International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 2(11), 74-77, November (2013) Int. Res. J. Biological Sci. International Science Congress Association 74 Brine Shrimp Lethality Assay of the Ethanolic Extracts of Three Selected Species of Medicinal Plants from Iligan City, PhilippinesLilybeth F. Olowa and Olga M. Nuñeza Dept. of Biological Sci, College of Sci. and Mathematics, Mindanao State University-Iligan Institute of Technology, Iligan City, PHILIPPINESAvailable online at: www.isca.in, www.isca.me Received 21st July 2013, revised 2nd August 2013, accepted 2nd October 2013Abstract The present study was conducted to test for in vivo Brine Shrimp Lethality Assay (BSLA) of the ethanolic extracts of Lantana camara, Chromolaena odorata, and Euphorbia hirta and correlate cytotoxicity results with known pharmacological activities of the plants. Novel cytotoxic, antitumor, and pesticidal compounds can be isolated from potential plant sources through the assessment of cytotoxic activity against brine shrimps. Cytotoxicity was evaluated in terms of LC50 (lethality concentration). Ten nauplii were added into three replicates of each concentration of the plant extract. After 24 hours the surviving brine shrimp larvae were counted and LC50 was assessed. Results showed that the extracts of L. camara, C. odorata, and E. hirta were potent against the brine shrimp with LC50 values of 55, 10, and 100 ppm (g/mL), respectively. It indicated that bioactive components are present in these plants that could be accounted for its pharmacological effects. Thus, the results support the uses of these plant species in traditional medicine. Keywords: Brine shrimp lethality assay, Lantana camara, Chromolaena odorata, Euphorbia hirta, LC50, potent, cytotoxicity. IntroductionMany of today’s drugs have been derived from plant resources. Historically, medicinal plants have provided a source of inspiration for novel therapeutic drugs, as plant derived medicines have made large contributions to the health and well being of humans. According to the World Health Organization (WHO), nowadays, 80% of the world’s population rely on plants for their primary health care. The medicinal value of plants is due to the substances that it contains which produce a physiological action on the human body. Some examples of these substances are alkaloids, essential oils, tannins, resins, and many others. Moreover, some negative effects obtained in the use of local plants as source of medicine are basically due to over-dosage and lack of adequate knowledge of other detrimental by-products contained in some plants. For centuries, Lantana camara L. have been used by folk healers in Asia and South America to treat various dermatological and gastrointestinal diseases, tetanus, malaria, and tumors. Leaf extracts of L. camara have been found to have an antimicrobial, fungicidal, insecticidal, and nematicidal potential. Lantana oil is used for treating skin itches, as an antiseptic for wounds, and sometimes used externally for leprosy and scabies. Chromolaena odorata is mainly used by some tribal groups for the treatment of cuts and wounds where young leaves are crushed. C. odorata has an antihelminthic, antimalarial, anti-inflammatory, antimicrobial, fungicidal, insecticidal, and wound healing properties. Further, Euphorbia hirta is considered to have an antiasthmatic, antibacterial, antifungal, antimalarial, antihelminthic, antitumor, diuretic, and hemostatic activities. In the Philippines, the leaves of E. hirtaare mixed with Datura metel leaves and flowers in the preparation of “asthma-cigarettes”. Some of the traditional medicine involves the use of crude plant extracts which may contain an extensive diversity of molecules, often with indefinite biological effects. However, most of the available information regarding the medicinal potential of these plants is not provided with credible scientific data. For this reason, several researches have been conducted to determine the toxicity of medicinal plants. A general bioassay that appears capable of detecting a broad spectrum of bioactivity present in plant crude extracts is the Brine Shrimp (Artemia sp.) Lethality Assay (BSLA). BSLA is used as an indicator for general toxicity and also as a guide for the detection of antitumor and pesticidal compounds. The low cost and ease of performing the assay and the commercial availability of inexpensive brine shrimp eggs makes BSLA a very useful bench top method10. This assay has been noted as a useful tool for the isolation of bioactive compounds from plant extracts11. In this present study, ethanolic extracts of the three selected medicinal plants collected in Iligan City were tested in vivo for their cytotoxic effect against the brine shrimp nauplii and relate toxicity results with their known ethno-pharmacological activities. In vivo lethality test has been successfully used as a preliminary study of cytotoxic and antitumor agents12. Thus, the findings of this present work would give baseline information on the most promising plant species that could be use as a basis for the development of new tools of great therapeutic importance. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(11), 74-77, November (2013) Int. Res. J. Biological Sci. International Science Congress Association 75 Material and Methods Plant Materials: Fresh leaves of Lantana camara and Chromolaena odorata and whole plant of Euphorbia hirta were collected randomly from different areas in Iligan City. These plants were selected because of their availability (common in wasteland and road side) in the area aside from their medicinal potentials. The plants were identified by comparing it with the herbarium specimens at the Botany Museum of the College of Science and Mathematics of the Mindanao State University-Iligan Institute of Technology, Iligan City. The plants were washed with water and air dried in shade for about one to two weeks. Then air-dried leaves were pounded and kept (at 20°C) in closed plastic containers. Preparation of Plant Extracts: Twenty grams of the fine powder of each plant samples were weighed and added into an Erlenmeyer flask containing 250 mL of 95% ethanol. The solution was covered and shaken every 30 min for about six (6) hours and allowed to stand for about 48 hours in room temperature. Then, it was shaken and filtered using Whatman filter paper (No.1). After filtration, the solvent was removed by evaporation using a rotary evaporator under reduced pressure at temperature below 55°C. An alternative dilution procedure developed by McLaughlin et al.13 were adopted in the preparation of the different dilutions of the plant extracts for BSLA where 20 mg of each extract was dissolved in 2 mL of the solvent. The final concentrations were 1000, 100, 10, and 1 ppm (g/mL). There were three (3) replicates in each concentration. A control test was also prepared. Brine Shrimp Lethality Assay (BSLA): Brine shrimp eggs were obtained from the New Aqua Laboratory in Naawan, Misamis Oriental, as a gift sample for the research work. Filtered, artificial seawater was prepared by dissolving 38 g of sea salt in 1 liter of distilled water for hatching the shrimp eggs. The seawater was put in a small plastic container (hatching chamber) with a partition for dark (covered) and light areas. Shrimp eggs were added into the dark side of the chamber while the lamp above the other side (light) will attract the hatched shrimp. Two days were allowed for the shrimp to hatch and mature as nauplii (larva). After two days, when the shrimp larvae are ready, 4 mL of the artificial seawater was added to each test tube and 10 brine shrimps were introduced into each tube. Thus, there were a total of 30 shrimps per dilution. Then the volume was adjusted with artificial seawater up to 5 mL per test tube. The test tubes were left uncovered under the lamp. The number of surviving shrimps were counted and recorded after 24 hours. Using probit analysis, the lethality concentration (LC50) was assessed at 95% confidence intervals. LC50 of less than 100 ppm was considered as potent (active)14. As mentioned by Meyer and others, LC50 value of less than 1000 g/mL is toxic while LC50 value of greater than 1000 g/mL is non-toxic. The percentage mortality (%M) was also calculated by dividing the number of dead nauplii by the total number, and then multiplied by 100%. This is to ensure that the death (mortality) of the nauplii is attributed to the bioactive compounds present in the plant extracts. Results and Discussion The ethanolic extracts of the three plants tested showed good brine shrimp larvicidal activity. The lethality concentration (LC50) of Lantana camara, Chromolaena odorata, andEuphorbia hirta extracts were 55 ppm (g/mL), 10 ppm, and 100 ppm respectively (table 1). The degree of lethality was directly proportional to the concentration of the extract. Maximum mortalities (100%) were observed at a concentration of 1000 ppm in both Lantana camara and Euphorbia hirtaextracts while that of Chromolaena odorata was at 100 and 1000 ppm. Based on the results, the brine shrimp lethality of the three plant extracts were found to be concentration-dependent. The observed lethality of the three plant extracts to brine shrimps indicated the presence of potent cytotoxic and probably antitumor components of these plants. According to Meyer et al., crude plant extract is toxic (active) if it has an LC50 value of less than 1000 g/mL while non-toxic (inactive) if it is greater than 1000 g/mL. Table-1 The number of shrimp nauplii that survived after treating with the three plant extracts and the percentage mortality Plant Extracts Concentration (ppm or g/mL) Number of Surviving Nauplii After 24 h Total Number of Survivors % Mortality T1* T2 T3 Lantana camara1 7 8 8 23 23% 10 5 6 5 16 47% 100 4 5 5 14 53% 1000 0 0 0 0 100% Chromolaena odorata1 7 7 8 22 27% 10 4 6 5 15 50% 100 0 0 0 0 100% 1000 0 0 0 0 100% Euphorbia hirta 1 10 9 8 27 10% 10 7 8 6 21 30% 100 6 5 4 15 50% 1000 0 0 0 0 100% *T=Trial International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(11), 74-77, November (2013) Int. Res. J. Biological Sci. International Science Congress Association 76 The results showed that the ethanolic extracts of the three selected medicinal plants were potent or active against brine shrimps where Chromolaena odorata was the most active at 10 ppm. In the study conducted by Vital and Rivera15, ethanolic extract of C. odorata leaves was observed as having an antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, and Salmonella typhimurium and more effective than ampicillin even at small amount. According to them, the possible antimicrobial potential of C. odorata could be due to its ability to bind to the cell wall of the bacteria, thereby inhibiting its synthesis probably because of the flavonoids and tannins present in the plant. The flavonoids, triterpenes, chalcones, and steroids isolated from C. odorata through phytochemical screening might be responsible for its pharmacological activities. Lantana camara ranks as the second potent or active plant species against brine shrimps at 55 ppm in this present study. Fatope et al.16 screened for the activity of the leaves, stems, and roots extracts of L. camara on brine shrimp larvae and was able to isolate oleanonic acid, lantadene A, and oleanolic acid which exhibited significant toxicity on the larvae. Zani and others17also found out that L. camara (root extracts) were active in the BSLA. Moreover, they also noted that L. camara inhibit the development of the phytopathogenic fungus Cladosporium sphaerospermum. This could be attributed to the presence of camaraside and lantanoside cytotoxic substances which were already isolated from L. camara18. Previous related studies showed that the essential oil from this plant is active against fungi18 and bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus19. The findings of Medeiros and et al20 indicated that the major compound of L. camara essential oils from leaves and stems are sesquiterpenes which were cytotoxic to V79 mammalian cells and also to Artemia salina, showing 50% lethal concentration (LC50) values from 0.23 g/mL. Their in vitro data suggested that essential oil of this plant may also be effective in treating yeast infection. Further, Dibua et al.21 exhibited that L. camara (and Allium sativum) has an antitubercular activity on multiple-drug-resistant Mycobacterium species which could be associated to the presence of cardiac glycosides enhancing myocardiac contraction, and exerting hypotensive effect by inhibiting Na+ and K ions21. Also the alkaloids, flavonoids, saponins, and oils isolated from this plant might be the cause of its observed activity against the isolates. Alkaloids interferes with the cell division process while flavonoids exert an antioxidant activity against the superoxide radical, thereby inhibiting low density lipid oxidation22,23. The result on the lethality of Euphorbia hirta on brine shrimps is in agreement with other studies where its LC50 values are 71.15 g/mL24 and 118.88 g/mL25. In contrast, the study of Amutha et al.26 have recorded an LC50 values of 0.71, 0.66, 0.41 and 0.03 mg/mL for the stems, leaves, roots, and flowers of E. hirta, respectively against brine shrimps. They also observed that E. hirta extracts showed antibacterial (against Staphylococcus aureus and Micrococcus sp.) and anticandidal (against Candida albicans) activities. The presence of alkaloids, tannins, and flavonoids could be accounted for its antibacterial properties. In the other hand, some studies have shown that E. hirta extracts exhibited selective cytotoxicity against several cancer cell lines. In one study, the extract of this plant species was noted as having an anti-proliferative activity against the normal mouse fibroblast cells. Furthermore, the methanolic extract of E. hirta leaves showed an anti-proliferative activity against Hep-2 cells obtained from human epithelioma of the larynx. Thus, the results on Euphorbia hirta support its use in traditional medicine as well as that of Lantana camara and Chromolaena odorata. Conclusion The leaf extracts of Lantana camara and Chromolaena odorataand whole plant extract of Euphorbia hirta exhibited cytotoxic activity against the brine shrimp and considered as containing active or potent components. This is because their LC50 values are less than 1000 ppm or g/mL. The ethno-pharmacological activities of these plant species are due to the different bioactive compounds present in these plants. Although, BSLA is inadequate in determining the mechanism of action of the bioactive substances in the plant, it is very useful by providing a preliminary screen that can be supported by a more specific bioassay, once the active compound has been isolated. Thus, some useful drugs of therapeutic importance may develop out of the research work. Acknowledgements The present research work was thankful to Dr. Roberto Malaluan of the Chemical Technology Department for the rotary evaporator, Prof. Buenaflor Jimenez of the Department of Biological Sciences for providing brine shrimp eggs, and Mr. Solaiman Olowa for assisting during the sample collection. References1.Okigbo R.N. and Ramesh P., Effects of plants and medicinal plant combinations as anti-infectives, African Journal of Pharmacy and Pharmacology, 2(7), 130-135 (2008)2.Nwachukwu C.U., Ume N.C., Obusi M.N., Nzewuihe G.U. and Onyirioha C.U., The qualitative uses of some medicinal plants in Ikeduru L.G.A. of Imo State, Nigeria, New York Science Journal, 3(11), 129-134 (2010)3.Sasidharan S., Badakhshan M.P., Rameshwar N.J. and Ramanathan S., Comparative study: antimicrobial activity of methanol extracts of Lantana camara various parts, Pharmacogn Res, , 348-351 (2009)4.Sankaran K.V., Lantana camara, Asia-Pacific Forest Invasive Species Network (APFISN), India (2012) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(11), 74-77, November (2013) Int. Res. J. Biological Sci. International Science Congress Association 77 5.Chakraborty A.K., Rambhade S. and Patil U.K., Chromolaena odorata (L.): An overview, Journal of Pharmacy Research, 4(3), 573-576 (2011)6.Stuart G. and Santiago A.S., List of Philippine herbal medicinal plants, www.stuartxchange.org (2012)7.Konan N.A., Bacchi E.M., Lincopan N., Varela S.D. and Varanda E.A., Acute, sub acute toxicity and genotoxic effect of a hydroethanolic extract of the cashew Anacardium occidentale L.), J Ethnopharmacol, 110, 30–38 (2007)8.Pisutthanan S., Plianbangchang P., Pisutthanan N, Ruanruay S, and Muanrit O., Brine shrimp lethality activity of Thai medicinal plants in the family Meliaceae, Naresuan University Journal, 12(2), 13-18 (2004)9.Meyer B.N., Ferrigni N.R., Putnam J.E., Jacobsen L.B., Nichols D.E., and McLaughlin J.L., Brine shrimp: A convenient general bioassay for active plant constituents, Plant Med, 45, 31-34 (1982)10.McLaughlin J.L., Chang C.J., and Smith D.L., Benchtop bioassays the discovery of bioactive natural products: an update, Nat Prod Chem, 383-397 (1991)11.Sam T.W., Toxicity testing using the brine shrimp Artemia salina in: Bioactive natural products detection, isolation, and structural determination, CRC Press, Boca Raton: FL., 442-456 (1993)12.Ramachandran S., Vamsikrishna M., Gowthami K.V., Heera B., and Dhanaraju M.D., Assessment of cytotoxic activity of Agave cantula using brine shrimp (Artemia salina) lethality bioassay, Asian Journal of Scientific Research, , 90-94 (2010)13.Mclaughlin J.L., and Rogers L.L., The use of biological assays to evaluate botanicals, Drug Information Journal, 32, 513–524 (1998)14.Gupta M.P., Monge A., Karitas G., Lopez de Cerain A., Solis P.N., Leon E., de Trujillo M., Surez O., Wilson F., Montenegro G., Noriega Y., Santana A.I., Correa M., and Sanchez C., Screening of Panamanian medicinal plants for brine shrimp toxicity, crown gall tumor inhibition, cytotoxicity and DNA interaction, Int. J. Pharmacol, 34, 123-127 (1996)15.Vital P.G. and Rivera W.L., Antimicrobial activity and cytotoxicity of Chromolaena odorata (L. f) King and Robinson and Uncaria perrottetii (A. Rich) Merr. Extracts, Journal of Medicinal Plants Research, 3(7), 511-518 (2009)16.Fatope M.O., Salihu L., Asante S.K., and Takeda Y., Larvicidal activity of extracts and triterpenoids from Lantana camara, Pharmaceutical Biology, 40(8), 564-567 (2002) 17.Zani C.L., Alves T.A., Silva A.F., Brandão M., Grandi T.M., Smânia E.A., and Smânia A. Jr., Biological screening of Brazilian medicinal plants, Mem Inst Oswaldo Cruz: Rio de Janeiro, 95(3), 367-373 (2000)18.Mahato S.B., Sahu N.P., Roy S.K., and Sharma O.P., Potential antitumor agents from Lantana camara: structures of flavonois and phenylpropanois glycosides, Tetrahedron, 50, 9439-9446 (1994)19.Ross S.A., El-Keltawi N.E., and Megalla S.E., Antimicrobial activity of some Egyptian aromatic plants, Fitoterapia51, 201-205 (1980) 20.Medeiros L.B.P., Rock M.S., Lima S.G., de Sousa Júnior G.R., das G. L. Citó A.M., da Silva D, Lopes J.A.D., Moura D.J., SAFFI J., Mobin M., and Coast J.G.M., Chemical constituents and evaluation of antifungal and cytotoxic activity of Lantana camara L. essential oils, Brazilian Journal of Pharmacognosy, 22, 1259-1267 (2012) 21.Dibua U.E., Odo G.E., Udengwu S., and Esimone C., Cytotoxicity and antitubercular activity of Allium sativum and Lantana camara against mycobacterial isolates from people living with HIV/AIDS, The Internet Journal of Infectious Diseases, 8(1), 1-12 (2010)22.Knekt P., Jarvinen R., and Reunanen A., Flavonoid intake and coronary mortality in Finland: a cohort study, Biomed. , 312, 478-481 (1996) 23.Hertog M.G.L., Feskens E.J.M. and Holman D.C.H., Dietary antioxidant flavonoids and risk of coronary heart disease, The Zutphen Ederly Study, Lancet, 342, 1007-1011 (1993)24.Patil S.B. and Magdum C.S., Determination of LC50 Values of Extracts of Euphorbia hirta Linn and Euphorbia nerifolia Linn Using Brine Shrimp Lethality Assay, Asian J. Res. Pharm. Sci., 1(3), 69-70 (2011)25.Patil S.B. and Magdum C.S., Brine shrimp lethality activity of Euphorbia hirta Linn, International Journal of Pharmacy and Pharmaceutical Sciences, 4(3), 347-348 (2012)26.Amutha S., Rajeh M.B., Zuraini Z., Sasidharan S., and Latha L.Y., Assessment of Euphorbia hirta L. leaf, flower, stem and root extracts for their antibacterial and antifungal activity and brine shrimp lethality, Molecules, 6008-6018 (2010)