Materials
All the chemicals and standard antibiotics were purchased from Hi-Media, Mumbai, India; and al the solvents used were of analytical grade. Precoated silica gel 60 F254 TLC plates and standard phytoconstituents were purchased from Merck, Germany and Sigma Chemicals, USA, respectively.
Bacterial cultures
Reference bacterial strains viz. Enterococcus faecalis (MTCC 439), Staphylococcus aureus (MTCC 96), Escherichia coli (MTCC 119), Klebsiella pneumoniae 1 (MTCC 109), K. pneumoniae 2 (MTCC 530), Pseudomonas aeruginosa 1 (MTCC 647), P. aeruginosa 2 (MTCC 741), Salmonella typhi (MTCC 531), Salmonella typhimurium 1 (MTCC 98), S. typhimurium 2 (MTCC 1251) and Shigella flexneri (MTCC 1457) were obtained from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh. These were maintained on nutrient agar slants except Enterococcus faecalis which was maintained on trypticase soya agar (TSA). All the isolates were sub cultured regularly and stored at 4°C as well as at -80°C by making their suspension in 10% glycerol.
Inoculum preparation
A loopful of isolated colonies was inoculated into 4 ml of peptone water, incubated at 37°C for 4 h. This actively growing bacterial suspension was then adjusted with peptone water so as to obtain a turbidity visually comparable to that of 0.5 McFarland standard prepared by mixing 0.5 ml of 1.75% (w/v) barium chloride dihydrate (BaCl2. 2H2O) with 99.5 ml of 1% (v/v) sulphuric acid (H2SO4). This turbidity is equivalent to approximately 1–2 × 108 colony forming units per ml (CFU/ml).
Plant materials
Seeds of different plants viz. Anethum graveolens, Foeniculum vulgare and Trachyspermum ammi were obtained from the local market of Amritsar. All the plant materials have been deposited vide accession number 6419 (V, VI and VII) in Herbarium, Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, Punjab, India.
Preparation of seed extracts
Aqueous/organic extracts of seeds were prepared by taking the weighed amount of each sample in known volume of water/organic solvent to get the desired concentration (200 mg/ml). Seeds of different plants were surface sterilized using 1% mercuric chloride (HgCl2) and crushed using pestle and mortar. Aqueous extracts of seeds were prepared in three different ways as described earlier [15]. Organic extracts of seeds were prepared using four different solvents with increasing polarity-hexane, ethyl acetate, acetone, and ethanol. Weighed amount of each sample was extracted in known volume of the solvent for 24 h with intermittent shaking. Each extracted material was filtered through Whatman filter paper No. 1 and centrifuged at × 10 000 g for 15 min and the supernatant was used for antibacterial testing.
Effect of grinding
To assess the effect of grinding on antibacterial activity, the seeds were shade dried; powdered using electric blender and their aqueous and organic extracts were prepared using the same methods as described above.
Determination of antibacterial activity by agar diffusion method
Sensitivity of different bacterial strains to various extracts was measured in terms of zone of inhibition using agar diffusion assay (ADA) [16]. The plates containing Mueller-Hinton/Nutrient agar were spread with 0.2 ml of the inoculum. Wells (8 mm diameter) were cut out from agar plates using a sterilized stainless steel borer and filled with 0.1 ml of the extract. The plates inoculated with different bacteria were incubated at 37°C up to 48 h and diameter of any resultant zone of inhibition was measured. For each combination of extract and the bacterial strain, the experiment was performed in duplicate and repeated thrice. The bacteria with a clear zone of inhibition of more than 12 mm were considered to be sensitive. The antibacterial activity of different plant extracts was compared with eight commonly employed antibiotics viz. ampicillin (10 μg/disc), cefixime (5 μg/disc), chloramphenicol (30 μg/disc), co-trimoxazole (25 μg/disc), gentamicin (10 μg/disc), imipenem (10 μg/disc), pipericillin/tazobactam (10 μg/disc) and tobramycin (10 μg/disc).
Minimum inhibitory concentration (MIC)
Minimum inhibitory concentration of the effective seed extracts was worked out by agar dilution method [17]. Nutrient agar plates containing varying concentrations (10–100 mg/ml aqueous extract; 1–50 mg/ml acetone extract) of different seed extracts were prepared and inoculated with 0.1 ml of the inoculum. The plates were incubated at 37°C for 24 h and the lowest concentration of the extract causing complete inhibition of the bacterial growth was taken as MIC. The results were compared with that of control using sterilized distilled water/acetone. The experiment was performed in duplicate and repeated three times.
Bactericidal activity
Bactericidal activity of hot water seed extracts prepared at 40°C at a concentration of 200 mg/ml was measured by viable cell count method [18]. Five ml of 4 h grown inoculum was serially diluted to 10-3 with double strength nutrient broth. Equal volumes of the diluted inoculum and the extract to be tested were mixed and incubated at 37°C. At different time intervals viz. 0, 1, 2, 3 ..., 24 h, 0.1 ml of the mixed suspension was spread on two separate nutrient agar plates and incubated for 24 h at 37°C. The mean number of colonies were obtained and compared with that of control in which the seed extract was replaced with sterilized distilled water. Each experiment was repeated thrice. The results were expressed as number of viable cells as a percentage of control.
Phytochemical screening
The powdered seeds were evaluated for qualitative and quantitative determination of major phytoconstituents i.e. alkaloids, flavonoids, tannins, saponins and cardiac glycosides; which were further confirmed by thin layer chromatography.
Qualitative screening
Alkaloid detection was carried out by extracting 1 g powdered sample with 5 ml methanol and 5 ml of 2N HCl; and then treating the filtrate with Meyer's and Wagner's reagents. The samples were scored positive on the basis of turbidity or precipitation. Flavonoids were tested by heating 1 g powdered sample with 10 ml ethyl acetate over a steam bath (40–50°C) for 5 min; filtrate was treated with 1 ml dilute ammonia. A yellow colouration demonstrated positive test for flavonoids. The presence of tannins was confirmed by boiling 0.5 g powdered sample in 20 ml distilled water, followed by addition of 3 drops of 5% FeCl3 to the filtrate. Development of brownish-green or blue-black colouration was taken as positive for the presence of tannins. Saponins content was determined by boiling 1 g powdered sample in 10 ml distilled water for 15 min and after cooling, the extract was shaken vigorously to record froth formation. Cardiac glycosides were identified by extracting 2 g sample in 10 ml methanol. Five ml of this methanolic extract was treated with 2 ml glacial acetic acid containing 1 drop of 5% FeCl3 solution. This solution was carefully transferred to surface of 1 ml conc. H2SO4. The formation of reddish brown ring at the junction of two liquids was indicative of cardenolides/cardiac glycosides [19].
Thin layer chromatography (TLC)
Identification of major phytoconstituents was further carried out by TLC using pre-coated silica gel 60 F264 plates [20]. Different screening systems were used to obtain better resolution of the components. Standard markers such as atropine, rutin, catechin, glycyrrhizic acid and lanatoside C were co-chromatographed for alkaloids, flavonoids, tannins, saponins and cardiac glycosides, respectively. The developed plates were observed under visible as well as UV light (254 nm and 356 nm). Rf value of each spot was calculated as – Rf = Distance travelled by the solute/Distance travelled by the solvent
Bioautography of extracts
Qualitatively isolated group of compounds which were subjected to thin layer chromatography [20], were also assessed for their antibacterial potential using agar disc diffusion assay. Alkaloids were isolated by mixing 1 g powdered sample with 1 ml of 10% (v/v) ammonia solution and extracted with 5 ml methanol for 10 min on water bath (40°C). It was then filtered through Whatman filter paper No. 1 and the filtrate was concentrated using rotary evaporator. Isolation of flavonoids was achieved by heating 1 g powdered sample with 5 ml methanol on water bath at 40°C for 10 min. The filtrate was then concentrated using rotary evaporator to 1/4th of its original volume. For saponins, one gram powdered sample was extracted with 5 ml methanol by heating on a water bath at 40°C for 10 min. The extract was filtered and evaporated to 1 ml, mixed with 0.5 ml water and then extracted thrice with 3 ml n-butanol. The n-butanol phase was evaporated and concentrated to approximately 1 ml. Tannins were obtained by treating 1 g powdered sample with 10 ml 2 M hydrochloric acid (HCl) and hydrolyzing in boiling water bath for 30 min. The solution was filtered, mixed thoroughly with 1 ml ethyl acetate, and ethyl acetate layer was then discarded. Five drops of amyl alcohol were added and shaken thoroughly. Alcoholic layer was retained and used for antibacterial testing. Cardiac glycosides were isolated by extracting 1 g powdered sample with 5 ml of 50% (v/v) methanol and 10 ml of 10% (w/v) lead (II) acetate solution by heating on water bath at 40°C for 10 min. The filtrate was cooled to room temperature and then extracted twice with 10 ml dichloromethane/isopropanol (3:2). The combined lower phases were filtered over anhydrous sodium sulphate and evaporated to dryness. The residue was dissolved in 1 ml dichloromethane/isopropanol (3:2) and this solution was further used for antibacterial investigations. Sterile discs (4.5 mm) cut out from Whatman filter paper No. 1 were saturated with all the five isolated group of compounds, air dried and used for antibacterial activity testing.
Quantitative analysis
Alkaloids were quantitatively determined according to the method of Harborne [19]. Two hundred ml of 10% acetic acid in ethanol was added to 5 g powdered sample, covered and allowed to stand for 4 h. The filtrate was then concentrated on a water bath to 1/4th of its original volume. Concentrated ammonium hydroxide was added drop wise to the extract until the precipitation was complete. The whole solution was allowed to settle; collected precipitates were washed with dilute ammonium hydroxide and then filtered. The residue was dried, weighed and expressed as the alkaloids.
To estimate flavonoids quantitatively, 10 g powdered sample of each plant material was extracted twice with 10 ml of 80% aqueous methanol at room temperature. The whole solution was filtered through Whatman filter paper No.1, the filtrate was later transferred into crucibles, evaporated to dryness on a water bath to a constant weight [21, 22].
Quantitative determination of saponins was done according to Obadoni and Ochuko [23]. Twenty gram of each powdered sample was added to 100 ml of 20% aqueous ethanol and kept in a shaker for 30 min. The samples were heated over a water bath for 4 h at 55°C. The mixture was then filtered and the residue re-extracted with another 200 ml of 20% aqueous ethanol. The combined extracts were reduced to approximately 40 ml over water bath at 90°C. The concentrate was transferred into a 250 ml separatory funnel, extracted twice with 20 ml diethyl ether. Ether layer was discarded while aqueous layer was retained and 60 ml n-butanol was added to it. Then n-butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath and after evaporation the samples were dried in oven (40°C) to a constant weight. The saponin content was calculated as percentage of the initial weight of sample taken.
Tannin determination was done according to the method of Van-Burden and Robinson [24] with some modifications. Distilled water (50 ml) was added to 500 mg of the sample taken in a 500 ml flask and kept in shaken for 1 h. It was filtered into a 50 ml volumetric flask and made up to the mark. Then 5 ml of the filtrate was pippetted out into a test tube and mixed with 2 ml (10 fold diluted) of 0.1 M FeCl3 in 0.1 N HCl and 0.008 M potassium ferrocyanide. The absorbance was measured at 605 nm within 10 min.
Statistical analysis
All values have been expressed as mean ± standard deviation and the comparison of the antibacterial activity of the samples with standard antibiotics was evaluated by applying t-test. P ≤ 0.05 values were considered to indicate statistically significant difference.
