GC MS analysis of bioactive compounds and antipathogenic activity in freshwater ampullariidae Pila virens

Transcription

1 Journal of Environment and Life Sciences J Environ Life Sci. September 2017; Vol. 2 (Issue 3): ISSN Research Article GC MS analysis of bioactive compounds and antipathogenic activity in freshwater ampullariidae Pila virens M. Gayathri*, Ramasamy M., Santhiya N. P.G & Research Department of Zoology and Biotechnology, A.V.V.M Sri Pushpam College, [Autonomous], Poondi, Thanjavur, Tamil Nadu, India *For correspondence M. Gayathri, Research Scholar P.G & Research Department of Zoology and Biotechnology, A.V.V.M Sri Pushpam College, Poondi, Thanjavur, Tamil Nadu, India. gayuphd@gmail.com ABSTRACT Objective: Molluscs are highly delicious food and they are also very good source for biomedically imported products. Among the molluscs some have pronounced pharmacological activities or other properties which are useful in biomedical area. The present work has been undertaken for isolation and purification of bioactive compounds extracts from the snail Pila virens and antipathogenic activity. Methods: The chemical compositions of the whole animal methanol extract of P. virens were investigated using GC SHIMADZU QP2010 GC MS. P. virens was screened against five human pathogenic bacteria strains for antibacterial activities. Control discs were with water and solvents to assess the effect of water and solvents on pathogens. The plates were incubated at 37 0 C for 24 h and the antimicrobial activity was measured accordingly based on the inhibition zone around the disc impregnated with gastropod extract. Results: GC-MS analysis of Pila virens whole animal methanol extract revealed the existence of the compound Benzaldehyde, 4-Methyl [9.844], Benzene, 1,3-Bis[1,1-Dimethylethyl]- [14.573], Phenol, 2,4-Bis[1,1- Dimethylethyl]- [21.337] Hexadecanoic Acid, Methyl Ester [30.613]. The inhibition zone ranged from 9 mm to 11 mm. Maximum inhibition zone was 11 mm against P. aeruginosa, S. aureus, E. faecalis and minimum zone was 9mm in S. typhi and 10mm in K. pneumonia. Water extract of P. virens zone ranged from 9 mm to 10 mm. The maximum inhibition zone was 10mm in S. aureus, E. faecalis and the minimum inhibition zone was 9mm in K. pneumonia, P. aeruginosa, S. typhi. Received: 01 August 2017 Revised: 26 August 2017 Accepted: 31 August 2017 Conclusions: Commercial antibiotics are highly effective to kill the bacterial and fungal pathogens involved in common infection. It is worthy to note that the product from natural source is good for health and to avoid side effects. Keywords: Pila virens, Bioactive compounds, GC-MS analysis, Benzaldehyde, 4-Methyl, Antipathogenic activity, Pseusdomonas aeruginosa 80

2 Introduction Many classes of molluscs exhibits bioactive compounds like antitumor, antileukemic, antibacterial and antiviral properties have been reported worldwide. 1-3 Among the molluscs some animals exhibited pharmacological activities or other properties which are useful in the biomedical area. In recent years, great attention has been paid to study the bioactive of natural products because of their potential pharmacological utilization. Most homeopathic medicines are either from plant or animal origin. 4 More than 1,100 antibiotic substances have been isolated from invertebrates. Among these, 50 have found widespread use in the prevention and treatment of bacterial diseases in animal and man. 5 Freshwater gastropods extract are usually complex mixtures of bioactive molecules, including proteins and peptides. In this study, the bioactive compounds of Pila virens have been evaluated using GC-MS. The experimental approach consists of a systematic investigation about the action of crude and purified extract on physiological preparations of whole animals to outline the profile of their biological activities and mechanism of action. The present work has been undertaken for isolation and purification of bioactive compounds and action of the extracts of the snail for its biological activity. Among the molluscs, oysters and mussels are very good source for bioactive compounds considering the importance of the group and paucity of information in this line present study has been undertaken to ascertain the antibacterial activity of extracts from P. virens against various pathogenic bacteria. Materials and Methods Sample collection and extraction Live specimens of freshwater gastropod P. virens was randomly collected by hand picking from In the Coleroon river, Lower anicut reservoir is one of the major fresh water fishery resource areas in Thanjavur District of Tamil Nadu [ N latitude and E longitude] which is selected for the present study. The collected fresh molluscs were preserved with ice and transported to the laboratory and identified by the standard literature of Rao. 6 The methanolic extract of flesh was prepared by the method of Chellaram et al. 7 GC MS analysis sample preparation The specimen was brought to the laboratory and their soft bodies were removed by breaking the shell. The flesh sample was dried using hot air oven at 60 0 C and powdered. 25grams sample was soaked in methanol and maintained for 3 days. The extract was filtered through Whatman No.1 filter paper. The resultant extract was concentrated by using rotary vacuum evaporator with reduced pressure. The resultant extract were then kept in airtight container and stored at 4 0 C for further analysis. Antimicrobial activity sample preparation The P. virens tissue samples was extracted with water and ethanol to 5g of tissue sample, 5 ml of water and solvent were added and ground well with mortar and pestle water and ethanol extract were centrifuged at rpm for 30 min and the supernatants were stored at C until use. In the most of the publications concerning antimicrobial activity in Mollusca, either single body compartment alone, like haemolymph and egg masses, or extracts of whole bodies have been tested for activity. 8 Identification of components Interpretation of mass spectrum GC-MS was conducted using the database of National Institute of Standard and Techniques [NIST11s] and WILEY8 having more patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST11s and WILEY8 library. The name, molecular weight, molecular formula and structure of the component of the test material were identified. 81

3 Antibacterial activity of gastropod extracts In vitro antibacterial activity was determined by agar well diffusion technique, 100 mg of lyophilized powder was dissolved in 1 ml of water. 9 This agar plates were incubated at 37 C for 24 hours. Three replicates were formed for every bacterium, inhibition zone was measured [mm]. 10 Antimicrobial antibiotics disc: Ciprofloxacin were used as antibacterial standards. Gastropod crude extract was tested for inhibition of bacterial and fungal growth against human pathogenic bacteria. Microbial assay was carried out by disc diffusion technique followed by Kelman et al. 11 Five species of pathogenic bacteria namely Klepsiella pneumonia, Pseusdomonas aeruginosa, Salmonella typhi, Staphylococcus aureus, Enteococcus faecalis were obtained from Research Laboratory, Thanjavur was used for screening the antibacterial activity of the gastropod extracts. Pathogenic bacterial strains were inoculated in sterile nutrient broth and incubated at 37 0 C for 24 h. Pathogens were swabbed on the surface of the Muller Hinton agar plates and discs [Whatman No. 1 filter paper with 3 mm diameter] impregnated with 50 µl of gastropod extracts placed on the surface. Control discs were with water and solvents to assess the effect of water and solvents on pathogens. The plates were incubated at 37 0 C for 24 h and the antimicrobial activity was measured accordingly based on the inhibition zone around the disc impregnated with gastropod extract. Results and Discussion Freshwater gastropods are found to be a vital source of useful bioactive substances. These bioactive compounds are involved in various biological functions such as communication, infection, reproduction and self- defense. The studies on the active principles in the P. virens whole animal methanol extract by GC-MS analysis clearly showed the presence of four compounds [Table 1]. The active principles with their retention time [RT], molecular Formula, Molecular weight [MW], and concentration [peak area%] are presented in Table 1. The GC-MS chromatogram of the four peaks of the compounds detected was shown in Figure-1. The mass spectrum and structure of the compounds identified were presented. The total number of compounds identified in methanol extracts. The results revealed that Benzaldehyde, 4-Methyl [9.844], Benzene, 1,3-Bis[1,1- Dimethylethyl]- [14.573], Phenol, 2,4-Bis[1,1- Dimethylethyl]- [21.337] Hexadecanoic Acid, Methyl Ester [30.613]. In the present study a total of two crude extracts from the P. virens was screened against five Figure 1: GC-MS chromatogram of P. virens methanol extracts. 82

4 Table 1: Bioactive compounds identified in the Pila virens. Peak Retention Time Peak Area% Molecular Formula Molecula r Weight Name C8H8O 120 Benzaldehyde, 4-Methyl C14H Benzene, 1,3- Bis[1,1- Dimethylethyl] C14H22O 206 Phenol, 2,4- bis[1,1- Dimethylethyl] C17H34O2 270 Hexadecanoic Acid, Methyl Ester Compound Structure Table 2: Zone inhibition (in mm) of different extracts in freshwater snail [P. virens]. Species Positive control Different extracts of P.virens [Ciprofloxacin] Aqueous Ethanol K. pneumonia 29 mm 9 mm 10 mm P. aeruginosa 30 mm 9 mm 11 mm S. typhi 26 mm 9 mm 9 mm S. aureus 24 mm 10 mm 11 mm E. faecalis 27 mm 10 mm 11 mm human pathogenic bacteria strains for antibacterial activities. After the evaporation of the solvents the extracts were brown [water], yellow [ethanol] in colour. These were used for further determination of antimicrobial activity. The inhibition zones of extracts against the specific test organisms were measured. The extract restricted the growth of pathogens on the media around the impregnated discs. Ethanol extract of P. virens was found active against five species of pathogens. The inhibition zone ranged from 9 mm to 11 mm. The maximum inhibition zone was 11 mm against P. aeruginosa, S. aureus, E. faecalis. The minimum zone was 9 mm in S. typhi and 10 mm in K. pneumonia. Water extract of P. virens zone ranged from 9 mm to 10 mm. The maximum inhibition zone was 10 mm in S. aureus, E. faecalis and the minimum inhibition zone was 9 mm in K. pneumonia, P. aeruginosa, S. typhi. The positive control [Ciprofloxacin] showed activities against to five pathogen bacterial strains. The inhibition zone ranged from 24 mm to 30 mm. Conclusions An attempt has been made to outline the most important aspects of the empirical approach to find new lead compounds from natural product mixtures such as animals. In the present study sixteen chemical constituents have been identified from methanol extract of the whole animal of Pila virens by Gas chromatogram Mass spectrometry analysis. The presence of various bioactive compounds justifies the use of whole animal for various ailments by traditional practitioners. Many antimicrobial screening studies have shown that Gram-negative bacteria are more sensitive than Gram- positive bacteria. Nevertheless in our screening study, water and ethanol extract were highly effective against both groups of bacteria, including several human pathogens, as an interesting activity has been discovered in these molluscs, the bioactive compound nature needs to be specified through more purification, steps and should lead to further studies relating to their antibacterial modes of action otherwise, a microbial origin of 83

5 these active compounds cannot be ruled out. Commercial antibiotics are highly effective to kill the bacterial and fungal pathogens involved in common infection. Water, ethanol and methanol extracts of gastropods used in the present study showed significant antibacterial activity compare with other solvents extraction. It is worthy to note that the product from natural source is good for health and to avoid side effects. Funding: No funding sources Conflict of interest: None declared References 1. Kamiya H, Muramoto K, Goto R, Sakai M, Endo Y, Yamazaki M. Purification and characterization of antibacterial and antineoplastic protein secretion of a sea hare, Aplusia Juliana Toxicon, 1989;27: Anand TP, Edward JKP. Screening for antibacterial activity in the opercula of gastropods. Phuket Mar Biol Centre Spl. 2001;25: Rajaganapathi J, Singh KK. Purification of Anti-HIV protein from purple fluid of the sea hare Bursatella leachii de Blainville. Ar Biotechnol. 2002;4: Bensemir A, Blume M, Schroder S, Lindequist U. Screening of cultivated seaweeds for antibacterial activity against fish pathogenic bacteria. Aquaculture; 2006;252: Gale GO, Kiser JS. Antibiotic resistance theory and practice. Trans NY, Acad Sci Ser. 1967;11: Rao SNV. Indian seashells [Part: 1] Polyplacoptera and Gastropoda. Zoological Survey of India, Occasional. 2003;192: Chellaram C, Gnanambal KM, Edward JKP. Antibacterial activity of the winged oyster, Pteria chinensis [Pterioida: Pteridae]. Indian J Mar Sci. 2004;33: Haug T, Stensverg K, Olsen O, Sandsdalen E, Styrovld OB. Antibacterial activities in various tissue of the horse mussel Modiolus modiolus. Invertebr Pathol. 2003;85: Perez C, Pauli M, Bazerque P. An antibiotic assay by the agar-well diffusion method. J. Actabiologiae. 1990;15: Cruickshank R, Duguide GP, Marmion BP, Sawin RH. Medical Microbiology. 1975;2: 12th Ed., Living Stone. Edinburgh, UK. 84