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Eur opean Rev iew for Med ical and Pharmacol ogical Sci ences 2015; 19: 1895-1899 Antibacterial activity of ovary extract from sea urchin Diadema setosum K. MARIMUTHU, P. GUNASELVAM, M. AMINUR RAHMAN 1, R. XAVIER, J. AROCKIARAJ 2, S. SUBRAMANIAN 3, F.M. YUSOFF 4, A. ARSHAD 4 Department of Biotechnology, AIMST University, Semeling, Bedong, Kedah Darul Aman, Malaysia 1Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, UPM Serdang, Selangor, Malays ia 2Division of Fisheries Biotechnology and Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University, Kattankulathur, Chennai, India 3School of Biological Sciences, Universiti Sains Malaysia (USM), Penang, Malaysia 4Laboratory of Marine Biotechnology, Institute of Bioscience and Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia Abstract. – OBJECTIVE: Sea urchin gonad is Introduction considered as a highly prized delicacy in several countries. It is also rich in valuable bioactive In the modern world, the techniques on the de - compounds including polyunsaturated fatty acids (PUFAs) and β-carotene. This study was velopment of drugs and therapeutic agents are undertaken to examine the antimicrobial proper - greatly improved by various new discoveries and ties of the ovary extract from sea urchin Diadema formulations of medicines from various natural setosum against selected Gram-negative and resources. However, throughout the world, infec - Gram-positive bacteria. tious diseases are still a limiting factor for public MATERIALS AND METHODS: The ovary ex - tract was obtained using two different solvents health. To combat this, many researches have such as methanol and chloroform. The obtained been undertaken to find out an effective method extract was used to examine its potential antimi - to prevent or cure diseases. Pharmacological in - crobial properties against the following 11 bac - dustries have developed a huge number of drugs terial species using the disc diffusion method: and antibiotics. In general, bacteria have the ge - Gram-negative bacteria ( Salmonella typhi, Sal - monella typhimurium, Shigella flexneri, netic ability to transmit and acquire resistance to Pseudomonas aeruginosa, Aeromonas hydrophi - drugs, which are utilized as therapeutic agents. la, Acinetobacter sp, Citrobacter freundii and Hence several attempts have been made to ex - Klebsiella pneumonia ) and Gram-positive bacte - plore new antimicrobial drugs from natural re - ria ( Bacillus subtilis, Staphylococcus epidermidis Staphylococcus aureus sources of land and water. But, till date, these and ). The activity was drug discoveries are not well explored from ma - measured in terms of zone of inhibition (mm). RESULTS: The methanol extract exhibited a rine resources due to lack of knowledge on the higher zone of inhibition against all the bacteria medicinal qualities of the marine resources in - taken for examination. Whereas, the ovary ex - cluding plants and animals. tract obtained by chloroform did not show any Microbial populations in seawater and sedi - S. typhi S. epider - antimicrobial activity against , ments may be as high as 10 6 and 10 9/mL, respec - midis , C. freundii and K. pneumonia . The results 1 indicated that the ovary extract obtained by tively . Marine invertebrates are, therefore, con - methanol extracts are capable of inhibiting the stantly exposed to high concentrations of bacte - growth of pathogenic microbes taken for analy - ria, fungi and viruses, many of which are patho - sis. Moreover, the result indicates the presence genic. The survival of these organisms depends of antimicrobial agents in sea urchin ovary. on the efficient antimicrobial mechanisms to pro - CONCLUSIONS: The study suggests that the ovary extract of D. setosum may be a potential tect themselves against various microbial infec - source of antimicrobial agent for pathogenic mi - tions. During the last decade, there has been an croorganisms. increase in research on marine crustaceans, mol - luscs and echinoderms, with particular interest Key Words: on their secondary metabolites which have desir - Antimicrobial activity, Sea urchin, Diadema 2,3 setosum , Ovary. able antimicrobial properties . Among the echinoderms, D. setosum is one of the most Corresponding Author: M. Aminur Rahman, D.Sc; e-mail: [email protected] 1895 K. Marimuthu, P. Gunaselvam, M. Aminur Rahman, R. Xavier, J. Arockiaraj, et al. widely distributed sea urchin. In the Indo-West methanol or chloroform in a shaker (90 rpm/min Pacific Ocean, where it occurs from the Red Sea at 10°C for 24 h). Then, the crude extract was (Gulf of Suez, Gulf of Aqaba, northern and centrifuged (12.000 g for 5 minutes at 4°C) and southern Red Sea) and the east coast of Africa to the supernatant was collected, followed by pass - Japan and Australia 4. This species can be avail - ing through a 0.2 µm millipore filter and collect - able in both tropical waters and temperate zones ed in a Beckman tube, and stored at –20°C. This including Malaysia 5. sterile filtrate was used for the antimicrobial as - Sea urchin gonad is considered as a highly say through agar disc diffusion method. prized delicacy in Asia, Mediterranean and Western Hemisphere countries including Barba - Bacteria dos and Chile 6. The brown gonads are colored Gram-negative bacteria ( Salmonella typhi, due to the presence of carotenoids and polyhy - Salmonella typhimurium, Shigella flexneri, droxylated naphthoquinones such as Pseudomonas aeruginosa, Aeromonas hydrophi - echinochrome A, which has a potent antioxidant la, Acinetobacter sp, Citrobacter freundii and activity. Diadema setosum has not yet been used Klebsiella pneumoniae ) and Gram-positive bac - as a common edible species in Malaysia. Howev - teria ( Bacillus subtilis, Staphylococcus epider - er, it is reported that an indigenous tribal people midis and Staphylococcus aureus ) were used for “Bajau Laut” from Sabah region (Malayisa) eat the antimicrobial assays. These bacteria were D. setosum roe with boiled rice 7. Gonads of D. grown at room temperature in their respective setosum also are rich in various bioactive com - nutrient broth. pounds including polyunsaturated fatty acids (PUFAs) and β-carotene 7. PUFAs consisted of Antimicrobial Assay eicosapentaenoic acid [(EPA, C20:5) (n-3)] and Antibacterial activity of the extract was tested docosahexaenoic acid [(DHA C22:6) (n-3)], have against various bacterial species using disc diffu - a significant preventive effects on arrhythmia, sion method 10 . In brief, 20 mL sterile nutrient cardiovascular diseases and cancer 8. Moreover, agar was poured into Petri dishes, and then al - β-carotene and some xanthophylls from D. seto - lowed them to solidify at 37°C. Then, 100 µL of sum contains strong pro-vitamin A activity, a 24 h broth cultured bacteria was inoculated. which prevents the tumour cell development 9. Discs of Whatman (No. 1) filter paper were cut Recently Lawrence 10 observed that D. Setosum using an office punching machine and autoclaved contains high level of arachidonic acid (AA) and at 121°C for 15 min. Each sterile disc was then EPA. These existing findings encouraged re - dipped in 100 µL of the ovary extract and placed searchers to explore the medicinal values of sea on the agar plate using flame sterilized forceps. urchin species. Hence, in this study, we attempt - After 30 min, the plates were inverted and incu - ed to examine the antimicrobial properties of sea bated at 37°C for 16-18 h. The diameter of the urchin through its ovary extract due to the pres - zone of inhibition was measured in millimetre ence of valuable bioactive compounds in the (mm). Clear zone of inhibition around the discs ovary. indicated the antimicrobial activity. The assay was performed in three replicates and the data were presented as average of three replicates ± Materials and Methods standard deviation. One hundred µg of strepto - mycin, ampicillin, cephalexin and gentamicin Ovary Collection and Maintenance were used as positive control, whereas the same Matured adults of D. setosum were collected volume of nuclease-free de-ionized water used as from Tanjung Dawai, Sungai Petani, Kedah a negative control. Darul Aman, Malaysia and transported to the laboratory. The ovaries were dissected out and Minimum Inhibitory Concentration stored at –20 ºC for further analysis. (MIC) and Minimum Bactericidal Concentration (MBC) Preparation of Extract and The methanolic extract of sea urchin ovary Protein Quantification showed better antimicrobial activity. Thus, it was Ovary extract was obtained as described by further subjected to examine the minimum in - Abubakar et al 11 . Four grams of ovary was ho - hibitory concentration (MIC) to obtain the lowest mogenized and extracted with 40 mL of 70% concentration of ovary extract, which can inhibit 1896 Antibacterial activity of ovary extract from sea urchin Diadema setosum - i f the growth of bacteria. One hundred µL of the i n c a c c a b e f g d e n g 0 8 8 5 8 6 8 9 0 8 9 i sea urchin ovary extract was serially two-fold di - i 0 5 5 1 5 7 5 2 0 5 2 c s . y 0 0 0 1 0 0 0 0 0 0 0 e luted with 70% methanol. The well plate was in - r m ± ± ± ± ± ± ± ± ± ± ± a a 0 0 7 3 7 7 3 3 0 7 3 n cubated for 18-24 h. The growth of inhibition t 0 3 6 3 6 1 3 8 0 6 8 . n m 6 7 5 6 7 8 7 8 0 4 5 u e was observed visually based on the turbidity of l 2 3 2 2 3 2 1 2 2 G o c the mixture. Minimum bactericidal concentration h c (MBC) was determined to identify the lowest a e b b d d a e e f f d c concentration at which the ovary extract kills the e 5 0 0 7 8 8 8 0 0 8 8 n h i t 1 0 0 1 5 5 5 0 0 5 5 . x bacteria.