IJTID Vol 6 No 4 Jan-Maret 2017.indd 88 Vol. 6. No. 4 January–April 2017 Research Report THE ANTIBACTERIAL EFFECT OF ROSELLE (Hibiscus sabdariffa) EXTRACT AGAINST Staphylococcus epidermidis IN VITRO Terrence Timothy Evan Lusida1a, Bambang Hermanto2, Sudarno3 1 Faculty of Medicine, Universitas Airlangga, Surabaya 2 Department of Pharmacology, Faculty of Medicine, Universitas Airlangga, Surabaya 3 Department of Biochemistry, Faculty of Medicine, Universitas Airlangga, Surabaya a Corresponding author: terencelusida@gmail.com ABSTRACT Infection of Staphylococcus epidermidis is still a common problem in many hospitals. Factor determining biofilm formation makes it harder for atibiotics to cure the infection. Roselle (Hibiscus sabdariffa), a well known traditional medicine plant, is a potential candidate as a drug againts infectious disease. The purpose of this research is to investigate the antibacterial effect of ethanol extract from Roselle (Hibiscus sabdariffa) calyx againts the growth of Staphylococcus epidermidis. Assessment for antibacterial effect is performed using broth diffusion method. The extract is made by maceration of the calyx of Roselle in 96% ethanol. Extracts with concentration of 125, 62.5, 31.25, 15.63, 7.81, 3.90, 1.95, 0.97, 0.48, 0.24 mg/mL are added into separated Mueller-Hinton broths (MHB), which have already been inoculated by Staphylococcus epidermidis. As for bacterial growth control, we used MHB with bacterial inoculation, while sterility control we used mixture of extract and MHB. Then from each broth, the solutions are added into separated nutrition agar plates. Replications are done three times. Clarity and bacterial growth are observed after 24 hours of incubation. However, clarity cannot be observed in 36 broth, but bacterial growth is observed on the plate for concentration 0.97, 0.48, and 0.24 mg/mL. Therefore Minimum inhibitory concentration (MIC) cannot be determined because the extract’s color interfere the observation. While minimum bactericidal concentration (MBC), the last concentration before the concentration where the bacteria are still viable, is 1.95 mg/mL. Based on the result of the research, the Roselle calyx ethanol extract (Hibiscus sabdariffa) through dilution method with a concentration of 1.95 mg / mL can kill Staphylococcus epidermidis and in order to find MIC in collored and turbid solution (before being incubated in incubator), we can consider using agar dilution methode or microdilution methode. Keywords: Hibiscus sabdariffa, antibacterial, Staphylococcus epidermidis, biofilm, flavonoids ABSTRAK Infeksi Staphylococcus epidermidis masih merupakan masalah umum yang ditemukan di banyak rumah sakit. Kemampuan bakteri untuk membuat biofilm mempersulit atibiotik untuk menyembuhkan infeksi. Rosella (Hibiscus sabdariffa), tanaman obat tradisional yang umum beredar di masyarakat, adalah bahan yang berpotensial untuk dikembangkan menjadi obat untuk mengatasi infeksi. Tujuan dari penelitian ini adalah untuk mengetahui efek antibakteri dari ekstrak etanol kelopak Rosella (Hibiscus sabdariffa) terhadap pertumbuhan Staphylococcus epidermidis. Penelitian ini merupakan penelitian eksperimental laboratorik dengan metode dilusi. Ekstrak Rosella dibuat dengan cara maserasi dari tampuk Rosella dengan menggunakan etanol 96%. Kemudian dilakukan pengenceran sebanyak 10 kali didalam 10 tabung. Konsentrasi yang didapatkan adalah 125, 62.5, 31.25, 15.63, 7.81, 3.90, 1.95, 0.97, 0.48, 0.24 mg/mL. Masing-masing tabung sudah berisi bakteri. Sebagai kontrol tumbuh bakteri digunakan campuran bakteri dengan Mueller-Hinton broth dan kontrol sterilitas menggunakan cairan ekstrak dengan Mueller-Hinton broth. Kemudian dari ke-12 tabung, dilakukan streaking pada media nutrient agar plate untuk melihat pertumbuhan dari bakteri. Replikasi percobaan dilakukan sebanyak 3 kali hasil percobaan diamati setelah inkubasi selama 24 jam. Hasil penelitian yang didapat, dari 36 tabung tidak dapat diamati kejernihan dari tabung. Hal ini disebabkan warna dari ekstrak mengganggu dari kejernihan pengamatan sehingga tidak dapat ditentukan nilai dari konsentrasi hambat minimal (KHM). Kemudian dari nutrient agar plate, didapatkan pertumbuhan bakteri pada 89Lusida, et al.,: The Antibacterial Effect of Roselle With the increase in Staphylococcus resistance and Roselle medical potential, we need further research regarding the antibacterial effect of this plant. In this study, researcher aims to investigate the antibacterial effect of Roselle extract on the growth of Staphylococcus epidermidis in vitro. MATERIAL AND METHOD Plant Material Extraction The flowers of Hibiscus sabdariffa were purchased from UPT Materia Medica in Batu small town, east java. The plant materials were taxonomically identified by a botanist at the same location. Calyces of the plant were separated and ground to a fine powder. About 250 g dried powder was taken and soaked with 96% ethanol. The wet powder is put in a jar and as much as 1500 mL 96% ethanol was poured into the jar. The jar was then closed tightly for 24 hours and placed on the shaker with 50 rpm. After that the suspension was filtered and was placed into erlenmeyer. The sediment from the filtration was re-maceration with the same technique (1500 mL 96% ethanol). The ethanolic extract was evaporated with rotary evaporator for 2 hours and water bath for 1 hour. The final result from the extraction was 77 mL extract with concentration 250 g/mL. Antibacterial Assay Prior to the experiment, a colony of S. epidermidis was subcultured in Mueller Hinton Agar (MHA) and incubated for 24 hours at 37 ºC. The bacteria were then adjusted by adding normal saline to be equivalent to 0.5 McFarland standard which comprised 1.0 x 108. Susceptibility Testing Procedure The experiment was repeated 3x. The extract was dispensed in 1 mL volume in each sterile tube with decreasing concentration starting from 125 g/mL. Each tube was then inoculated by 1 mL volume of diluted S. epidermidis. The growth control tube consists of 1 mL inoculum and extract free medium while the sterility control contains 1 ml extract and 1 ml medium. All tubes were incubated at 37 ºC and MICs were read after 24 hours of incubation. INTRODUCTION Bacterium is the microorganism that is most frequently found in human body. This microorganism often causes infection and medical problems. One of them is Staphylococcus epidermidis. Normally, this bacterium is present in healthy people and doesn’t cause infectious disease, however in certain condition like immunodeficiency syndrome, it can cause infectious disases.1 Because of this condition, Staphylococcus epidermidis is a common cause of nosocomial infections. As many as 40-90% of nosocomial infections associated with hospital tools are caused by this bacterium.2 This increases the patient’s health expenditure and duratiuon of staying in hospital. In America, 50-70% of the 16,000 cases of bacteremia by catheter infection in ICU are caused by S. epidermidis with an additional cost of 37.000-39.000 US$ for each person.3 Besides the high incidence rate, many strains of Staphylococcus have antibiotics resistance like methicillin and vancomycin. This resistance is also associated with the bacteria’s ability to make biofilms.4 Biofilm is an ability possessed by a certain kind of bacteria to bind and create a complex structure which is formed by its colonization. It has the ability to allow bacteria to develop resistance to host immune responce and antibiotics.5 Therefore, the discovery of treatments to infectious diseases, in particular caused by S. epidermidis is very important. In addition to drug development, the use of medical plants as natural antimicrobial agents is gaining popularity. Roselle (Hibiscus sabdariffa) is a tropical and sub-tropical plant with a potential candidate in herbal medicine. It is commonly used to make form drink and pickle and it is used in folk medicine infor treatment of hypertention, liver disease and fever.6 Several studies have been conducted on Rosella and have shown various benefits for medical purpose. A research was conducted by Majorie et al. Bioactive substances in Hibiscus like alkaloids, flavonoids, phenolics and biterpenoids ) may have antibacterial effect agains Esherichia coli.7 Anthocyanin and protocatechuic acid compounds also isolated from dried flower of Hibiscus sabdariffa demonstrate protective effects against oxidative agents.6 Moreover, a research also conducted found that the protocatecuic acid also inhibited the growth of methicillin- resistant S. aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, and Acinetobacter baumanniliu, tsao, yin).8 konsentrasi 0.97, 0.48, dan 0.24 mg/mL. hal ini berarti bahwa konsentrasi bunuh minimal (KBM) dari ekstral Rosella adalah 1.95 mg/mL. Berdasarkan hasil penelitian yang dilakukan, ekstrak tampuk Rosella dengan menggunakan metode dilusi dapat membunuh bakteri Staphylococcus epidermidis pada konsentrasi1.95 mg / mL dan untuk memperoleh hasil KHM pada larutan yang berwarna dan keruh (sebelum diinkubasi dalam inkubator). dapat dipertimbangkan untuk menggunakan metode dilusi agar atau mikrodilusi. Sehingga Rosella memiliki efek antibakterial dan memiliki potensi yang besar sebagai bahan antimikroba. Kata kunci: Hibiscus sabdariffa, antibacterial, Staphylococcus epidermidis, biofilm, flavonoids 90 Indonesian Journal of Tropical and Infectious Disease, Vol. 6. No. 4 January–April 2017: 88–91 RESULT A total of 30 tubes were tested for MIC. The result of the MIC is shown in Table 1. Overall, by using macrodilution methode the MIC couldn’t be determined. This result happens because the extract’s red color and turbidity interfere with the observation and the assessment. The same conditon could also be seen in sterility conttrol tube because of that it couldn’t be used as a comparation to determine sterility. so in order to determine the extract’s efficacy, each of the 30 tubes was streaked into nutrient agar plate to determind MBC value which was shown in Table 2. From the experiment, it can be determind that the MBC of Roselle extract against S. epidermidis is 1.95 mg/mL (1.56%). DISCUSSION Roselle is a great plant to be used for medical purpose. First, it is easily grown in tropical country like in Indonesia and has many properties. The time required to grow is around 4 to 8 months with the lowest temperature 20°C, 13 hours lighting, and 130 to 250 mm of rainfall for each month.9 With these condition, people can easily get the plant and cultivate it. Second, it is known to have many good effects. Roselle has antimicrobial, anti-parasite, anti- cancer, anti-pyretic, anti-inflamation, anti-oxidant, nephro- protective, hepato-protective, diuretic, anti-cholesterol, antidiabetic, and antihypertensive.6, 10-11 From the result, the MIC in the experiment couldn’t be indentified because of the extract’s red colour and turbidity. To find out the result of the MIC, extract Roselle can be tested with another dilution methode. In Nigeria, research was conducted by Mary12. She did MIC testing by agar dilution methode. She examined the antimicrobial effect of Roselle against Staphylococcus aureus, Bacillus stearothermophilus, Micrococcus luteus, Serratia mascences, Clostridium sporogenes, Escherichia coli, Klebsiella pneumoniae, Bacillus cereus, and Pseudomonas fluorescence with MIC 0.30 ± 0.2- 1.30 ± 0.2 mg/mL. A similar research was conducted by Sulistyani13 and her research group with microdilution methode. They tested antimicrobial activity against mouth pathological bacteria that could make biofilm. These bacteria were Streptococcus mutans, Streptococcus sanguinis, Lactobacillus casei, Actinomyces naeslundii, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia with MIC and MBC 7.2 mg/mL to 28.8 mg/ mL and 14.4 to >57.6 mg/mL. Interestingly, Roselle extract also has the ability to inhibit biofilm formation on the concentration of the MIC.13 The formation of biofilm is also found in S. epidermidis.4 The overal mechanism how Roselle extract has antibacterial effects is still not completely comprehanded. In USA, Marjorie, Janak, Jacqueline, Shurrita, and Leonardo were conducted a research about the antimicrobial activity of Hibiscus sabdariffa against E. coli. By using disk diffusion method, they concludedgain conclusion that all concentration (10%, 5%, and 2.5%) of H. sabdariffa could Table 1. Roselle extract’s MIC Extract Concentration 100% (1) 50% (2) 25% (3) 12.5% (4) 6.25% (5) 3.12% (6) 1.56% (7) 0.78% (8) 0.39% (9) 0.19% (10) G+ S- 1 X X X X X X X X X X X X 2 X X X X X X X X X X X X 3 X X X X X X X X X X X X G+: Growth control S-: Sterility control X: Can't be assessed Table 2. Roselle extract’s MBC Extract Concentration 100% (1) 50% (2) 25% (3) 12.5% (4) 6.25% (5) 3.12% (6) 1.56% (7) 0.78% (8) 0.39% (9) 0.19% (10) G+ S- 1 - - - - - - - + + + + - 2 - - - - - - - + + + + - 3 - - - - - - - + + + + - G+: Growth control S-: Sterility control +: Viable bacteria -: No viable bacteria 91Lusida, et al.,: The Antibacterial Effect of Roselle REFERENCES 1. Madigan MT, Martinko JM, Bender KS, Buckley DH, Stahl DA. Brock biology of microorganisms. 14th ed. Boston: Benjamin Cummings; 2014. 2. Du X, Zhu Y, Song Y, Li T, Luo T, Sun G, et al. Molecular Analysis of Staphylococcus epidermidis Strains Isolated from Community and Hospital Environments in China. Yam W-C, editor. PLoS One. 2013 May 13;8(5):e62742. 3. Lyte M, Freestone PPE, Neal CP, Olson BA, Haigh RD, Bayston R, et al. Stimulation of Staphylococcus epidermidis growth and biofilm formation by catecholamine inotropes. Lancet (London, England). 2003 Jan 11;361(9352):130–5. 4. Vuong C, Otto M. Staphylococcus epidermidis infections. Microbes Infect. 2002 Apr;4(4):481–9. 5. Vu B, Chen M, Crawford RJ, Ivanova EP. Bacterial extracellular polysaccharides involved in biofilm formation. Molecules. 2009 Jul 13;14(7):2535–54. 6. Da-Costa-Rocha I, Bonnlaender B, Sievers H, Pischel I, Heinrich M. Hibiscus sabdariffa L. - a phytochemical and pharmacological review. Food Chem. 2014 Dec 15;165:424–43. 7. Fullerton M, Khatiwada J, Johnson JU, Davis S, Williams LL. Determination of antimicrobial activity of sorrel (Hibiscus sabdariffa) on Escherichia coli O157:H7 isolated from food, veterinary, and clinical samples. J Med Food. 2011 Sep;14(9):950–6. 8. Liu K, Tsao S, Yin M. In vitro antibacterial activity of roselle calyx and protocatechuic acid. Phytother Res. 2005 Nov;19(11):942–5. 9. Sallam MN, Plotto A. Post-harvest Operations FAO. Hibiscus Post- Production Manag Improv Mark Access. 2005;2–20. 10. W, Itharat A. Antipyretic Activity of the Extracts of Hibiscus sabdariffa L. Calyces in Experimental Animals. Songklanakarin J Sci Technol. 2007;29(SUPPL. 1):29–38. 11. HH, Aty OAA-E, Morgan EN, S.Youssaf SM, Mackawy AMH. Biochemical and Ultra Structure Studies of the Antioxidant Effect of Aqueous Extract of Hibiscus Sabdariffa on the Nephrotoxicity Induced by Organophosphorous Pesticide (Malathion) on the Adult Albino Rats. J Am Sci. 2011;7(12):407–21. 12. Tolulope M. Cytotoxicity and antibacterial activity of Methanolic extract of Hibiscus sabdariffa. J Med Plants Res. 2007;1(1):9–13. 13. Sulistyani H, Fujita M, Miyakawa H, Nakazawa F. Effect of roselle calyx extract on in?vitro viability and biofilm formation ability of oral pathogenic bacteria. Asian Pac J Trop Med. 2016 Feb;9(2):119–24. 14. Alshami I, Alharbi AE. Antimicrobial activity of Hibiscus sabdariffa extract against uropathogenic strains isolated from recurrent urinary tract infections. Asian Pacific J Trop Dis. 2014 Aug;4(4):317–22. inhibit E. coli activity in from food, veterinary, and clinical samples and showed that the most effective concentration was at 10%, whereas the least effective concentration was at 2.5%. They were stated that the antimicrobial effect of H. sabdariffa might come from its flavonoids chemical. The structure of flavonoids have the ability to form combined complex with bacterial walls. Besides that, the number of hydroxyl groups present on the phenolic ring helps the antimicrobial activity of the extract. Due to the increase of hydroxyl group, the hydroxylation would accelerate and cause the increase of antimicrobial activity.7 Issam and Ahmed alsowere stated a similar discussion that phenolic compounds including flavonoids and cyaniding contribute to antimicrobial activity. They added that flavonoid with its phenolic chain could decreases iron level and increases hydrogen level, which deactivates bacterial enzymes.14 Moreover Sulistyani et al. reported that Flavonoids are also thought to have the ability to inhibit the formation of bacterial biofilms. This capability is possible because the phenolic group in the extract capable to bind strongly to proteins and enzymes from the bacteria. This makes the bacteria unable to produce biofilms.13 This effect is important considering S. epidermidis and some gram- positive and gram-negative bacteria capable of producing biofilms. Based on the research that has been conducted, Roselle calyx extract can be used as a alternative treatment for infections caused by Staphylococcus epidermidis. CONCLUSION The Roselle calyx ethanol extract (Hibiscus sabdariffa) through dilution method with a concentration of 1.95 mg / mL can kill Staphylococcus epidermidis and in order to find MIC in collored and turbid solution (before being incubated in incubator), we can consider using agar dilution methode or microdilution methode.