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 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 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

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Bio-functional… Narlyn C. Castillo et al. particular, snails are relatively successful 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

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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].

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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 organisms. Zone of inhibition was observed against E. coli and S. aureus after 24 hours with an average of 18.16±1.91 mm and 18.66±1.90 mm, respectively. Statistical analysis also revealed significant difference with the negative control, suggesting that the extracts had inhibiting effect against the test organisms. This suggests that J. asperata is a potential candidate for the exploitation of novel antibacterial compounds.

Table 2: Zone of inhibition exhibited by J. asperata mantle extract against E. coli and S. aureus after 24 hours of incubation.

Treatments Zone of inhibition E. coli S. aureus Distilled Water (Negative control) 6.00±.00c 6.00±.00c Streptomycin (Positive Control) 36.32±2.54a 28.24±1.02b Jagora asperata extract 18.16±1.69b 18.66±1.90a Note: Means having the same letter of super script in the same column are significantly different from each other at 5% level of significance using Tukey’s multiple range test.

Figure 1: Zones of inhibition of Jagora asperata against (A) E. coli and (B) S. aureus after 24 hours of incubation with different treatments: (1) distilled water, (2) streptomycin and (3) Jagora asperata extract

The hemolymph and whole-body homogenates of snails have been reported to contain a variety of bio active compounds such as peptides, glycopeptides and glycans [13]. Mucin, epiphragm from molluscan groups [6], have been isolated and found to have strong inhibitory action against viruses, bacteria and protozoan mixed cultures. Cilia and Fratini [14] stated that mucus from snail, slugs and its protein and components have been subjected to some investigations on their antibacterial, antiviral, and antifungal

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Bio-functional… Narlyn C. Castillo et al. activity due to extensive traditional uses and for a future application in medicine. Moreover, it has also been reported that fresh water snail and crabs have good antimicrobial activity against pathogenic microbes [15]. Therefore, they can be used to treat many pathogenic infections.

Cytotoxic Activity of J. asperata: After 24 hours of exposure, LC50 is observed to be at approximately 2.09% concentration of Jagora asperata extract. Moreover, the nauplii placed in 100 percent extract concentration of the extract were recorded 77% mortality rate. On the contrary, the lowest percentage of mortality rate is evident in 50% concentration of the extract with 33% mortality rate. The results indicate that the Jagora asperata ethanolic extract has cytotoxic activity and this may be attributed to its chemical constituents like terpenoids and saponin. Bioassay is significantly used as a pre-screening tool for antitumor drug development due to the significant correlation between the brine shrimp assay and invitro growth inhibition of human solid tumor cell lines [16]. Several cytotoxic compounds have been isolated from snails. Franceschi et al. [17] have identified cytotoxic and immunocyte markers in cells from the freshwater snail Planorbarius corneus (L.).

90 80 70 60 50 40 30 20

10 Percentage Percentage Mortality 0 0 2.09 6.25 12.5 25 50 100

Percent Concentration

Figure 2: Shows the cytotoxicity lethality assay of ethanolic extract of Jagora asperata

CONCLUSION

Jagora asperata, being an endemic Philippine species, is a component of the local ecosystem, and investigation of its bioactivity and secondary metabolites is important. In this study, ethanol extracts of its mantle were found to contain various chemical constituents and possess antibacterial and cytotoxic activities. To the best of our knowledge, this study is the first to present information regarding the bio- functional activities of this endemic species. This preliminary investigation on the bioactivity of J. asperata suggests that these organisms can be further exploited and studied to better elaborate their capacity as novel natural resources of pharmacologically important compounds.

ACKNOWLEDGEMENT

The authors are grateful for the provision of materials and facilities by the Department of Biological Sciences of the Isabela State University, Echague, Isabela. 156 JCBPS; Section B; November 2020 –January 2021, Vol. 11, No. 1; 152-158. [DOI:10.24214/jcbps.B.11.1.15258.]

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Corresponding author: Ron Patrick C. Campos College of Arts and Sciences, Isabela State University, Echague, Isabela [email protected] Online publication Date: 18.01.2021

158 JCBPS; Section B; November 2020 –January 2021, Vol. 11, No. 1; 152-158. [DOI:10.24214/jcbps.B.11.1.15258.]