Bangladesh Journal of Pharmacology Volume: 18; Number 1; Year 2023 Cite this article as: Interino JO, Alombro NC, de Vera PJD. Cytotoxic activity of Centrosema molle leaf aqueous extracts. Bangladesh J Pharmacol. 2023; 18: 33-35. Cytotoxic activity of Centrosema molle leaf aqueous extracts Sir, Currently, about 39% of the approved drugs by the Food and Drug Administration are of natural origin (Boy et al. 2018). The biomedical importance of natural products especially in plants can be attributed to the presence of their diverse secondary metabolites (Atanasov et al., 2021). These secondary metabolites play a key role in biological activities, especially in their cytotoxic activity. Cytotoxic activity is a critical factor for the success of developing novel drugs from natural products. In drug development, the tolerable level of a cell from natural products should be determined before proceeding to experiments in animal models (Bácskay et al., 2018). Moreover, the toxicity test serves as a basis for dosage selection that may involve both in vitro and in vivo setups (Maheshwari and Shaikh, 2016). Plants such as Iphonia aucheri (Shah et al., 2020), Cyperus iria (de Vera et al., 2022) had shown to have cytotoxic activities. Centrosema molle Mart. ex Benth. is a perennial and a common climbing herb and does not have any recorded folkloric medicinal uses yet but it was noted that it has been utilized by the indigenous people in Maguindanao province, Philippines in treating wounds. In Laos, this weed had been used for treating scorpions and snakebites (Chima et al., 2013). In Nigeria, the leaves of this plant had been used for treating skin diseases (Ariwaodo et al., 2012). In addition, a study had been conducted that shows the potential of this plant for wound-healing activity (Ekpo et al., 2011). Thus, this plant shows potential for bioactive properties if proven to be non-toxic to cells. This study determined the in vitro cytotoxic activity of C. molle leaf aqueous extract using brine shrimp lethality assay (Meyer et al., 1982). This preliminary study will help establish the dosage or concentration that will be used for future studies involving the biological activities of C. molle extract in both in vitro and in vivo setups. This also explores as a potential source of novel drugs in the future. The C. molle plant leaves (1 kg) were collected from Pinaring, Sultan Kudarat, Maguindanao, and was authenticated at the Biology Department of Ateneo de Davao University, Davao City. C. molle extract was prepared by suspending 30 g of C. molle leaf powder in 100 mL of deionized water, then was heated at a 60°C water bath (Thermo Fisher Scientific, USA). The water bath temperature was regularly checked to maintain the desired temperature range. After 1 hour of heating, the suspension was filtered through cheesecloth and placed in a beaker. Centrifugation (Thermo Fisher Scientific, USA) at 3,000 rpm for 5 min was done, and the resulting supernatant liquid was placed in a clean amber glass bottle. C. molle extract concentrations were prepared by diluting different amounts of the collected supernatant liquid into different amounts of deionized water to make different concentrations. The presence of secondary metabolites was determined using standard methods by Harborne (1993). Brine shrimp lethality assay was conducted to deter- mine the cytotoxicity of the different concentrations of C. molle extract. The assay began by transferring three thousand microliters (3000 µL) of the different C. molle extract concentrations and control (artificial seawater) in their respective well using a Pasteur pipette. In every microwell, ten brine shrimps were pipetted. After 30  min, 6 hours, and 24 hours of exposure of brine shrimps to the samples and control, the number of them that were alive, impaired, and dead was determined. Probit analysis was employed to generate the median lethal concentrations (LC50) value and 95% confidence inter- vals of each time of exposure of C. molle extract to brine shrimps (Finley, 1952). LC50 was obtained using a regression line by plotting the concentration against the percent mortality on a probit scale. Percent mortality was calculated using the equation: The presence of different secondary metabolites present in C. molle extract was determined. Saponins were found to be absent in the C. molle extract when tested using the froth test (data not shown). On the other hand, carbohydrates, reducing sugar, tannins, flavo- noids, and alkaloids were present. The percent mortality rate of the brine shrimps exposed to C. molle extract increased in concentration and time dependent (Figure 1). Results showed that 10,000 µg/ mL concentration of C. molle extract had the highest percent mortality. Analysis of the results also indicated A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2023; 18: 33-35 Journal homepage: www.banglajol.info; www.bdpsjournal.org Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088; DOI: 10.3329/bjp.v18i1.62627 Letter to the Editor This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor that the correlation coefficient of the logarithm of the concentration to the percent mortality from brine shrimps was 0.992 for the C. molle extract at 24 hours of exposure. This value meets the required value of >0.99 which is used to indicate an almost perfect correlation and the relationship between the ordinate and axis (Akoglu, 2018). Thus, increasing the concentration of C. molle extract higher than 10,000 µg/mL might also increase the percent mortality of the brine shrimps. Data from the brine shrimp lethality assay (Table I) also shows an LC50 of 14,842.9 µg/mL concentration of C. molle extract at 24 hours of exposure. This indicates that a 14,842.9 µg/mL concentration of C. molle extract can kill 50% of the brine shrimps. The LC50 value at 14,842.9 µg/mL concentration of C. molle extract can be the basis for dosage or concentration selection in determining the other biological activities of C. molle extract such as its anti-inflammatory and anti-diabetic activities. The C. molle extract at a concentration of 10,000 µg/mL showed a higher percent mortality rate among the C. molle extract concentrations being tested. Data from brine shrimp lethality assay also shows an LC50 of 14,842.9 µg/mL C. molle extract concentration. The correlation coefficient of the logarithm of the C. molle extract concentrations to the percent mortality of the brine shrimps indicated that as the concentration of C. molle extract increases, the percent mortality rate may also increase. Thus, this implies that the LC50 value of C. molle extract can be used to establish the dosage or concentration to be tested for future studies that involve C. molle extract’s biological activities such as anti- inflammatory and anti-diabetic properties for both in vitro and in vivo setups. Jawhar O. Interino1, Nancy C. Alombro1 and Peter Jan D. de Vera 2 1 Natural Sciences Department, Notre Dame University, Cotabato City, Philippines; 2 Natural Sciences Department, College of Arts and Sciences, Mindanao State University, Maguindanao, Dalican, Datu Odin Sinsuat, Maguindanao, Philippines. Corresponding author: Email: peterjandevera0302@gmail.com References Akoglu H. User’s guide to correlation coefficients. 2018; 18: 91- 93. Ariwaodo JO, Chukwuma EC, Adeniji KA. Some medicinal plant species of Asamagbe stream bank vegetation, forestry research institute of Nigeria, Ibadan. Ethnobot Res Appl. 2014; 10: 541-49. Atanasov AG, Zotchev SB, Dirsch VM, Supuran CT. 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