ANTIBACTERIAL ACTIVITIES OF SELECTED THAI MEDICINAL PLANTS BEARING QUINONOIDS. Sciences, Chulalongkorn University, Bangkok 10330, Thailand

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1 ANTIBACTERIAL ACTIVITIES OF SELECTED THAI MEDICINAL PLANTS BEARING QUINONOIDS Kitthisak Chansukh, 1 Chanida Palanuvej, 1 Nijsiri Ruangrungsi 1,2 1 College of Public Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 2 Department of Phamacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand Abstract: The selected Thai medicinal plants bearing quinonoids were continuous extracted with petroleum ether and ethanol respectively. All of the extracts were investigated for antibacterial activity at 200 mg/ml concentrations by using the agar well diffusion method test against gram-positive, gram-negative bacteria and yeast. The organisms were Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Candida albicans. After incubation for 24 hours, the zone of inhibition was compared with standard of quinonoid compounds at 10 mg/ml. The result showed that petroleum ether and ethanol extracts has low active on gram-negative bacteria (Escherichia coli) or some extract not active tested. However petroleum ether and ethanol extracts for bulbs of Eleutherine americana (Aubl.) Merr. and root of Morinda citrifolia L. showed the high antimicrobial activity on gram-positive, gram-negative bacteria and yeast (Bacillus subtilis, Staphylococcus aureus and Candida albicans). Introduction: Historically, pharmacological screening of compounds of natural or synthetic origins have been the sources of innumerable therapeutic agents. Random screening as tool in discovering new biologically active molecules has been most productive in the area of antibiotics. Even now, contrary to common belief, drugs from higher plants continue to occupy an important niche in modern medicine. Thai medicinal plants namely: Xyris indica L., Cassia tora Linn., Rhinacanthus nasutus (L.) Kurz, Morinda elliptica Ridl., Ardisia elliptica Thunb., Eleutherine americana (Aubl.) Merr., Morinda citrifolia L, Morinda coreia Ham. and Nigella sativa L. are containing the group of Quinonoid compounds including Benzoquinone, Naphthoquinone, and Anthraquinone. There were studied on the antibacterial activity by using the agar well diffusion method in order to provide a knowledge on antibacterial among these plants. Methodology: Plant Materials: Different plant parts (root, fruit, seed, flower, aerial part and bulb) were collected from Thai Traditional drug stores in Bangkok and from various gardens in Thailand. All of the plants were authenticated by Ruangrungsi N. Voucher specimens were deposited at College of Public Health Sciences, Chulalongkorn University, Thailand. Plant extracts: All the plant materials mentioned above were dried in hot air oven at 60 o C and grounded to coarsely powder. Dried powdered of each were continuous macerated with petroleum ether at room temperature for 48 hours, the remaining marc extracted with ethanol 95 % sequentially and then filtered through Whatman number 1 using Buchner funnel. The extracts were further concentrated to dryness 37 th Congress on Science and Technology of Thailand 1

2 under reduced pressure at 50 o C, using Buchi rotary evaporator. The yields from the different extracs were weighed, recorded and dissolved in dimethyl sulfoxide (DMSO) to a concentration of 200 mg/ml. The samples were then stored at -20 o C and further used for antibacterial tests (table 1). Testing microorganisms: Microoganisms used in the determination of antibacterial activities of different plant extracts were as follow gram-positive bacteria Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538P, the gram-negative bacteria Escherichia coli ATCC and yeast Candida albicans ATCC They were obtained from the Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University. Screening extracts for antibacterial activity: The microorganisms inoculum with Mueller Hinton agar (MHA) for bacteria and Sabouraud Dextrose agar (SDA) for yeast. They were inoculated at 37 o C for 24 hours. The turbidity of the culture was adjusted with 0.85 % NaCl sterile at 625 nm to match 0.5 McFarland standard (1 x 10 8 CFU/ml). Antibacterial screening was by a modified agar well diffusion method 1-3, using a two layer agar technique. A 100 µl volume of standard suspension (1 x 10 8 CFU/ml) mixed with 3 ml of sterile seeds agar and poured it on sterile base agar. The plates allowed to dry at room temperature. Agar wells were cut from seeded agar plates by a cork borer (6 mm.) 4. Twenty microliters of plant extracts (200 mg/ml) were added in each well. Twenty microliters of DMSO per well was used as a negative control, alizarin, juglone, lapachol and lawsone (10 mg/ml) were used as positive controls. The agar plates were then covered with lids and incubated at 37 o C for 24 hours at which time the diameters of inhibition (in millimeters) were measured. These measurements included the well diameter. Each extract was tested in triplicate. Results, Discussion and Conclusion: The extraction yields from selected Thai medicinal plants bearing quinonoids showed in Table 1. The petroleum ether extracts from the seeds of Nigella sativa L. provided the highest percent of w/w. and lowest percent from the roots of Morinda citrifolia L. (0.363 w/w). Moreover, the ethanol extracts gave the highest percent from the root of Morinda citrifolia L. (8.743 w/w) and lowest percent from the aerial part of Rhinacanthus nasutus (L.) Kurz (2.317 w/w). The antibacterial activities of different various solvent extracts at the concentration of 200 mg/ml from selected Thai medicinal plants bearing quinonoids showed the means of inhibition zone in Table 2. The petroleum ether extracts from bulbs of Eleutherine americana (Aubl.) Merr. exhibited a great in vitro potential for Bacillus subtilis (17.7±0.6 mm), Staphylococcus aureus (21.0±0.0 mm) and Candida albicans (14.0±0.0 mm) whilst no activity against Escherichia coli. The antimicrobial activity of the ethanol extracts from the root of Morinda citrifolia L. revealed the highest potential for Bacillus subtilis (16.7±0.6 mm) and Candida albicans (13±0 mm). In addition, the ethanol extracts from the bulbs of Eleutherine americana (Aubl.) Merr. showed the highest potential for Staphylococcus aureus (15.0±0.0 mm). DMSO was used as a negative control. 37 th Congress on Science and Technology of Thailand 2

3 The extract of Eleutherine americana (Aubl.) Merr. and Morinda citrifolia L. contains the group of quinonoid compounds including naphthoquinone and anthraquinone respectively. Both solvent extract had great in vitro potential for grampositive bacteria and yeast, but it failed to inhibit the growth of gram-negative bacteria. The zones of inhibition were compared with alizarin, juglone, lapachol and lawsone 10 mg/ml using as positive controls which shown in Table 2. Table 1. Extract yields from Thai medicinal plants bearing quinonoids. Plant Part used Chemical compound % Yield (w/w) Petroleum ether Ethanol Xyris indica L. Flower Anthraquinone Cassia tora Linn. Seed Anthraquinone Rhinacanthus nasutus (L.) Kurz Root Naphthoquinone Aerial part Naphthoquinone Morinda elliptica Ridl. Root Anthraquinone Morinda citrifolia L. Root Anthraquinone Morinda coreia Ham. Root Anthraquinone Ardisia elliptica Thunb. Fruit Benzoquinone Eleutherine americana (Aubl.) Merr. Bulb Naphthoquinone Nigella sativa L. Seed Benzoquinone th Congress on Science and Technology of Thailand 3

4 Table 2. Antibacterial activities of different solvent extracts (200 mg/ml) from Thai medicinal plants bearing quinonoids, negative control and positive controls (10 mg/ml). Plant Part(s) used Solvent extracts Zone of Inhibition (mm.) B. subtilis S. aureus E. coli C. albicans Xyris indica L. Flower Petroleum ether 7.3±0.6 NA NA NA Ethanol 12.3± ± ± ±0.6 Cassia tora Linn. Seed Petroleum ether 8.33±1.52 NA 9.0±1.0 NA Ethanol NA NA NA NA Rhinacanthus nasutus (L.) Kurz Root Petroleum ether 16.0± ±0.0 NA 10.0±0.0 Ethanol 12.7± ± ±0.6 NA Aerial part Petroleum ether 14.7± ±0.6 NA NA Ethanol 13.0± ±0.6 NA NA Morinda elliptica Ridl. Root Petroleum ether 12.0± ±0.6 NA 8.7±0.6 Ethanol 11.0± ±0.0 NA 9.7±0.6 Morinda citrifolia L. Root Petroleum ether 14.3± ±0.6 NA 9.7±0.6 Ethanol 16.7± ±0.0 NA 13±0 Morinda coreia Ham. Root Petroleum ether 14.7± ±0.0 NA 12.7±0.6 Ethanol 12.7± ±0.6 NA 9.3±0.6 Ardisia elliptica Thunb. Fruit Petroleum ether 7.0±0.0 NA NA NA Ethanol 9.7± ± ± ±0.6 Eleutherine americana Bulb Petroleum ether 17.7± ±0.0 NA 14.0±0.0 (Aubl.) Merr. Ethanol 15.7± ±0.0 NA 10.0±0.0 Nigella sativa L. Seed Petroleum ether 12.0± ±0.6 NA NA Ethanol 10.0± ±0.6 NA NA Negative control DMSO NA NA NA NA Positive controls (10 mg/ml) Alizarin 11.0± ± ± ±0.6 Juglone 11.0± ± ± ±2.0 Lapachol 10.0± ±0.0 NA NA Lawsone 25.7± ± ± ±2.1 Data are means ± SD, NA = no activity, Ø 6 mm of well, the extract was tested in triplicate. References: 1. Mathabe, M.C.; Nikolova, R.V.; Lall, N. and Nyazema, N.Z. Antibacterial activities of medicinal plants used for the treatment of diarrhea in Limpopo Province, South Africa. Journal of Ethnopharmacology. 2006, 105, Okeke, M.I.; Iroegbu, C.U.; Eze, E.N.; Okoli, A.S. and Esimone, C.O. Evaluation of extracts of root of Landolphia owerrience for antibacterial activitiy Journal of Ethnopharmacology. 2001, 78, Anesini, C. and Perez, C. Screening of plants used in Argentine folk medicine for antimicrobial activity. Journal of Ethnopharmacology. 1993, 39, Bell, S.C. and Grundy, W.E. Preparation of Agar Wells for Antibiotic Assay. Applied Microbiology. 1968, 16(10), Ruangrungsi N, Phadungcharoen T, Suriyagan S, Sekine T and Fujii Y. Murakoshi I. Anthraquinones from Xyris indica. The Thai Journal of Pharmaceutical Sciences. 1993, 17(4), Choi, J.S.; Jung J.H.; Lee, H.J. and Kang, S.S. The NMR Assignments of Anthraquinones from Cassia tora. Archives of Pharmacal Research. 1996, 19(4), th Congress on Science and Technology of Thailand 4

5 7. Kodama, O.; Ichikawa, H. and Akatsuka T. Isolation and Identification of an Antifungal Naphthopyran Derivative from Rhinacanthus nasutus. Journal of Natural Products. 1993, 56(2), Siripong, P.; Yahuafai, J.; Shimizu, K.; Ichikawa, K.; Yonezawa, S.; Asai, T. et al. Induction of Apoptosis in Tumor Cells by Three Naphthoquinone Esters Isolated from Thai Medicinal Plant: Rhinacanthus nasutus Kurz. Biol. Pharm. Bull. 2006, 29(10), Ismail, N.H.; Ali, A.M.; Aimi, N.; Kitajima, M.; Takayama, H. and Lajis, N. Anthraquinones from Morinda elliptica. Phytochemistry. 1997, 45, Pongnaravane, B.; Goto, M.; Sasaki, M.; Anekpankul, T.; Pavasant, P. and Shotipruk, A. Extraction of anthraquinones from roots of Morinda citrifolia by pressurized hot water: Antioxidant activity of extracts. J. of Supercritical Fluids. 2006, 37, Kanchanapoom, T.; Kasaia, R. and Yamasakia, K. Iridoid and phenolic glycosides from Morinda coreia. Phytochemistry. 2002, 59, Phadungkit, M. and Luanratana, O. Anti-Salmonella activity of constituents of Ardisia elliptica Thunb. Natural Product Research. 2006, 20, Paramapojna, S.; Ganzerab, M.; Gritsanapana, W. and Stuppnerb, H. Analysis of naphthoquinone derivatives in the Asian medicinal plant Eleutherine Americana by RP- HPLC and LC MS. Journal of Pharmaceutical and Biomedical Analysis. 2008, 47, Muhtasib, H.G.; Roessner, A. and Stock, R.S. Thymoquinone: A promising anti-cancer drug from natural sources. The International Journal of Biochemistry & Cell Biology. 2006, 38, Acknowledgements: The authors are grateful to College of Public Health Sciences, Chulalongkorn University for providing research facility. Thanks are also due to Suan Sunandha Rajabhat University for granting K.C. in the form of his M.S. education. The authors wish to thank the Herbal Remedies and Alternative Medicine Task Force of STAR: Special Task Force for Activating Research under 100 Years Chulalongkorn University Fund for financial support. Keywords: Antimicrobial activity, Agar well diffusion, Quinones 37 th Congress on Science and Technology of Thailand 5