanol, 0.1 N HCl, 0.1 N NaOH, 0.9% NaCl, phosphate buffer solutions (pH 4, 7 & 10) and petroleum ether. Furthermore, it was most sensitive to Staphylococcus aureus (28 mm) followed by Trichophyton mentagrophytes (23 mm), Pseudomonas aeruginosa and Aspergillus niger (22 mm) while least sensitive to Candida albicans (8 mm). The formulated ointment passed the quality control test but needed further reformulation to improve product stability. Hence, future studies recommend the exploration of other excipients, formulation of other dosage forms and employment of additional methods of preparation. Key words: microbial assay, natural products, preformulation, pomegranate formulation studies from the extracts of Punica granatum L. 1. Introduction (Lythraceae) exocarp as an antimicrobial ointment. The Punica granatum L., (Lythraceae) fruit exocarp Moreover, this scientific work is a response to the call of has an antimicrobial property. Its fruit peel extract exhibits the Philippine Department of Science and Technology antibacterial activity against several bacteria like (DOST) for researches to reflect the National Unified Health Streptococcus mutans, Streptococcus sanguis, Streptococcus Research Agenda for 2017–2022. One of the six (6) themes mitis (Vasconcelos et al., 2006), Escherichia coli, comprising these research priorities is global competitiveness Psuedomonas aeruginosa and Staphylococcus aureus in research and innovation in health. DOST promotes (Khan and Hanee, 2011). Likewise, it is also very effective research as a tool for creating a novel solution to existing and against several fungi such as Candida albicans (Vasconcelos emerging health problems through technology development et al., 2006), Trichophyton mentagrophytes, Trichophyton and innovation in the fields of rapid advancement such as rubrum, Microsporum canis and Microsporum gypseum drug discovery and development (Philippine National Health (Foss et al., 2014). In fact, its antimicrobial activity is Research System, 2017). attributed to several phytoconstituents present in the fruit exocarp such as punicalagin (Foss et al., 2014), ellagitannins 2. Material and methods and punicalagin (Machado et al., 2002) and phenolic punicalagins, gallic acid, fatty acids, catechin, quercitin, rutin, 2.1. Materials flavonols, flavones, flavonones and anthocyanidins (Jurenka, This study was conducted at the University of Perpetual 2008). Considering the dearth of information on Punica Help System DALTA-College of Pharmacy, Las Piñas City, granatum, this study bridged the lack of scientific studies on Metro Manila, Philippines. The microorganisms used in the preformulated and formulated antimicrobial ointment this study were Staphylococcus aureus ATCC 29213, from the exocarp extract of this plant. Thus, the general Pseudomonas aeruginosa ATCC 27853, Aspergillus niger objective of the study was to conduct a preformulation and ATCC 16404, Candida albicans ATCC 10231 and 50 JAASP 2019; 8: 50–57 Tricophyton mentagrophytes ATTC 18748 which were Graduated Cylinder). A quantity of material sufficient to purchased from the University of the Philippines-Department complete the test was passed through a 1.00-mm (No.18) of Medical Microbiology. All chemicals and reagents used in screen to break up agglomerates that may have formed during the experiment were of analytical grade and were purchased storage. Into a dry 250 mL cylinder, approximately 100 g of from Merck (Darmstadt, Germany), Sigma (St. Louis, MO, the test sample (M) was introduced without compacting. It USA) and HiMedia (India). was weighed with 0.1% accuracy. The powder was carefully leveled without compacting, if necessary and the unsettled 2.2. Methods apparent volume, VO was read to the nearest graduated unit. The design of the study was experimental and methods The bulk density, in g per mL, was calculated by the formula: were derived from the United States Pharmacopeia 23/ (M) / (Vo). National Formulary 18 (1995), Philippine Pharmacopeia (2004) and Modified Irritation Test in Rabbits, OECD, 2.2.2.4. Solubility was determined with the use of a #404. Methods used include collection, extraction, physico- mechanical shaker (MRC, USA) using different solvents chemical evaluation, microbial assay, compatibility testing, such as water, 95% ethanol, 0.1 N HCl, 0.1 N NaOH, 0.9% formulation, quality control, sensitivity and stability test of NaCl, phosphate buffer solutions and petroleum ether. About antimicrobial ointment. All equipment used in this study were 1 mg of lyophilized extract was placed in an Erlenmeyer flask properly calibrated prior to use. and then 1 µL of solvent was added. The mixture was agitated at 100 rpm using an automatic shaker until the test sample 2.2.1. Collection of plant material was dissolved. If the lyophilized extract did not dissolve, a The whole plant was collected in Quezon Province, further 30 µL of solvent was added and its effect was noted. (Coordinates 13.3471° N, 122.5202° E) Philippines. It was Successive amount of the solvent was added until the air- dried and brought to the Research Center for Natural and compound exhibited solubility. If the test sample Applied Sciences for authentication. About 10 kg of ripe immediately dissolved with the solvent, the amount of the pomegranate fruits that have no spoilage were hand-picked solvent to be added was decreased. and washed with tap water. The exocarp was peeled off, chopped into pieces using a kitchen knife and dried in an oven 2.2.2.5. Melting point was determined using differential until crispy at 50˚C. It was pulverized using the Wiley Mill scanning calorimetry (DSC-4000 Perkin Elmer, USA). About Grinder. 5 mg of the sample was accurately weighed and placed inside the pan. The parameters used were the following: heat from 2.2.2. Extraction and physicochemical evaluation of 25˚C to 300˚C at 30˚C/min and nitrogen gas at 20 mL/min. lyophilized extract The powdered pomegranate peel was macerated in a 2 L 2.2.2.6. Screening test for tannins was determined Erlenmeyer flask for 24 hr using 80 percent ethyl alcohol. qualitatively using the gelatin test and ferric chloride test as The extract was filtered using Whatmann filter paper no.1, described by Guevara B. (2005). The formation of a jelly- concentrated in vacuo (Wilmad WG-EV311, Condenser precipitate indicates the presence of tannins. On the other H50-500 Lab-Tech, Rotavap RE-2000A) and lyophilized at hand, in the ferric chloride test, a blue-black color indicates Agcaoli Memorial Tissue Bank of the Department of the the presence of hydrolysable tannins while a brownish-green Orthopedics, University of the Philippines Manila-Philippine color may indicate the presence of condensed tannins. General Hospital. The lyophilized extract was weighed using Ohaus Analytical Balance (Serial no. 8329340171, USA), 2.2.3. Microbial assay of lyophilized extract stored in a clean amber bottle and kept inside the refrigerator Microbial assay was performed using the paper disk (4ºC) for succeeding tests. The following tests were diffusion method by Kirby Bauer. The size of the disk was 6 performed in triplicate. mm. Nutrient agar (HiMedia, India) plates for bacterial strains and Sabouraud glucose agar (Titan Biotech Ltd, India) 2.2.2.1. Physical characteristics were identified using the plates for fungi strains were prepared in triplicate. The organoleptic evaluation method by describing the appearance, media surface was inoculated with test microorganisms color, odor and taste. from broth culture that were previously standardized against 0.5 McFarland to obtain turbidity of approximately 1.5 × 108 2.2.2.2. The pH was measured by immersing the CFU/mL of the test organism. The plant extracts were electrodes of a pH meter (TS-1, Suntex) at 25ºC in a 5 mL prepared at different concentrations: 1 g/mL, 0.75 g/mL, solution of the lyophilized extract obtained by diluting 1 g of 0.50 g/mL and 0.25 g/mL. Nutrient broth and Sabouraud lyophilized extract with 10 mL of purified water. broth were used as solvents of the plant extract as well as negative control. Neomycin (Sigma-Aldrich) and Miconazole 2.2.2.3. Bulk density using Method 1 (Measurement in a (Coloplast A/S DK-3050 Humlebaek, Denmark) were used 51 Anna Muriel T. Jacinto et al. as positive controls. The inoculated petri dish were inverted 2.2.6.1. Organoleptic test: About five (5) g of the sample and placed inside the incubator (Model No. 10-140) with a was placed in the watch glass with white background and temperature of 35ºC for 24 hr for bacterial strain and fungi observed for their physical properties such as visual strain. Formations of a clear zone of inhibition were appearance, color, odor and smoothness. measured using a ruler and interpreted according to the following: < 10 mm expressed as inactive; 10–13 mm, 2.2.6.2.