Barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework Rachmadita et al. (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 micro- plates 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: Folin- Ciocalteu 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 Gram- negative 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:005- 0055 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. (2012). Effects of Saponins Against Clinical E. coli Strains and Eukaryotic Cell Line. Journal of Biomedicine and Biotechnology, 2012(286216), 1-6. doi:10.1155/2012/286216 Bobbarala, V. (2012). Antimicrobial Agents. Croatia. In Tech Published. Chaffin, W. L. (2008). Candida albicans Cell Wall Protein. Microbiology and Molecular Biology, 72(3), 495-544. doi: 10.1128/MMBR.00032-07 Elfirta, R.R., Syamsul, F., Dimas, A., & Tien, L. (2018). Identification of Active Compounds and Antifungal Activity of Toona sinensis Leaves Fractions Against Wood Rot Fungi. Journal of Biological Diversity, 19(4), 1313-1318. doi: 10.13057/biodiv/d190418 Harun, Aiza, Siti, Zaiton M.S., Norazian, M.H., & Neni K. C. M. R. (2014). Bioactive Terpenoid from The Stem Bark of Entada spiralis Ridl. (Sintok). Proceedings of the International Conference on Science, Technology and Social Sciences (ICSTSS) 2012, 605 – 615. Hidayat, J., Ay, L., Margaret, Hanna, Y., & Lies, K. W. (2014). Hexane Neem Leaf Extract More Potent than Ethanol Extract Against Aspergillus flavus. Universa Medicina, 33(3), 172-178. doi:10.18051/UnivMed.2014.v33.171-178 Joseph, N., Mirelle, A. F. R., Matchawe, C., Patrice, D. N., & Josaphat, N. (2016). Evaluation of The Antimicrobial Activity of Tannin Extracted from The Barks of Erythrophleum guineensis (Caesalpiniaceae). Journal of Pharmacognosy and Phytochemistry, 5(4), 287-291. https://doi.org/10.13057/biodiv/d190418 http://dx.doi.org/10.18051/UnivMed.2014.v33.171-178 Rachmadita et al. (2021) Journal of Pharmacy, 1(1), 45-53 madi Page 53 Liu, J., Wang, C., Wang, Z., Zhang, C., Lu, S., & Liu, J. (2011). The Antioxidant and Free-radical Scavenging Activities of Extract and Fractions from Corn Silk (Zea mays L.) and Related Flavone Glycosides. Food Chemistry, 126(1), 261–269. doi: 10.1016/j.foodchem.2010.11.014 Mohamad, Sharifah Nurul. (2017). Bioassay Guide Isolation and Characterization of Compounds with Antioxidant Activities from The Leaves of Entada spiralis Ridl. (Sintok). Malaysia: International Islamic University Malaysia. National Park. (2019). National Parks. Retrieved from https://www.nparks.gov.sg/ Ram, A. J., Bhakshu, L. Md., & Venkata Raju, R.R. (2004). In Vitro Antimicrobial Activity of Certain Plants from Eastern Ghats, India, Used for Skin Diseases. Journal of Ethonopharmacology, 90(2), 353-357. doi:10.1016/j.jep.2003.10.013 Reygaert, Wanda. (2017). Antimicrobial Mechanisms of Escherichia coli. Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications. London: Intech Open. Shankar, S., Singh, G., & Srivastava R. K., (2007). Chemoprevention by Resveratrol: Molecular Mechanisms and Therapeutic Potential. Frontiers in bioscience: a journal and virtual library, 12, 4839- 4854. doi:10.2741/2432 Swabha, T., (2018). Bioactive Compounds in Medicinal Plants: A Condensed Review. SEJ Pharmacognosy and Natural Medicine, 1(1), 1-35. Tsuchiya, H. (2015). Membrane Interactions of Phytochemicals as Their Molecular Mechanism Applicable to The Discovery of Drug Leads from Plants. Molecules, 20, 18923–18966. doi: 10.3390/molecules201018923 Widyawati, P.S., Wijaya, C.H., Harjosworo, P.S., & Sajuthi, D. (2011). Evaluation of Antioxidant Activity of Beluntas (Pluchea indica Less) leaf Extract Based on Differences in Leaf Segments. Recapitulation Journal of Food Technology, 5(1), 1-14. Zhang, Ping, Fei, W., Jianying, H. & Robert, S. (2013). Exploring the Relationship Between Drug Side- Effects and Therapeutic Indications. AMIA Annual Symposium Proceeding Archive, 2013, 1568-1577. http://doi.org/10.1016/j.foodchem.2010.11.014 http://doi.org/10.1016/j.foodchem.2010.11.014 https://doi.org/10.1016/j.jep.2003.10.013 https://www.intechopen.com/books/-i-escherichia-coli-i-recent-advances-on-physiology-pathogenesis-and-biotechnological-applications https://doi.org/10.2741/2432 https://doi.org/10.3390/molecules201018923