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Philippines Has Rich Floral Biodiversity Accompanied with an Abundant Source of Medicinal Plants Easily Accessible in the Locality

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Philippines Has Rich Floral Biodiversity Accompanied with an Abundant Source of Medicinal Plants Easily Accessible in the Locality EVALUATION OF THE CYTOTOXICITY POTENTIAL OF ETHANOLIC EXTRACT OF CHRYSOPHYLLUM Journal of Experimental Research CAINITO USING THE BRINE SHRIMP LETHALITY BIOASSAY March 2021, Vol 9 No 1 Lumogdang LP 1,2*, Bullong LQ 1,2, Nuñeza OM 3,4, Uy MM 3,4 Email: [email protected] 1*Department of Marine Biology, Southern Philippines of Agri-Business [email protected] and Marine and Aquatic School of Technology (SPAMAST), Poblacion Malita, Davao Occidental Philippines 2College of Arts and Sciences, Xavier University-Ateneo De Cagayan, Cagayan de Oro Philippines 3Department of Biological Sciences, Mindanao State University-Iligan Institute of Technology (MSU-IIT), Tibanga, Iligan City Philippines 4Department of Chemistry, Mindanao State University-Iligan Institute of Technology (MSU-IIT), Tibanga, Iligan City Philippines ABSTRACT Philippines has rich floral biodiversity accompanied with an abundant source of medicinal plants easily accessible in the locality. In terms of ethno-medical properties, Chrysophyllum cainito has been used to treat various diseases. In this study, C. cainito leaves were collected and evaluated for cytotoxicity using the Brine Shrimp Lethality Bioassay. The C. cainito leaves were extracted with water, 50:50 ethanol-water, and absolute ethanol to produce the decoction, hydro-ethanolic, and ethanolic extracts respectively. Four concentrations (10, 100, 500, 1000 μg/ml) of the extracts were prepared and tested. The mortality rates of the brine shrimp were observed after 6 and 24 hours. The results showed that all the prepared extracts exhibited active biological activities with the ethanolic and hydro-ethanolic extracts exhibiting greater activities compared to the decoction. The ethanolic and hydro-ethanolic extracts showed toxicity effects after 24-h exposures with LC50 values of 25.85μg/ml and 84.14μg/ml, respectively. The results indicate that the use of absolute ethanol and 50:50 ethanol- water may have successfully extracted the bioactive compounds in the C. cainito that have acted on the brine shrimp. The presence of active components in the extracts indicated the potential of C. cainito as an alternative medicine and hence requires further tests to qualitatively identify the bioactive compounds. Keywords: Alternative medicine, bioactive, ethno-medical, extraction, mortality rate An Official Publication of Enugu State University of Science & Technology ISSN: (Print) 2315-9650 ISSN: (Online) 2502-0524 1 This work is licenced to the publisher under the Creative Commons Attribution 4.0 International License. Lumogdang et al: cytotoxicity potential of ethanolic extract of Chrysophyllum cainito reaching to a height of 15 meters. Branches are the initial assessment of bioactive compounds usually few and slender; the young tips are present in plant extract (Sarah et. al. 2017). This conspicuously copper-colored and hid with assay has been effective as a bioassay template appraised hairs. The leaves of C. cainito can be in prescreening of active cytotoxic and characterized as leathery ovate or oblong with antitumor agents (Meyer et. al. 1982). an estimated 7.5 to 13 centimeters long Moreover, brine shrimp lethality assay provides appeared with a pointed tip, edgeless or a comprehensive analysis on the degree of rounded at the base and has covered underneath cytotoxicity. It is a simple test with no aseptic with golden-brown, silky and soft hairs techniques required. It can easily process a (Shailajan and Gurjar, 2014). large quantity of organisms for statistical C. cainito has several health benefits and validation with relatively minimal quantity of has been chemically tested and proven (Doan sample (Sarah et. al. 2017). and Le, 2020). Various studies were conducted The test uses Artemia salina (Leach) through extractions of its fruit, pulp, seeds, and reaction towards the extracts. The mortality is leaves. In 2002, polyphenolic antioxidants were quantified and the lethality assay is computed. isolated in the C. Cainito fruit (Luo et. al. 2002). The assay is highly capable to detect wide Fruit extracts were tested not only for their spectrum of bioactivity in crude extracts. The antioxidant activity but also for their anti- method has been proven to be predictive of diabetic effect (Hegde et. al. 2016) and gastro- cytotoxicity and pesticide activity (Mentor et. protective activity (Da Rosa et. al. 2019). al. 2014). Previously, the brine shrimp lethality Moreover, a study tested the pulp and seed assay has been utilized in determining the extracts for antimicrobial activity (Oranusi et. cytotoxicity potential of various medicinal al. 2015). The tests have proven that C. cainito plants such as Lantana camara, Chromolaena pulp and seed extracts also have great potential odorata, and Euphorbia hirta (Balinado and as antimicrobial agent for treating enteric Chan, 2017), Kleinhovia hospital (Morilla et. bacterial infections and other selected al. 2015), Acmella grandiflora (Elias et. al. pathogens. In addition, various studies also 2014), Ficus nota (Arquion et. al. 2015), reported on the extraction of the leaves which Phyllanthus niruri and Passiflora foetida lead to successful evaluations of the anti- (Balinado and Chan, 2017). diabetic activity (Koffi et. al. 2009), anti- In this study, the C. cainito Linn leaves inflammatory, anti-hypersensitivity effects were collected in Iligan City and their (Meira et. al. 2014), and also for wound healing decoction, hydro-ethanolic, and ethanolic (Shailajan and Gurjar, 2016). From all these extracts were prepared and tested for their assessments, the medicinal benefits of C. cytotoxicity potential against the brine shrimp cainito are supported. and correlated with the known medicative Furthermore, the root of C. cainito has properties of the plant. The data generated from been utilized as alternative medicine in curing the present work serve as baseline information sterility, sexual asthenia, and asthma; while in targeting specific bioactive compounds seeds were predominantly used to alleviate present in the C. cainito extracts. hemorrhoid and intestinal worms. The bark has been wielded for treating cough, icterus, yellow MATERIALS AND METHODS fever while the fruits are used to treat dental The protocols on performing the decay and avitaminosis. Moreover, the entire experiment were adapted from the modified plant can potentially treat ulcer and varicella protocol from the previous works of Olowa and (Houessou et. al. 2012). Nuñeza (2013). To further scientifically assess the medicinal importance of C. cainito Linn, a Botanical Source and Preparation of simple test on its cytotoxicity potential is highly Extracts desirable. Accordingly, cytotoxicity potential C. cainito Linnaeus, particularly the can be assessed by brine shrimp lethality leaves, was selected because of its known bioassay (BSLT). The Brine shrimp lethality ethno-pharmacological uses based on a (BSLT) assay is an indispensable technique on previous survey and interview with local folks An Official Publication of Enugu State University of Science & Technology ISSN: (Print) 2315-9650 ISSN: (Online) 2502-0524 2 This work is licenced to the publisher under the Creative Commons Attribution 4.0 International License. Lumogdang et al: cytotoxicity potential of ethanolic extract of Chrysophyllum cainito and traditional practitioners in the of C. cainito were prepared: 10 μg /mL, 100 μg communities. The fresh leaves of the plant were /mL, 500 μg /mL and 1000 μg /mL. By using the collected on February 2014 from a local source same amounts of the three extracts, the stock in Iligan City. The extracts from C. cainito solutions were prepared. Then, 36.5 mg, 25.18 leaves were prepared with various extraction mg and 35.4 mg were weighed and separately solvent namely: ethanol, hydro-ethanol (50:50) dissolved with plenty of solvent to prepare the and decoction. The decoction were prepared by 10,000-ppm stock solution. For the extraction cutting clean and fresh plant leaves into of alcohol-based extracts, ethanol was used as miniature sizes and heat to boil in distilled water extraction solvent and allowed to standby to be in a 1:2 ratios for a minimum of 5 minutes. Plant evaporated for two days. The same amounts of samples were gradually chilled at 25o C before Dimethyl sulfoxide (DMSO) were added to the subjected to the process of separation then extracts except for the decoction. From the allow to freeze-dried succinctly to dissipate prepared stock solutions, 1000 ppm, 500 ppm, excess amount of moisture. In preparing the 100 ppm and 10 ppm concentrations from the mixture of ethanolic extracts and hydro- stock solution were prepared by serial dilutions. ethanol, unwilt samples were cleaned in clean For each extract, three replicates were prepared water and the dirt and other foreign matter then while the 5 mL sterile filtered seawater served removed by final rinsing using the sterile water. as the control set up. The Ten nauplii were The cleaned samples were air-dried in a week or added to every prepared extracts whereas until the samples were already fragile upon another 10 nauplii were added to the control set picking. The dried samples were ground using up (sterile seawater). The test tubes were then a sterile electric blender. The powdered plant observed and examined the number of dead samples were weighed, and divided evenly into (non-motile) nauplii in each test tube and was two parts and placed into a glass container; it documented after 6 hours and 24 hours. was then filtered with
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