Online-ISSN 2565-1409 Journal of Widya Medika Junior Vol 3. No. 2 April 2021

ANTIBACTERIAL EFFECT OF GNETUM GNEMON L. LEAVES EXTRACT ON STAPHYLOCOCCUS AUREUS

Paul J Dayoh1), Endang Isbandiati2), Titien Rahayu3)

ABSTRACT

Introduction: Bacterial infections are common among doctors in Indonesia, leading to increased use of antibiotics. Increased use of antibiotics, if not accompanied by rational indication, can increase bacterial resistance. At present, alternative medicine from natural ingredients is widely studied, one of which is the leaves of melinjo (Gnetum gnemon L.). Melinjo leaves are thought to contain antibacterial compounds such as flavonoids, tannins, saponins, and alkaloids. Staphylococcus aureus is a round Gram-positive bacteria, that forms grape-like clusters. Staphylococcus aureus is a normal flora on the skin and human mucosa but can be opportunistic pathogens that cause mild to severe infections such as sepsis. Pathogenic Staphylococcus aureus can produce hemolysis blood, frozen plasma, and produce various extracellular enzymes and toxins. Purpose: This research aims to study the MIC and MBC of melinjo leaves extract on the growth of Staphylococcus aureus bacteria. This research is an experimental study with a post- test control group design. Method: Testing conducted using the microdilution method. Melinjo leaves used were extracted by maceration method using 96% ethanol. Samples were Staphylococcus aureus ATCC 25923 from BBLK Surabaya. Results: extract of melinjo leaves (Gnetum gnemon L.) had MIC on Staphylococcus aureus bacteria at a concentration of 640 mg/ml. MBC was not obtained because the extract of melinjo leaves clotted above 1280 mg/ml concentration. Conclusion: Melinjo leaves extract has an antibacterial effect against Staphylococcus aureus, the MIC is obtained at a concentration of 640 mg/ml, and the MBC cannot be determined.

Keywords: Antibacterial, Gnetum gnemon L., Staphylococcus aureus, Microdilution Minimum Inhibitory Concentration, Minimum Bactericidal Concentration

1)Student of Faculty of Medicine, Widya Mandala Surabaya Catholic University Indonesia, Jl. Kalisari Selatan No.1 Surabaya Email: [email protected] 2)Clinical Pharmacology Department, Faculty of Medicine, Widya Mandala Surabaya Catholic University Indonesia, Jl. Kalisari Selatan No.1 Surabaya 3)Clinical Pathology Department, Faculty of Medicine, Widya Mandala Surabaya Catholic University Indonesia, Jl. Kalisari Selatan No.1 Surabaya

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INTRODUCTION resistance, including the selection of In Indonesia, bacterial infections are antibiotics based on local sensitivity common among clinicians, both in patterns, the use of appropriate and careful hospitals and private practice, which also catheters, proper antiseptic techniques, causes an increase in the use of antibiotics. infection control procedures to isolate Data from the Proceedings of the National pathogens, patient compliance with Academy of Sciences of the United States antibiotic use, and hand sanitation, and of America (PNAS 2018) states that in always pay attention to the cleanliness of 5 2000-2015 there was an increase in the use the environment around the patient . of antibiotics in the world by 65%, Infection is the process of especially in the United States, France, and microorganisms entering the body, Italy in the broad-spectrum penicillins, multiplying and causing severe health cephalosporins, quinolones, and problems. Bacteria can enter the body macrolides class1. World Health through wounds on the mucous Organization (WHO 2015) states that membranes, respiratory tract, digestive 6 about 64% of antibiotics are purchased tract, or genital and urinary tracts . One of without using a prescription in Southeast the bacteria that can infect humans is Asia. High rates on the use of antibiotics Staphylococcus aureus. Staphylococcus is without a prescription in Southeast Asia round-shaped Gram-positive bacteria with are caused by low public awareness a diameter of 0.7 to 1.2 µm, that do not regarding appropriate use of antibiotics2. move, do not produce spores, and are 7 Irrational use of antibiotics can arranged in grape-like clusters . Some increase bacterial resistance rates. Staphylococcus species are normal flora on According to the Centers for Disease the skin and human mucosa, while other Control (CDC 2013), there are around species can cause pus, abscess formation, 2,049,442 cases of illness due to antibiotic various pyogenic infections, and even fatal resistance and about 23,488 cases of septicemia. Pathogenic Staphylococcus can death3. Some theories of resistance include produce hemolysis blood, freeze plasma, the production of enzymes that can and produce multiple extracellular deactivate antibiotics, changes in the enzymes and toxins. The toxin produced structure of target receptors, and decreased by Staphylococcus is an enterotoxin that is bacterial cell membrane permeability for stable to heat and can cause food 6 antibiotics4. The CDC has made several poisoning . recommendations for reducing antibiotic

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Indonesia is a country with a lot and cytoplasm of Enterobacter cloacae variety of natural resources8. According to cells, thus interfering with the exchange of the Republic of Indonesia Health Office nutrients and metabolites and ultimately (2007), an estimated 9,600 species grow in inhibiting the supply of energy for Indonesia and have medicinal properties. bacteria12,13,14,15,16. Anti-bacterial activity Still, only around 300 species have been against Staphylococcus aureus shows that used as traditional medicine. One of them flavone, a flavonoid compound, is melinjo plant. Melinjo leaves and fruit significantly suppresses the production of are often processed into vegetables, and staphyloxanthin and α-hemolysin17. melinjo fruit seeds are processed into Tannins are phenolic compounds that can chips. parts of melinjo seeds are often used interfere with iron absorption by forming in herbal medicine research as natural anti- complex bonds with iron, thereby reducing bacterial. According to Siswoyo (2007), the bioavailability of iron in bacteria18. anti-bacterial from melinjo plants can be Saponin has an anti-bacterial mechanism found in the seeds, skins, and leaves of by disrupting the balance of surface melinjo plants9. permeability of bacterial cell membranes. phytochemical examination of melinjo Cell membrane damage can occur and leaves found that melinjo leaves contain causes various important components to several anti-bacterial compounds including come out of the bacterial cell, such as flavonoids, tannins, saponins, and proteins, nucleic acids, and nucleotides19. alkaloids10,11. The alkaloid anti-bacterial effect has Flavonoids are very lipophilic several mechanisms, first by inhibiting the compounds that make it possible to synthesis of nucleic acids because they damage bacterial membranes. The anti- inhibit the enzyme dihydrofolate reductase, bacterial effect of flavonoids is suspected secondly by interrupting the Z-ring, to involve three mechanisms: First, by thereby inhibiting cell division and inhibiting nucleic acid synthesis. Second, inhibiting the synthesis of nucleic acids. by damaging the cytoplasmic membrane, Finally, penetration of the outer membrane For example, the addition of galangin to and distruption of the integrity of the Staphylococcus aureus bacteria cause bacterial cytoplasmic membrane, which potassium leakage. Finally, by inhibiting causes intracytoplasmic leakage20. the metabolism of energy in bacteria. Research on the use of melinjo leaves Examples of administration of apigenin as an anti-bacterial is still minimal in data and naringenin alter the outer membrane and scientific sources. Therefore we are

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interested in examining the anti-bacterial concentrated using Waterbath to get a thick effect of extracts of melinjo leaf (Gnetum extract. The thick extract was obtained gnemon L.) on Staphylococcus aureus. then the yield was calculated.

METHOD The Making of Extract concentrations This research is an experimental study The results of ethanol extract of with a post-test control group design. This melinjo leaves obtained after maceration, research was conducted at the then diluted with Dimethyl sulfoxide 2% Phytochemical-Pharmacogenomic (DMSO) to form concentrations of 40 Laboratory of the Faculty of Pharmacy mg/ml, 80 mg/ml, 160 mg/ml, 320 mg/ml, Widya Mandala Catholic University and 640 mg/ml. Surabaya, the Surabaya Health Laboratory Center (BBLK) in Faculty of Pharmacy Bacteria Preparation Research Laboratory in Widya Mandala Staphylococcus aureus ATCC 25923 Catholic University Surabaya. The bacterial culture was regenerated to obtain population of this research is bacterial culture in the growth phase. Staphylococcus aureus. The sample used Regeneration was carried out on Mueller- was the bacterium Staphylococcus aureus Hinton broth media and then incubated for ATCC 25923. The concentration of 24 hours at 37ºC in an incubator. melinjo leaf extract used was 40-640 Microscopic and biochemical tests mg/ml. (catalase tests, coagulase tests, and mannitol tests) were carried out to ensure The Making of Extracts that the bacteria used are not contaminated. Melinjo leaf powder that has been obtained from UPT Batu City Medical Antibacterial Test Material was taken to the Phytochemical- Antibacterial activity testing was carried Pharmacogenomic Laboratory of the out by the microdilution method. This Faculty of Pharmacy Widya Mandala study was divided into two groups, namely Catholic University Surabaya to be the control group and the treatment group. extracted. The extraction process was carried out by maceration method using 96% ethanol solvent for 24 hours, then remaseration was carried out. The ethanol liquid extract obtained was then

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K1-5 = The control group consisted of RESULTS K1 = Mueller-Hinton Broth Optical density (OD) values on K21-5 = Mueller Hinton Broth + melinjo spectrophotometer readings (Table 1) leaves extract (Gnetum gnemon L.) at indicate that optical density values do not concentration 40-640 mg/ml show linear results. K3 = Mueller Hinton Broth + Staphylococcus aureus Tabel 1. Optical density result K4 = Mueller Hinton Broth + DMSO 2% + Optical density Concentration Mean value Staphylococcus aureus 0,3885 K5 = Mueller Hinton Broth + Penicillin 0,3947 0,3884 P1 0,4012 0,12 µg/ml + Staphylococcus aureus 0,3716 P1-5 = Mueller Hinton Broth + 0,3861 Staphylococcus aureus + melinjo leaves 0 ,3018 0,2897 0,3058 extract (Gnetum gnemon L.) at P2 0,3101 concentration 40-640 mg/ml. 0,3197 0,3080 0,6208 Then the microplate was incubated for 0,5963 0,60524 P3 0,6007 24 hours at 37oC. After waiting 24 hours, 0,6021 the microplate was observed with turbidity 0,6063 using a microplate reader. The MIC was 0,4011 0,3993 0,3994 determined based on the percentage P4 0,3964 inhibition of ≥ 90%. The MIC is measured 0,4013 0,3989 by calculating the percentage of resistance 0,1712 from the absorbance value or optical 0,2005 P5 0,1673 0,1819 density (OD) obtained from the microplate 0,1719 reader/microplate spectrophotometer. 0,1986

MBC was observed by subculture by Because the optical density value streaking or streaking a small portion of obtained was not linear, it was entered into the results of microplate incubation tubes the formula to get the MIC (Minimal into gelatine. The results of streaking were Inhibitory Concentration). not found in the growth of bacteria to be determined as MBC.

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Tabel 2. Percentage of bacterial inhibition DISCUSSION per concentration MIC Test (minimum inhibitory Optical density concentration) Concentration Mean value The MIC test in this study was carried 64,19% 63,36% out by reading the spectrophotometry from P1 62,46% 64,21% the results of microdilution incubation. In 66,52% 64,54% Table 1, the results of spectrophotometric 71,42% readings are found to be non-linear. This is 73,08% P2 70,28% 70,86% due to the bias of the turbidity of the 68,95% extract used, so the optical density values 70,57% 72,24% obtained cannot be used as data in analysis. 75,61% These values must be converted into a P3 75,01% 74,38% percentage. After receiving the percentage, 74,81% 74,23% the data was then processed in SPSS. 78,08% The percentage of inhibition in the 78,34% P4 78,73% 78,32% microdilution test showed MIC at a 78,06% concentration of 640 mg/ml. This 78,39% 95,97% concentration was obtained after the first 91,96% microdilution trials with sequential P5 96,51% 96,16% 95,87% concentrations of 5, 10, 20, 40, 80 mg/ml 92,22% not achieving MIC and MBC. Hence, we

increased the concentration to 640 mg/ml Table 2 shows that the higher the in the second trial. concentration of melinjo leaf extract, the There are no studies with higher the percentage of inhibition to microdilution methods similar to this Staphylococcus aureus bacteria. The study. Previous research was conducted by highest percentage of inhibition was at a Fitri (2017) with a diffusion test to concentration of 640 mg/ml, which is determine the diameter of the resistance above 90%. zone. The results of Fitri's research (2017)

showed inhibition zone diameter in ethanol

extract of melinjo leaves (Gnetum gnemon

L.) against Staphylococcus epidermidis at

concentrations of 10%, 20%, 30%, 40%,

50%, and 100% w/v, respectively.

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Obtained diameter inhibitory zones concentration could inhibit bacterial respectively of 2.65, 22.02, 22.33, 25.09, growth by 99.9%. 26.15, and 26.79 mm21. In that study, The MBC value in this study cannot clindamycin antibiotic was used as a be determined because, at a concentration positive control with a zone of inhibition of of 1280 mg/ml, the percentage of growth 42.85 mm. The largest inhibitory zone inhibition of the Staphylococcus aureus diameter in Fitri's study (2017) was 26.79 bacteria was found to be above 90% but mm at a concentration of 100% w/v does not inhibit bacterial growth to 99.9%. (equivalent to 100 g / 100 ml or 1000 at concentrations above 1280 mg/ml, the mg/ml). The results of her study indicate melinjo leaf extract does not become a that the ethanol extract of melinjo leaves liquid form as in concentrations below has an inhibitory effect on the growth of 1280 mg/ml. Instead, it remains in Staphylococcus epidermidis bacteria21. If semisolid form, so that concentration compared with results of this study, cannot be made. Microdilution tests cannot inhibition was also found in ethanol extract be carried out. Semisolid form occurs of melinjo leaves against Staphylococcus because of the hygroscopic nature of aureus bacteria. At concentrations of 640 melinjo leaves. At high concentrations (in mg/ml and 1280 mg/ml extract of melinjo this study above 1280 mg/ml), it is using the microdilution method, suspected that the volume of the solvent Staphylococcus aureus bacterial growth used cannot compensate for the inhibition was greater than 90%, but hygroscopic nature of melinjo leaves, so because it did not kill 99.9% of bacteria, the extract remains in semisolid form. only MIC was obtained. This reinforces In previous studies, no studies have previous research on the antibacterial used microdilution or macro dilution effect of melinjo leaf extract. methods to determine the MBC of melinjo leaf extract, so researchers cannot compare MBC Test (minimum bactericidal current research results with previous concentration) studies. The MBC test in this study was carried out by streaking from the microplate CONCLUSION incubation results of the treatment group Based on the study results, it can be into blood agar media. Visual observations concluded that melinjo leaf extract has an showed that all plates still contained antibacterial effect against Staphylococcus bacterial growth, which indicated that no aureus bacteria with a minimum inhibitory

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concentration at a concentration of 640 New York : McGraw-Hill; 2008: 671- mg/ml, while a minimum bactericidal 72 p. concentration cannot yet be determined. 6. Brooks GF, Jawetz E , Aldeberg EA. Jawetz, Melnick & Adelberg’s REFERENCES medical microbiology. 26th edition. New York: Mcgraw-Hill Medical; 1. Laxminarayan R, Goossens H, Klein 2013. 1-817 p. EY, Gandra S, Pant S, Levin SA, et al. 7. Bhatia R, Ichhpujani RL. Essentials of Global increase and geographic Medical Microbiology. 4th edition. convergence in antibiotic consumption New Delhi: Jaypee Brothers Medical between 2000 and 2015. Proc Natl Publishers; 2008. 1-489 p. Acad Sci. 2018;115(15):1-8 p. 8. Departemen Kesehatan Republik 2. WHO. Worldwide country situation Indonesia. Kebijakan Obat Tradisional analysis : Response to Antimicrobial Nasional. Departemen Kesehatan Resistance. WHO Libr Publ data Republik Indonesia. 2007: 1-42 p. [Internet]. 2015; Available from: 9. Siswoyo TA. Free Radical Scavenging http://www.who.int/drugresistance/doc Activity and Phenolic Content of uments/situationanalysis/en/ Melinjo Tree (Gnetum gnemon L.). 3. U.S. Department of Health and Human 2007: 221 p. Services. Antibiotic resistance threats 10. ITIS. Gnetum gnemon L. [Internet]. in the United States. [Internet]. 2010 [cited 2019 Apr 9]. 2013;114. Available from: 11. Alvita LR. Aktivitas Antibiofilm Dari http://www.cdc.gov/drugresistance/thr Bakteri Escherichia coli Oleh Ekstrak eat-report-2013/index.html Air Daun Singkong, Pepaya Dan 4. Trevor AJ, Katzung BG, Kruidering- Melinjo Secara In Vitro. 2015;9–10 p. Hall M, Masters SB. Pharmacology 12. Panche AN, Diwan AD, Chandra SR. Examination & Board Review Tenth Flavonoids: an overview. J Nutr Sci. Edition. 10th ed. New York: McGraw 2016;5:1–15 p. Hill Medical; 2013: 381 p. 13. Nagula RL, Wairkar S. Recent 5. Goodman LS, Gilman A, Brunton LL, advances in topical delivery of Parker KL, Blumenthal DK, Buxton flavonoids: A review [Internet]. Vol. LO. Goodman and Gilman's Manual 296, Journal of Controlled Release. of Pharmacology and Therapeutics. Elsevier B.V; 2019. 190-201 p. Available from:

129 Online-ISSN 2565-1409 Journal of Widya Medika Junior Vol 3. No. 2 April 2021

https://doi.org/10.1016/j.jconrel.2019. materials with tannins for enhancing 01.029 antibacterial properties. Process 14. Ulanowska K, Tkaczyk A, Konopa G, Biochem [Internet]. Elsevier Ltd; Wȩgrzyn G. Differential antibacterial 2010;45(7):1072–81 p. Available activity of genistein arising from from: global inhibition of DNA, RNA and http://dx.doi.org/10.1016/j.procbio.201 protein synthesis in some bacterial 0.03.022 strains. Arch Microbiol. 19. Purbowati O. Pengaruh Campuran 2006;184(5):271–8 p. Ekstrak Tanaman Binahong (Anredera 15. Cushnie TPT, Lamb AJ. Detection of cordifolia (Ten.) Steenis) dan galangin-induced cytoplasmic Sambiloto (Andrographins paniculata membrane damage in Staphylococcus Nees) terhadap Kadar Glukosa Darah aureus by measuring potassium loss. J Tikus Putih (Rattus norvegicus L.) Ethnopharmacol. 2005;101(1–3):243– Jantan. Departemen Biologi Fakultas 8 p. Matematika dan Ilmu Pengetahuan 16. Eumkeb G, Chukrathok S. Synergistic Alam Universitas Indonesia. 2011 activity and mechanism of action of 20. Cushnie TPT, Cushnie B, Lamb AJ. ceftazidime and apigenin combination Alkaloids: An overview of their against ceftazidime-resistant antibacterial, antibiotic-enhancing and Enterobacter cloacae. Phytomedicine antivirulence activities. Int J [Internet]. Elsevier GmbH.; Antimicrob Agents [Internet]. Elsevier 2013;20(3–4):262–9 p. Available B.V.; 2014;44(5):377–86. Available from: from: http://dx.doi.org/10.1016/j.phymed.20 http://dx.doi.org/10.1016/j.ijantimicag. 12.10.008 2014.06.001 17. Kong C, Neoh HM, Nathan S. 21. Hidayah FA, Setiawan NCE. Targeting Staphylococcus aureus EFEKTIVITAS EKSTRAK ETANOL toxins: A potential form of anti- DAUN MELINJO ( Gnetum gnemon virulence therapy. Toxins (Basel). 2016;8(3):1–21 p. L .) TERHADAP BAKTERI 18. Widsten P, Heathcote C, Kandelbauer Staphylococcus epidermidis. 2017;4–7 A, Guebitz G, Nyanhongo GS, p Prasetyo EN, et al. Enzymatic surface functionalisation of lignocellulosic

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