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Bio-Functional Activities of Jagora Asperata (Lamarck, 1822) Mantle Ethanolic Extracts

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Bio-Functional Activities of Jagora Asperata (Lamarck, 1822) Mantle Ethanolic Extracts JCBPS; Section B; November 2020 –January 2021, Vol. 11, No. 1 ; 152-158, E- ISSN: 2249 –1929 [DOI: 10.24214/jcbps.B.11.1.15258.] Journal of Chemical, Biological and Physical Sciences An International Peer Review E-3 Journal of Sciences Available online atwww.jcbsc.org Section B: Biological Sciences CODEN (USA): JCBPAT Research Article Bio-functional Activities of Jagora asperata (Lamarck, 1822) Mantle Ethanolic Extracts Narlyn C. Castillo, Noviel Delos Santos, James Kennard S. Jacob, Ron Patrick C. Campos* and Jose B. Abucay Jr. College of Arts and Sciences, Isabela State University – Main, Echague, Isabela 3309 Philippines Received: 20 December 2020; Revised: 06 January 2021; Accepted: 18 January 2021 Abstract: Jagora asperata is an endemic freshwater snail species found in the Philippines. Currently, there is little research elaborating the secondary metabolites of the snail and few extensive studies have been conducted to assess the bioactivity of this gastropod. The study was conducted to assess the bio-functionality and secondary metabolite constituents of Jagora asperata (locally called agurong). Results indicate the presence of saponins, tannins, cardiac glycosides, flavonoids, terpenoids and steroids in Jagora asperata ethanol extract. The extracts also showed promising antibacterial activities against Escherichia coli and Staphylococcus aureus, with mean zones of inhibition of 18.16 mm and 18.66 mm respectively. Cytotoxicity test also revealed low toxicity to brine shrimp. With these, Jagora asperata can be a candidate for exploitation and isolation of novel natural products with pharmacological importance. Keywords: antibacterial, cytotoxicity, Jagora asperata, mollusc, secondary metabolites INTRODUCTION Mollusks are highly successful animal group in terms of ecology and adaptation and they are found in nearly all habitats ranging from deepest ocean trenches to the intertidal zone, freshwater and land where they occupy a wide range of habitats [1]. Among mollusks, gastropods represent the most abundant class. In 152 JCBPS; Section B; November 2020 –January 2021, Vol. 11, No. 1; 152-158, [DOI:10.24214/jcbps.B.11.1.15258.] Bio-functional… Narlyn C. Castillo et al. particular, snails are relatively successful animals due to their capacity to adapt to different environments and to reach dry land [2]. This is an indication that snails possess special adaptive chemicals with which they are able to utilize for survival in their environment. Snails have been used as food and their mucus secretions have been utilized as treatment for a variety of medicinal conditions. In the last decade, numerous studies on composition and bioactive components of snails have clarified many aspects of its functional properties, however, much is still to be investigated and revealed from these organisms [3]. The Philippine archipelago, being highly fragmented and isolated, is one of the most biogeographically diverse areas for mollusk research [4]. One of these Mollusk groups is represented by the endemic Philippine freshwater gastropod named Jagora asperata Lamarck, 1822. Locally known as ‘agurong’, it is commonly found in agricultural systems and collected for food [5]. At present, relatively few extensive investigations have been made elucidating the bioactivity of this gastropod, compared to other snails where whole-body homogenates of some have been reported to contain a variety of antimicrobial and antioxidant compounds [3]. There is an increasing need for the development of new and more effective alternative antibiotics and novel compounds from readily available materials such as antimicrobial proteins produced by some animals, an example of which is mucin produced by snails [6]. As of yet, no reports of the bio-functional activities and mantle composition of J. asperata have been reported. With its endemic distribution and potentially promising properties, it is worth determining the antibacterial and cytotoxic activities of compounds extracted from these animals. In this study, the mantle extracts of J. asperata were evaluated for its composition, antibacterial properties and cytotoxic activity. EXPERIMENTAL Collection and Extraction of Samples: Fresh samples of Jagora asperata were collected from Saguday, Quirino, Philippines (16.5549° N, 121.5969° E). Samples were washed thoroughly and crushed to obtain its mantle then oven dried at 40˚C for 48 hours. After oven drying, the samples were pulverized using a mechanical grinder. The pulverized samples were soaked in 1L of 95% laboratory grade ethanol for 48 hours then subjected to steam distillation through the use of rotary evaporator under 60˚C at 150 revolution per minute for 3 hours. The remaining mixtures are refluxed for 3 hours at 70˚C-80˚C until a sticky residue was obtained. Evaluation of Secondary Metabolite Components: The secondary metabolite constituents of J. asperata mantle was evaluated and carried out using ethanol extracts in the study using the standard procedures to identify the various constituents described by Sofowara [7]. Chemical constituents namely alkaloids, saponins, tannins, cardiac glycosides, terpenoids and sterols were evaluated using standard protocols with minor modifications. Antibacterial Properties of Mantle Ethanolic Extract: Bacterial strains of Escherichia coli and Staphylococcus aureus were used in the study. The test organisms were obtained from the bacterial culture collection of Microbiology and Bio-Industry Laboratory, Department of Biological Sciences, Isabela State University, Echague, Isabela. The antibacterial assay was carried out using disc diffusion method of Bauer et al. [8], which involved the use of filter paper discs as carrier for the antimicrobial agents. Sterile Whatman filter discs with 6 mm diameter were impregnated with liquid treatments. At the same time, Petri plates containing 20 mL MHA 153 JCBPS; Section B; November 2020 –January 2021, Vol. 11, No. 1; 152-158. [DOI:10.24214/jcbps.B.11.1.15258.] Bio-functional… Narlyn C. Castillo et al. were seeded with 24-hour culture of the bacterial strains. Afterwards, 50 μl of each liquid treatment was carefully pipetted in each disc then placed equidistantly on the surface of the medium. Zones of inhibition were measured using a calibrated digital Vernier caliper. The assay was conducted in triplicates with the treatments as distilled water (negative control), streptomycin (positive control), and J. asperata ethanolic extract. Cytotoxicity test using Brine Shrimp Lethality Assay: Brine shrimp (Artemina salina) was used as test organism in this study. The cytotoxic property of J. asperata mantle extract was evaluated following the methods described by Mclaughlin and Roger [9]. In Elisa wells, 150 brine shrimps were placed using an air displacement pipette. Three individual brine shrimps were distributed per well and the set-up containing the extracts isolates at different concentrations were left uncovered under a lamp. After 24 hours, total number of brine shrimps were monitored using the stereomicroscope. The experiment was employed using the Probit Analysis and data were computed to get the LC50. For the cytotoxicity, the % concentration crude extract of Jagora asperata are (T4); 6.25, (T3); 12. 50, (T2); 25, 50 and (T1); 100. Statistical Analysis: Statistical analysis was laid out using Completely Randomized Design (CRD) with three replicates per treatment. The results presented were the mean difference of three replicates. The recorded data were treated statistically using one-way Analysis of Variance (ANOVA). The means were compared by Tukey’s Honest Significant Difference test at p< 0.05 using IBM™ SPSS v25. RESULTS AND DISCUSSION Secondary Metabolites of J. asperata Mantle Extract: The ethanolic extract of Jagora asperata mantle was subjected to various chemical tests to determine the presence of the alkaloids, saponins, tannins, glycosides, terpenoids, flavonoids, and steroids. Table 1 shows the different constituents of the mantle extract. Table 1: Secondary metabolites of J. asperata mantle ethanolic extract Constituents Ethanol extract Alkaloids - Saponins + Tannins + Glycosides ++ Flavonoids ++ Terpenoids ++ Steroids + Note: (-) negative; (+) traceable amount; (++) appreciable amount Results indicate the presence of alkaloids, flavonoids, glycoside, terpenoid, saponins, steroids and tannins in the mantle of J. asperata. Biochemical constituents like glycosides, flavonoids and terpenoids were present in appreciable amount while saponins and steroids were both appeared to be present in traceable amounts. However, alkaloids were found to be absent in the ethanolic extract. These classes of compounds (such as alkaloids, saponins, tannins, and flavonoids) are known to have curative activity against several pathogens and therefore could suggest the use traditionally for the treatment of various illnesses [10]. 154 JCBPS; Section B; November 2020 –January 2021, Vol. 11, No. 1; 152-158. [DOI:10.24214/jcbps.B.11.1.15258.] Bio-functional… Narlyn C. Castillo et al. Moreover, according to Siqueira [11], tannins possess antidiarrheal and antimicrobial activities. Flavonoids possess a variety of biological activities, including antioxidant, anti-allergic, anti-inflammatory, antiviral, anti-proliferative, and anti-carcinogenic activities [12]. With the presence of these biochemical constituents, J. asperata shows promising potential to have various bioactive properties. Antibacterial Properties of J. asperata: Results revealed presence of antibacterial activity against both test
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