Tongkat Ali) Root Extract

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by UMP Institutional Repository In Vitro Antibacterial Activity of Eurycoma Longifolia Jack (Tongkat Ali) Root Extract Faisal GGa, Zakaria SMb, Najmuldeen GFc aDepartment of Basic Medical Science, Kulliyyah of Dentistry, International Islamic University Malaysia, bDepartment of Biomedical Science, Kulliyyah of Allied Health Science, International Islamic University Malaysia, cFaculty of Chemical and Natural Resources Engineering, University Malaysia Pahang, Malaysia. ABSTRACT Introduction: Currently, researchers are aiming to explore herbal plants to replace synthetic drugs because herbal plants contain high active compounds and fewer side effects. Our study was done to determine the antibacterial activity of Eurycoma longifolia Jack (E. longifolia) root using ethanol based extract. Methods: Five types of pathogenic bacterial strains were used; Gram-positive (Staphylococcus aureus and Bacillus cereus) and Gram-negative (Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa). Disc diffusion assay and Minimum Inhibitory Concentration (MIC) tests were used to determine the inhibition zone and turbidity of suspension which reflects the antibacterial activity of the extract.Results: The ethanolic extract of E. longifolia Jack root extract showed positive results against Gram-positive bacteria (S. aureus and B. cereus) and Gram- negative (S. typhi). B.cereus and S.typhi showed inhibition zone values of 11.76mm and 14.33mm at the extract concentration of 150mg/ml that were higher than the positive control values (9.00, 12.67mm) respectively. However, E. coli and P. aeruginosa did not show any inhibition by the ethanol-based extract. Conclusion: From the results we can conclude that E.Longifolia root extract possesses antibacterial activity that can be further explored to produce new medicinal products. KEYWORDS: Eurycoma longifolia Jack, antibacterial, root extract INTRODUCTION There is an increasing demand especially from pharmaceutical company to search for new Exploring herbal plant is becoming an ultimate aim medicine to treat diseases, while reducing side effects to cure health problems.1 Herbal and traditional associated with synthetic drugs. Hence, plant-based medicines have already been used since thousands medicine is a good choice as it contains a lot of of years ago and showed improvement in health. secondary metabolites with the greatest effects on Currently researchers are exploring the medicinal biological activities.8 According to Nadia et al. benefits of herbal plants because they contain high (2012),9 isolation of secondary metabolites from the amounts of biologically active compounds and produce root of E. longifolia Jack showed the presence of less side effects2 and also to overcome the emergence eurycomanone, eurycomanol, eurycomalactone, of multidrug bacterial resistance.3 Eurycoma longifolia cathine-6-one alkaloid, phenolic components, tannins, Jack (E. longifolia) also known as Tongkat Ali, Bidara quassionoids, and tripertenes. In this study, the main Pahit, Bba binh, and Hau phat (Vietnamese) can be goal was to explore the antibacterial activity of E. found in Malaysia, Cambodia, Sumatra and Borneo. It longifolia Jack root using an ethanol based extraction belongs to family Simaroubacae and has a very bitter method. taste. E. longifolia Jack root is known to have many medical properties such as aphrodisiac,4,5 anticancer6 MATERIALS AND METHODS and antimalarial.7 Plant materials: The E. longifolia Jack root used in this study was purchased from a certified Corresponding author: supplier. The sample was dried in the oven at 40oC until constant weight was achieved. After that, the dried plant Asst. Prof. Dr. Ghasak Ghazi Faisal materials were grinded into powder using a dry Kulliyyah of Dentistry grinder and 35.7404 g was obtained. The active Basic Medical Sciences Unit compounds of the sample were extracted with Jalan Sultan Ahmad Shah absolute ethanol around its boiling point at 60 to 65 Indera Mahkota ºC using Soxhlet extraction method. It was done for Kuantan approximately 24 hours. Then, the extracts were Pahang, Malaysia dried under vacuum and reduced pressure using rotary Telephone: +60105442293 evaporator at 60°C and at a pressure of 175 mbar. This Email: [email protected] was done to evaporate the solvents and to obtain the crude extracts. The weight of final extract was 11.8 g and the percentage yield of ethanol extract of E. Volume 14 Number 1, June 2015 77 IMJM THE INTERNATIONAL MEDICAL JOURNAL Malaysia longifolia Jack was 33%. The residue obtained were photometer. Finally, the resultant broth suspensions placed in universal bottle and stored in freezer at -4°C contained 107 CFU/ml for bacterial spp. was used for all until further test was conducted. Finally, the crude experiments performed. Suspensions were always extracts of root E. longifolia Jack materials were agitated thoroughly before OD measurement and used in the antibacterial assay. The working stocks for inoculation. the crude extract of root of E. longifolia Jack were prepared at 50 mg/ml, 100 mg/ml and 150 mg/ Agar Disc Diffusion Assay: The Kirby-Bauer Disc ml by dissolving 100 mg, 200 mg and 300 mg of the Diffusion method was chosen to determine the crude extracts into 2 ml of 25% ethanol respectively. anticipated antimicrobial activity of E. Longifolia Erythromycin and Ciprofloxacin containing discs were Jack root.11 Five pathogenic bacterial strains were used as positive controls for treatment of Gram- swabbed into the MH agar to grow the bacteria for a positive and Gram-negative bacteria respectively. night in the incubator at 37 oC. The filter paper discs loaded with crude extract of E. longifolia Jack root and Bacterial strains: Reference strains of human reference antibiotic-containing disc (5 µg/disc pathogens were used, including Gram-positive ciprofloxacin and 5 µg/disc erythromycin) were laid bacteria, Bacillus cureus (ATCC 25923) and down on the inoculated agar plates using sterile Staphylococcus aureus (ATCC 14778), while Gram– forceps with gentle pressing to ensure a good negative bacterial strains were Pseudomonas adherence to the agar surface. The discs were aeruginosa (ATCC 27853), Escherichia coli (ATCC distributed to be at least 15mm from the edge of 25922), and Salmonella thypi (from Institute of the plate and no closer than 24mm from center to Medical Research Health Ministry, IMR). For the anti- center. Finally, the plates were inverted upside downward bacterial assay, the bacteria were grown on the Muller and incubated at 37oC for 24 hour. After the incubation Hinton (MH) broth and agar and put in the incubator at period, the zone of inhibition (mm) around each 37 oC and observed the turbidity. disc was measured using ruler and compared with reference antibiotics used. Adjustment of Microorganism Number and Inoculums Preparation: Direct Colony Broth Microdilution test (MIC): Microdilution suspension method was used to prepare inoculation method12 was used in this study to identify the suspension.10,11 Barium Sulfate (0.5 McFarland) of inhibition growth of test organisms through the minimal standard suspension was prepared by adding 0.5 part concentration of root extract. Serial double-fold of 0.048M BaCl2 to 99.5 parts of 0.18M H2SO4 and dilutions were carried out in sterile 96-well plate. agitated vigorously until a homogenous suspension was First, all wells to use were filled with 180 µl of Muller- obtained. The turbidity of the suspension was verified Hinton broth containing microorganisms. Then, 20 µl by measuring the optical density at 600 nm (OD600) by of sample extract were transferred to the first well the spectrophotometer. Proper dilutions were done and mixed. Three fold serial-dilution was performed to get an absorbance value of 0.008 - 0.10 which by transferring 100 µl of the mixture in the first well corresponded to 0.5 McFarland standards. Under into the next consecutive wells, until the end of aseptic condition, five colonies isolated by the row. The last well, 100 µl of the mixture were ignition-sterilized inoculation loop from 24 hour discharged, so the total volume of each well was 100 cultivated agar plates of each microbe were suspended µl. The microplate was incubated at 37 oC for 18 – 24 separately in 20 ml pre-warmed (37oC) broth medium hours. The MIC value was determined by comparing (Nutrient broth (NB) for bacterial strains and kept in the turbidity of the mixture in test wells with blank screw-cap bottles and incubated at 37oC. During the wells. To decrease the occurrence of bias, the test was incubation period, aliquots of 1 ml were taken from the assessed by two examiners who were blinded to the culture at hourly intervals and optical density (OD600 for type of mixture. The test of all sample and control bacterial suspension) were measured using spectro- were performed in triplicates. RESULTS Table I. Antibacterial activity profile of ethanol extract ofEurycoma longifolia Jack root using disc diffusion method _____________________________________________________________________________________________ Zone of inhibition (mm) (Mean ± SD) ________________________________________________________________________________________________________ Extract concentration Positive Control Negative Control Bacteria 50 mg/ml 100 mg/ml 150 mg/ml Erythromycin Ciprofloxacinc 25% Ethanol S. aureus 5.67 ±2 .05 8.78 ± 1.13 10.67 ± 0.82 11.78 ± 0.79 - - B. cereus 5.89 ± 1.80 9.44 ± 1.42 11.67 ± 1.50

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