African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12487

Full Length Research Paper

Isolation and partial characterization of the active metabolite of ascidian, Polyclinum madrasensis from the Palk Bay Region, Southeast coast of India

Kathirvel Iyappan
  • Kathirvel Iyappan
  • Tunicate Biology Laboratory, Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai - 608 502, TN, India.
  • Google Scholar
Gnanakkan Ananthan*
  • Gnanakkan Ananthan*
  • Tunicate Biology Laboratory, Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai - 608 502, TN, India.
  • Google Scholar


  •  Received: 19 June 2014
  •  Accepted: 18 November 2014
  •  Published: 28 November 2014

 ABSTRACT

Ascidians are rich source of bioactive agents which could be used for novel antimicrobial drugs. The present investigation inspects the antibacterial potential of ascidian, Polyclinum madrasensis collected from Mandapam, the Palk Bay region, Southeast coast of India. The crude extracts were tested for inhibition of bacterial growth against human pathogens.  Antibacterial assay was carried out by agar well diffusion method. The maximum inhibition zone (12.0±0.5 mm) was observed against the Staphylococcus aureus in crude methanol extract. The consequent zone of 6.5±0.1 mm was observed against S. aureus in ethanol extract and minimum inhibition zone (3.2±0.5 mm) was noticed with Pseudomonas aeruginosa. Molecular weight of tissue protein was determined through sodium dodecyl sulphate polyacryalamide gel electrophoresis (SDS-PAGE) and active metabolites were characterized by Fourier Transform Infrared spectroscopy (FTIR) analysis. The protein bands were at 36.5, 20.5, and 10.5 kDa, in SDS-PAGE and O-H stretch carboxylic acid compounds identified at the peak 3533.59 cm-1. It could be concluded from the present study that crude extract of the ascidian, P. madrasensis has potential antimicrobial effect against human pathogens.

 

Key wards: Polyclinum madrasensis, bioactive compounds, Fourier Transform Infrared spectroscopy (FT-IR), sodium dodecyl sulphate polyacryalamide gel electrophoresis (SDS-PAGE).


 INTRODUCTION

It is a real fact that the importance of marine organisms as a source of new substances is growing. With marine species comprising approximately a half of the total global biodiversity, the sea offers an enormous resource for novel compounds (Blunt et al., 2011). A very different kind of substances have been obtained from marine organisms among other reasons because they are living in a very exigent, competitive and aggressive surrounding and are very different in many aspects from the terrestrial environment, a situation that demands the production of quite specific and potent active molecules (De Vries, 1995). Peptidic compounds analyzed are obtained from very different marine organisms exhibiting different chemical structures and displaying a large variety of pharmacological effects on specific targets (Aneiros and Garateix, 2004). In marine invertebrates so far, appro-ximately 7,000 marine natural products have been reported (Venkataraman and Wafer, 2005). Ascidians have attracted attention as a source of antimicrobial proteins because they are marine sessile, filter feeding invertebrate which hold a phylogenetically strategic position close to the origin of the vertebrate line. The group of ascidians is one of the most compelling sources of metabolites both of chemical and biomedical interest in the marine environment (Blunt et al., 2011). Antitumor, antiviral and immunosuppressive active compounds are primarily isolated from tunicates (Schmitz et al., 1993).

Bacterial infection cause high rate of mortality in human population (Kandhasamy and Arunachalam, 2008). During the last few decades, the number of upcoming infectious disease is increasing in the developing countries (Murugan and Mohan, 2011). Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, are well known to be causative agents for boils skin infections, abscesses, dysentery and diarrhoea (Levin, 1987; Ananthanarayanan and Paniker, 2008). Presently, various ascidians such as Botryllus sp., and Didemnum sp. have been reported for producing anti-cancer drugs (Azumi et al., 1990). Halocyamine A, an antimicrobial substance was isolated from haemocytes of the solitary ascidians Halocynthia roretzi (Azumi et al., 1990). Ascidian-based products may be a new boon to the development of antibiotics effective against antibiotic resistant strain. Then, attention has focused on ascidians because of their biologically active metabolites and the chemistry of ascidians has become one of the most active fields of marine natural products; it has been amply demonstrated that these sea creatures are prolific producers of unusual structures with significant bioacti-vities (Faulkner, 2002; Blunt, 2010).

In the arena of marine habitat the colonization process is affected by organic metabolites produced by the host organism. Such metabolites may affect bacteria in a number of ways, ranging from the induction of chemo tactic responses to the inhibition of bacterial growth or cell death (Bell and Mitchell, 1972). In this study, we evaluated the antimicrobial potential of the ascidian, Polyclinum madrasensis and partially characterized its biologically active metabolites through sodium dodecyl sulphate polyacryalamide gel electrophoresis (SDS-PAGE) and Fourier Transform Infrared spectroscopy (FT-IR).


 MATERIALS AND METHODS

Collection

The ascidian, P. madrasensis was collected from Mandapam (Lat.  0957’ N: Long79 11’ E) Palk Bay region, Southeast coast of India.  The sample was collected by hand-picking during low tide season and  transported  to  the  laboratory with  safe  condition.  Molluscan  shell, calcrete rock fragments attached to the foot of the animal was carefully removed. They were identified using key to identification of Indian ascidians (Meenakshi, 1997).

Preparation of ascidian extract

The fresh tissues of P.madrasensis were freezed at -20°C and used for the extraction of antimicrobial metabolites. Their soft bodies were removed by breaking the outer layer. The whole body tissues of the sample (50 g) were cut into small pieces and the tissue sample was used for extraction using methanol and ethanol. The extracts were cold steeped over night at -18°C and filtered with What man No. 1 filter paper. The filtrate was evaporated to dryness in rotary evaporator (Becerro, 1994; Wright, 1998). The dried crude extracts were used for antibacterial assay against human pathogens (P. aeruginosa, E. coli, S. aureus, Salmonella typhi, Vibrio cholera, Vibrio parahaemolyticus, Klebsiella pneumoniae and Proteus mirabilis). All the pathogenic bacterial strains were obtained from the Department of Clinical Pathology, Raja Muthiah Medical College, Annamalai University, Tamil Nadu, India.

Antibacterial assay

The ascidian, P. madrasensis crude extracts were tested for inhibition of bacterial growth against human human pathogens. Antibacterial assaywas carried out by agar well diffusion technique (El-Masry et al., 2000). Human pathogens were inoculated in sterile nutrient broth and incubated at 37°C for 24 h. Pathogens were swabbed on the surface of the Muller Hinton agar plates and wells were punched out using a sterile cork borer (6 mm diameter). About 50 µl of the different solvent of ascidian, P. madrasensis extracts, were transferred in to each well. For each pathogen, controls were maintained where pure solvents were used instead of the ascidian extract.

The plates were incubated at 37°C for 24 h (Mtolera and Semesi, 1996). The results were obtained by measuring the inhibition zone diameter for each well and expressed in millimetre (Mohamed et al., 2010).

Muscle protein molecular weight determination by SDS-PAGE

Molecular weight of the muscle protein of P. madrasensis was determined using SDS-PAGE following the procedure by Sambrro et al. (2006). Glass plates were assembled and 20 ml of 15% resolving gel was prepared and poured immediately to the notch plate. It was over laid with butanol. After polymerization was completed, it was poured off and the top layer was washed with deionized water. Then it was over laid with 8 ml of stock gel. Approximately 1 ml of 1% SDS gel loading buffer and sample were taken and it was heated at 100°C for 3 min. Then, it was carefully fixed in electrophoresis apparatus. 15 µL of samples with different molecular weight markers (14.0 to 97.4 kDa) were loaded, respectively in the well, run in the gel and stained with coomassive brilliant blue.

Fourier transform infrared (FT-IR) spectral analysis

The lyophilized methanol extract of P. madrasensis (10 mg) were mixed with 100 mg of dried potassium bromide (KBr) and com-pressed to prepare as a salt disc. The disc was then read spectro-photometrically (Bio-Rad FTIR-40-model, USA). The frequencies of different components present in sample were analyzed.


 RESULTS

Antibacterial activity

Two crude extracts of ascidian, P. madrasensis were screened against eight human bacterial pathogens for testing their antibacterial activities. The inhibition zones of methanol and ethanol extracts against the specific test organisms were shown in Table 1. The methanol extracts showed high antibacterial activity against S. aureus (12.0±0.5 mm), followed by E. coli (8.2 ±0.5 mm), P. aeruginosa (6.5±0.2 mm), V. parahaemolyticus (6.5±0.8 mm), V. cholera, (5.5±0.6 mm) K. pneumonia (5.5±0.8 mm) P. mirabilis (5.5 ±0.8 mm) and S. typhi (4.3±0.7 mm). The minimum inhibition zone (3.2±0.5 mm) was noticed with P. aeruginosa in ethanol extract. However, maximum zone of inhibition range between 5.0±0.1 - 5.0±0.8 mm against E. coli, S. aureus and V. parahaemolyticus in ethanol crude extract. Other indicated organisms were recorded as having meagre activity in ethanol extract.

 

 

Muscle protein molecular weight determination by SDS-PAGE

Crude protein sample of ascidian, P. madrasensis yielded three bands ranging from 25.0 to 110.0 kDa with well defined. The bands were at 36.5, 20.5 and 10.5 kDa, respectively. Ascidian, P. madrasensis sample was com-pared with the standard protein molecular weight marker (14.4 to 97.4 kDa) (Banglore Genei, India) (Figure 2).

 

FT- IR spectral analysis

The FTIR spectrum of the ascidian methanol extracts revealed characteristics of functional groups (Figure 1) such as O-H stretch carboxylic acid compounds, identified at the peak 3533.59 cm-1 and C-H stretching peak of alkanes group at 2954.95, 2922.16 and 2850.79 cm-1, respectively. The –C (triple band) C–stretch peaks of alkenes were at 1641.42 and 1631.78 cm-1. The peaks of 1458.18 and 1402.25 cm-1 indicate C-C stretch aromatic. Peak values were recorded at 1014.56 and 1083.99 cm-1. Absorption indicated C-N stretch aliphatic amines.

 


 DISCUSSION

Marine organisms have been recognized as a rich source of novel compounds that are of potential interest to man-kind (Faulkner, 2000). They produce secondary meta-bolites and other compounds to repel and deter predators

(Pawlik et al., 2002). Most of these pristine resources have not been explored for bio prospecting and microbial ecological studies. The ascidians are a potential chemical, ecological phenomenon, which provides sustainable source of supply for developing novel pharmaceutical leads. Antibacterial activity has been previously reported from extracts of some ascidians. Overall, ascidian extracts caused growth inhibition in gram positive and gram negative bacteria, indicating that these extracts do not selectively inhibit one group of microorganism (Thompson et al., 1985).                      

In the present study, a pronounced antimicrobial effective has been observed against some human patho-gens. Both methanol and ethanol crude extract of P. madrasensis inhibit growth of human clinical pathogens. The maximum activity was observed against S. aureus (12.0±0.5 mm) and minimum activity was observed against P. aeruginosa (3.2±0.5 mm). Similarly, maximum activity was exposed by methanol extract in S. aureus and minimum activity was showed by ethanol extract in P. aeruginosa which was collected at Tuticorin coast (Bragadeeswaran et al., 2010). Selva Prabhu et al. (2012) have reported that the crude methanol extract of P. madrasensis of the maximum inhibition zone of against E. coli and the minimum of P. aeroginosa and ethanol extract produced E. coli and P. aeruginosa. It supports this work. In this present study, we used -18°C cold steep extract.

In the present investigation, the tissue extraction from P. madrasensis showed antimicrobial activity and it was subjected to SDS-PAGE in order to estimate the molecular weight. After electrophoresis, clear bands were detected in the gel which represented molecular weight of proteins ranging 11 to 97 kDa. Green et al. (2003) reported that the molecular weight of protein from hemolymph of the solitary ascidian, Styela plicata from Australian waters is 43 kDa. The endoderm specific alkaline phosphate proteins with molecular masses of 86 and 103 kDa were likewise reported by Kumano et al. (1996). The amount of carbohydrate, protein, lipid and minerals such as phosphorous and calcium contents in these ascidians were previously reported by Rajesh and Ali (2008) and Natarajan et al. (2011). FT-IR analysis reveals the presence of bioactive compound signals at different ranges. Previously, aliphatic chain was identified as the peak of 3394.43 and 2920.40 cm-1 in ethanol extract of P. madrasensis by Meenakshi et al. (2013). FT-IR spectroscopy is proved to be a reliable and sensitive method for detection of biomolecular composition (Komal Kumar and Devi Prasad, 2011). This study shows that the medicinal value of the ascidian P. madrasensis tissue may be due to high quality of bioactive metabolite.

The present study revealed that the species of P. madrasensis showed antibacterial activities against the human pathogens. So, they possess potential pharmacological action.  


 CONFLICT OF INTEREST

The author(s) have not declared any conflict of interests.


 ACKNOWLEDEGMENT

The authors thank to Dean and Director of CAS in Marine Biology and authorities of Annamalai University for provi-ding  the  facilities. Also, authors  gratefully  acknowledge the financial assistance of major research project (F.No.42-603/2013 (SR) dt.22.03.2013) by the University Grants Commission, New Delhi, India.



 REFERENCES

Ananthanarayanan R, Paniker CKJ (2008). Text book of Microbiology. Universities Press Pvt. Limited. Hyderbad. pp:192-201,319-323.
 
Aneiros A, Garateix A (2004). Bioactive peptides from marine sources: pharmacological properties and isolation procedures. J. Chromatogr. B 803:41-53.
Crossref
 
Azumi K, Yoshimizu M, Suzuki S, Ezura Y, Yokosswa H, (1990). Inhibition effect of halocyamine, an antimicrobial substance from ascidian hemocytes, on the growth of fish viruses and marine bacteria. Cell Mol. Life. Sci. 46(10):1066-1068.
Crossref
 
Becerro MA, Lopez NI, Turon X, Uriz MJ (1994). Antimicrobial activity and surface bacterial film in marine sponges. J. Exp. Mar. Biol. Ecol. 179:195–205.
Crossref
 
Bell W, Mitchell R (1972) Chemotactic and growth responses of marine bacteria to algal extracellular products. Biol. Bull. (Woods Hole) 143:265–277.
Crossref
 
Blunt JW (2010). Structure determination: Molecules under the microscope. Nature Chem. 2: 799-800.
Crossref
 
Blunt JW, Copp BR, Munro MHG, Northcote PT, Prinsep MR (2011). Marine natural products. Nat. Prod. Rep. 28:196e, 268 and previous issues in this series.
Crossref
 
Bragadeeswaran S, Ganesan K, Sri Kumaran N, Thangaraj S, Suganthi K (2010). Antibacterial and Cytotoxic Activities of Ascidians Polyclinum madrasensis Sebestian, 1952 and Phallusia nigra Savigny, 1816 from Tuticorin Coast of India. World Appl. Sci. J. 9(12):1387-1391.
 
El-Masry HA, Fahmy HH, Abdelwahed ASH (2000). Synthesis and antimicrobial activity of some new benzimidazole derivatives. Molecules 5:1429-1438.
Crossref
 
Faulkner DJ (2000). Marine natural products. Nat. Prod. Rep. 17:7-55.
Crossref
 
Green PL, Nair SV, Raftos DA (2003). Secretion of a collection-like protein in tunicates is enhanced during inflammatory responses. Dev. Comp. Immunol. 27:3-9.
Crossref
 
Kandhasamy M, Arunachalam KD (2008). Evaluation of in vitro antibacterial property of seaweeds of southeast coast of India. Afr. J. Biotechnol. 7(12):1958-1961.
 
Komal Kumar J, Devi Prasad AG (2011). Identification and comparison of biomolecules in medicinal plants of Tephrosia tinctoria and Atylosia albicans by using FTIR. Rom. J. Biophys. 21(1):63-71.
 
Kumano G, Yokosawa H, Nishida H (1996). Biochemical evidence for membrane-bound endoderm-specific alkaline phosphatase in larvae of the ascidian, Halocynthia roretzi. Eur. J. Biochem. 240:485-489.
Crossref
 
Levin MM (1987). Escherichia coli that cause diarrhea: enterotoxigenic, enterohemorrhagic and enteroadherent. J. Infect. Dis. 155(3):377-389.
Crossref
 
Meenakshi VK (1997). Ph.D thesis. Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India.
 
Meenakshi VK, Veerabahu C, Roselin KF (2013). GC-MS and IR Studies of ethanolic extract of colonial ascidian- Polyclinum madrasensis Sebastian, 1952. Int. J. Pharm. Bio. Sci. 4(4):(B) 1187- 1198.
 
Mohamed Elanwar H, Osman, Atef M, Abushady Mostafa E, Elshobary (2010). In vitro screening of antimicrobial activity of extracts of some macro algae collected from Abu-Qir bay Alexandria, Egypt. Afr. J. Biotechnol. 9(12): 7203-7208.
 
Mtolera MSP, Semesi AK (1996). Antimicrobial activity of Extracts from Six Green Algae from Tanzania. Current and Trends Marin Botany East African Regional. pp. 211-217.
 
Murugan M, Mohan VR (2011). Evaluation of phytochemical analysis and antibacterial activity of Bauhinia purpurea L. and Hiptage benghalensis L. Kurz. J. Appl. Pharm. Sci. 1(9): 157-160.
 
Natarajan K, Ramanathan S, Sathish R, Amudhdevi C (2011). In Vitro cytotoxic study on crude Extract of marine invertebrate animal Polyclinum Madrasensis Sebastian. Asian J. Pharm. Tech. (1)4:152-154.
 
Pawlik JR, McFall G, Zea S (2002). Does the odor from sponges of the genus Ircinia protect them from fish predators? J. Chem. Ecol. 28:1103-1115.
Crossref
 
Rajesh M, Ali HAJ (2008). Nutritional value and antimicrobial activity of marine ascidian species. Ascidian News 7:206-210.
 
Sambrro JE, Fritsch E, Maniatis T (2006). Appendix-8. In: Russel T. (ed.) Molecular cloning. New York: Cold Spring Harbour Laboratory Press.
 
Schmitz FJ, Bowden BF, Toth SI, Attaway DH, Zaborsky OR (1993). Pharmaceuticals and Bioactive Natural Products. Marine Biotechnology, (Eds.), vol. 1, Plenum Press, New York, p. 270.
 
Selva Prabhu A, Ananthan G, Sathish kumar R (2012). Antibacterial activity of marine ascidian Polyclinnum madrasensis (Sebastian, 1952) against human clinical isolates. Int. J. inst. Pharm. life Sci. 2(3):53-61.
 
Thompson JE, Walker RP, Faulkner DJ (1985). Screening and bioassays active substances from forty marine sponge species from San Digo, California. USA. Mar. Biol. 88:11-21.
Crossref
 
Venkataraman K, Wafer M (2005). Coastal and marine biodiversity of India. Indian J. Mar. Sci. 34(1):57-71.
 
Wright AE (1998). Isolation of marine natural products. In: Cannell RPJ. (ed.) Methods in biotechnology, natural products isolation. New Jersey: Humana Press Inc. pp. 305-408.
Crossref

 




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