Abstract: The antibacterial screening of hexane, chloroform and alcohol extracts of leaves of Finlaysonia obovata was carried out against fresh water fish pathogenic bacteria viz., Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio alginolyticus, Staphylococcus aureus, Escherichia Coli, Edwardsiella tarda and Micrococcus Sp. (multidrug resistant strain) by disc-assay method. The hexane and chloroform extracts were found active against four and five pathogens, respectively. Chromatographic purification of active extracts improved the activity profiles. The GCMS analysis of extracts analysis revealed the presence of triterpenes and hydrocarbons as major constituents. The fatty acids composition of the leaf is also studied by FAME analysis. It contains palmitic acid as a major constituent (54.65%). Two important poly unsaturated fatty acids i.e., Linoleic acid (1.54%) and 9,11-Octadecadienoic acid (3.25%) are also present.
INTRODUCTION
Latex bearing plants are found in some 20 botanical families. The most prominent families include Sapotaceae, Moraceae, Compositae, Apocynaceae, Asclepiadaceae and Euphorbiaceae. In total there are about 18,000 species of latex-producing plants. These are divided into three categories i.e., rubber latex plant, petro corp plant and latex plant showing bioactivity. The plant under investigation is in the third category of latex plant showing antimicrobial activity against fish pathogens.
Latex bearing plants were found to show Anthelmintic activity (Amorin et al., 1999), anti-inflammatory activity (Kumar and basu, 1994) and other medicinal activities (Bhatt et al., 2002). Latex-bearing plants are the renewable sources of energy and chemicals (Kalita et al., 2004).
Finlaysonia obovata is a lofty climber with stout branches and obovate or oblong leaves with a common name Lata Rai and belongs to the family Periplocaceae. It is a mangrove plant exuding white latex and found in the tidal flats in India, Burma and Malay, the leaves of which are reported to be eaten as salad in Moluccas.
Mangroves have been a source of several bioactive compounds. Mangrove plants have been used in folklore medicines and extracts from mangrove species have proven activity against human, animal and plant pathogens. Secondary metabolites like alkaloids, phenolics, steroids, terpenoids have been characterized from mangroves and have toxicological, pharmacological and ecological importance (Bandaranayake, 2002; Kokpal et al., 1990).
Taking into account the ever increasing interest of research in the field of natural products we evaluated the efficacy of mangroves and marine algae (Choudhury et al., 2005) for their antibacterial activity against some fish pathogenic bacteria. This paper presents the antimicrobial activity of F. obovata against fish pathogens.
MATERIALS AND METHODS
Plant material: F. obovata is collected from Bhitarkanika area of Orissa (during late winter season) and was identified by Mr. K.S. Murthy, I/C SMP unit, Central Research Unit (AY), Bhubaneswar.
Extraction of plant material: The leaves of the plant (2 kg) were cut, shade dried, powdered and extraction was carried out with different solvents (1:2 vol./vol., thrice) in the increasing order of polarity like; hexane, chloroform, ethyl acetate and alcohol sequentially by soaking overnight at ambient temperature. The extracts were freed from solvent under reduced pressure. The residue thus obtained are finally dried under vacuum and used for in vitro screening of antibiotic activity.
Antibiotic activity testing of extracts/fractions /compounds: The antibacterial assay of extracts (500 μg/6 mm disc) were carried out against seven fresh water fish pathogenic bacteria viz., Micrococcus sp. (multidrug resistant strain), Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio alginolyticus, Staphylococcus aureus, Escherichia coli, Edwardsiella tarda by disc-assay method (Acar, 1980). The test bacterial pathogen cultures were obtained from the stock cultures maintained in the Pathology Laboratory of Central Institute of Fresh Water Aquaculture, ICAR, Bhubaneswar (Vimala et al., 2000).
Briefly, each extract, 500 μg/50 μL of appropriate solvent was applied to sterile filter paper discs (6 mm in diameter). After solvent evaporation the discs were placed on nutrient Agar (Himedia, India) test plates inoculated with the overnight culture of the test pathogen (106 cfu mL-1) in Brain Heart Infusion (BHI) broth. The plates were incubated for 48 h at 37°C. Discs loaded with respective solvent (50 μL) used for dissolution were taken as control after evaporation of the solvent. The zone of inhibition around the disc (average of three experiments) was measured.
Among all extracts, the chloroform extract was found highly active against all except E. coli and E. tarda. So, this extract was further taken up for fractionation, further screening and isolations of secondary metabolites.
Fractionation of chloroform extract and isolation of pure compounds: The active crude chloroform extract was chromatographed on a column packed with silica gel (100-200 Mesh) and the fractions were monitored by Thin Layer Chromatography (TLC). The antibacterial screening of column fractions of chloroform extract (200 μg/20 μL/6 mm disc) was carried out by the same method as above.
The active column fractions were again rechromatographed to find out the sub fractions which contain maximum purity with major components. These sub fractions are again taken to study the antibacterial activity at 200 μg mL-1.
GC and GCMS analysis of chloroform, hexane extracts and FAME of lipids: The powdered material of dry leaves (10 g) was homogenized and successively extracted three times with chloroform-methanol(2:1,v/v) to isolate lipids (Christie, 1982). Crude lipid extracts were purified by folch wash to remove non lipid contaminants (Folch et al., 1957). The chloroform phase was separated from the combined extract, dried over anhydrous sodium sulphate and concentrated under nitrogen atmosphere. The total lipid (10 mg) were dissolved in 4 mL of 5% hydrochloric acid in methanol and 0.5 mL benzene and then the mixture was refluxed in a silicone bath at 80-100°C for 2 h. After cooling, the methyl esters were extracted with petroleum ether, simultaneously neutralized and dried over sodium sulphate-sodium bicarbonate mixture. The solvent was evaporated to dryness at reduced pressure at 40°C in a water bath. These fatty acid methyl esters were analyzed by GC and GCMS for identification.
The hexane extract, chloroform extract and FAME were analyzed on a Shimadzu GC-17A gas chromatographed equipped with FID and a 25 mx0.25 mm, 0.25 μm film thickness, WOT column coated with 5% diphenyl dimethyl siloxane, supplied by J and W (DB-5). Helium was used as the carrier gas at a flow rate of 1.2 mL min, at a column pressure of 42 Kpa. Component separation was achieved following a linear temperature program (120-300°C at 2°C min-1 for 90 min), with a total run time of 120 min. The percentage composition was expressed as mean value of three experiments. The samples were then analyzed on a Shimadzu QP-5000 GCMS fitted with the same column and following the same temperature program as above, using 70 eV ionization voltage (EI). Peak identification was carried out by comparison of the mass spectra with those available in the NIST and WILEY libraries.
RESULTS AND DISCUSSION
Results of the screening showed that the chloroform extract and hexane extract exhibited strong antibacterial activity against the fish pathogens (Table 1). The phytochemical screening of this extract showed positive results towards steroids and terpenoids. So, the antimicrobial activity is due to any of these components. The antibacterial screening of column fractions of chloroform extract (200 μg/20 μL/6 mm disc) was carried out by the same method as above is given in Table 2.
The active column fractions were again rechromatographed to find out the sub fractions which contain maximum purity. These sub fractions are again taken to study the antibacterial activity at 200 μg mL-1 and results are also presented in Table 2.
The results of present study showed that the chloroform extract of leaf has shown strong antibacterial activity against fish pathogens compare to other organic extracts. Literature reveals that the hexane and chloroform extract of the plants are found to show strong antimicrobial activity (Kunle et al., 2003; Katerer et al., 2005; Elzaawely et al., 2005), anti-inflammatory activity (Ebrahimzadeh et al., 2006) etc.
The GCMS analysis of the chloroform extract, hexane extract and FAME of Folch
extract are given in the Table 3-5, respectively.
Hexane extract is found to contain saturated hydrocarbons, unsaturated hydrocarbons
(3.33%), sesquiterpens (3.76%), triterpenes (10%).
| Table 1: | Antibacterial activity screening of leaf extracts of F. obovata leaves (Zone of inhibition in mm including 6 mm disc) |
| Table 2: | Antibacterial activity screening results of column fractions/subfractions of chloroform extract of leaves of F. obvoata leaves. (Zone of inhibition in mm including 6 mm disc) |
| EtA = Ethyl acetate; H = Hexane; (-) = No zone, FR = Fraction, SFR = Sub fraction | |
| Table 3: | GCMS analysis of chloroform extract of leaf |
| RT- Retention time | |
Chloroform extract is found to contain mostly saturated hydrocarbons, unsaturated hydrocarbons, alcohols (10%), sesquiterpens (4.45%), triterpenes (30%). Longiborneol acetate, sesquilavandulyl acetate which are also found in essential oils (Asekun et al., 2003) are present in hexane and chloroform extract. Triterpenes are found in latex and resins of some plants and physiological function of these compounds is generally believed to be a chemical defense against certain pathogens causing human and animal diseases.
The widespread reports in recent years on useful biological activities of tritepenes,
indicate their potential. Triterpenes are found to show antitumor, anticancer,
antiviral, antimicrobial, anti-inflammatory activity (Mahato et al.,
1997). In the present investigation hexane and chloroform extracts of F.
obovata were found to contain triterpene hydrocarbon, urs-12 ene (>30
and 10%, respectively).
| Table 4: | GCMS analysis of hexane extract of leaf |
| RT- Retention time | |
Ursolic acid was reported to be cytotoxic against A-549, L-1210 and KB tumour
cells (Yamagishi et al., 1988). 23-Hydroxy-3-oxo-urs-12-en-28-oic acid
was found to exhibit anti-ulcer properties (Fourie et al.,
1989). So, the antimicrobial activity of present study may be due to terpenes
and sesquiterpenes (Bryon and Eric, 2003). The fatty acids composition of the
leaf is also studied by FAME analysis. It mainly contains palmitic acid as major
constituent (54.65%).
| Table 5: | GCMS analysis of FAME of Folch extract |
| RT- Retention time | |
Two important poly unsaturated fatty acids i.e., Linoleic acid (ω-6, 1.54%) and 9,11-Octadecadienoic acid (3.25%) are also present along with arachidic acid (2.56%).
The non polar extracts of mangroves have shown antibacterial activity against fish pathogens earlier (Choudhury et al., 2005). The present study provides enough data to show the potential of mangrove extracts for the development of anti-pathogenic agents against aquaculture pathogens.
ACKNOWLEDGMENTS
The authors thank Director, RRL, Bhubaneswar for facilities, Mr. K.S. Murthy, I/C SMP Unit, Central Research Institute (AY), Bhubaneswar for identification of the plant. One of us (PMM) thanks DOD for research fellowship. We express our thanks to Dr. Subhashree Choudhury for helping in antibacterial screening.