In vitro antibacterial activity and phytochemical analysis of hexane extract of Vicia sativa

Vicia sativa is traditionally used medicinal plant in skin infections, asthma, bronchitis, urinary diseases and also used as antiseptic, anti-poison, aphrodisiac, anti rheumatic and antipyretic. In the present study n-hexane extract of V. sativa was evaluated for the antibacterial activity against pathogenic bacteria Staphylococcus aureus, Bacillus atrophaeus, Escherichia coli and S. epidermidis by disc diffusion method. Minimum inhibitory concentration of the n-hexane extract against all bacteria was determined by broth dilution method. Preliminary phytochemical analysis and HPLC analysis showed the presence of a number of bioactive constituents which exhibits antibacterial activity. So the current study showed that V. sativa possesses the significant antibacterial activity. Article Info Received: 3 February 2014 Accepted: 19 March 2014 Available Online: 18 April 2014

plant material was filtered and the filtrate was evaporated in rotary evaporator until the solid mass extract was obtained which was stored at 4°C.
Test microorganisms: Antibacterial activity of n-hexane extract of V. sativa was evaluated against pathogenic bacteria Staphylococcus aureus, Bacillus atrophaeus, Escherichia coli and S. epidermidis. These strains were obtained from the Department of Microbiology, Saffron Pharmaceuticals Pvt Ltd.
Antibacterial assay of plant extracts: Antibacterial activity was determined by standard agar disc diffusion method (Heatley, 1944). Sterile filter paper discs were loaded with different concentrations of extract (300 mg/mL, 200 mg/mL and 100 mg/mL) and placed on the surface of inoculated agar plates of bacteria S. aureus, E. coli, S. epidermidis and B. atrophaeus by using sterile forceps. A standard disc of tetracycline was also placed along with extract discs to check the comparison of inhibition of bacterial growth. These plates for antibacterial assay were incubated in incubator at 37°C for 24 hours. After 24 hours, the plates were observed for zones of inhibitions, the diameters of zone of inhibition were determined by digital varnier caliper in mm.

Determination of minimum inhibitory concentration:
Minimum inhibitory concentration of extract against S. aureus, E. coli, S. epidermidis and B. atrophaeus was determined by broth dilution method using 96 microwel plate (Mothana et al., 2009).

Preliminary phytochemical analysis:
The phytochemical screening of the plant extracts were performed by standard procedures as given below: Test for alkaloids: 0.2 g of the extract was added in 2N HCl (5 mL) then heated on the boiling water bath. The mixture was filtered after cooling and the filtrate was divided in the two equal halves. Few drops of Mayer's reagent were added in one portion and the Dragendeoff's reagent was added in other portion. Turbidity of the formed precipitate in the both reagents indicates the presence of alkaloids (Mojab et al., 2003;Sharma et al., 2010) Test for tannins: 0.2 g extracts was dissolved in 10 mL of distilled water and warm on water bath. The mixture was filtered and 5%solution of ferric chloride was added to filtrate. The formation of dark green solution indicates the presence of tannins (Mojab et al., 2003).
Test for saponins: 0.2 g of extract was mixed in test tube with 5 mL of distilled water and warm on water bath until it begins to boil. Formation of foam that persists for 10 min indicates the presence of saponins (Mojab et al., 2003;Sofowora, 1993).
Test for terpenoids: 0.2 g of extract was shaken separately with 2 mL of chloroform (CHCl3), and then 3 mL of concentrated H2S04 was added carefully to form a layer. The formation of reddish brown color at inert face of the solution indicates the presence of terpenoids (Sofowora, 1993;Sharma et al., 2010).
Test for flavonoids: 0.2 g of extract was taken separately and dissolved in 5 mL diluted NaOH then 1M of 5 mL HCl was added. A yellow color solution that changes into colorless solution indicates the presence of flavonoids (Sofowora, 1993).
Test for anthraquinones: 0.5 g of extract was boiled with 10% of HCl in water bath for few min and then filtered. The filtrate was cooled and then equal amount of CHCl3 was added in the filtrate. Few drops of 10%NH3 was added in the mixture and heated. The formation of rose -pink coloration indicates the presence of authraquinones (Mojab et al., 2003;Sharma et al., 2010).
Test for glycosides: 1.2 g of extract was hydrolyzed with 10 mL of 1%HCl and then neutralized by 10% of NaOH solution. Few drops of Fehling's solution A and B were poured in it. Formation of red color precipitates shows the presence of glycosides (Mojab et al., 2003;Sharma et al., 2010).

HPLC of the plant extracts:
Flavonoids and phenolics present in n-hexane extract of V. sativa was determined by some modification in procedure as determined by (Sultana et al., 2008). Sample of plant extract (n-hexane) was prepared by adding small amount of extract in 5 mL distilled water, mix it well then 12 mL methanol was added in it, shake it well then stay for 5 min. 6 mL distilled water was added and again stay for 5 min. 10 mL of 15 M HCl was added to it and place this in digital drying oven for 2 hours. Filter this with syringe filter and analyze it by HPLC. Kaempferol and phenolics were separated using a shim-pack CLC-ODS (C-18) column, 25 cm × 4.6 mm, 5 µm. mobile phase used was acetonitrile and acetic acid at a flow rate of 1 mL/min. Detector used was gradient HPLC detector, range was bipolar, 1250 mV, 10 samples per sec. Threshold was set at 0.005 Mv. Length of column used was 100 mm. Width of peak was 0.2 min. Ethanol was used for calibration. Samples were analyzed by UV-visible detector at 280 nm for phenolics at 248 nm for kaempferol at room temperature.

Results
The results of antibacterial activity of n-hexane extracts of V. sativa are tabulated in Table I, and zones of inhibitions are shown in Figure 1. n-Hexane extract of V. sativa showed maximum antibacterial activity at 100 mg/mL concentration against E. coli (40.7 mm ± 0.8) while tetracycline positive control showed zone of (23.3 ± 0.5) and MIC was 1.6 mg/mL. V. sativa showed maximum antibacterial activity against other bacteria at 100 mg/mL concentration; B. atrophaeus (32.2 mm ± 0.2), S. epidermidis (31.3 mm ± 0.6), S. aureus (25.8 mm ± 0.2) and their MIC were 3.1 mg/mL, 6.3 mg/mL, 6.3 mg/ mL respectively.
HPLC analysis of V. sativa (n-hexane) extract was carried out and HPLC chromatograms obtained is shown in Figure 2 and Figure 3. HPLC chromatogram indicated the peaks of constituents which are present in extract, peaks of chromatotropic acid, gallic acid, quercitin, vanillic acid, syringic acid, vitamin C, trans-4hydroxy-3-methoxy cinamic acid and kaempferol were observed. These constituents with their retention time and quantity are given in Table II.

Discussion
V. sativa traditionally used as antiseptic (Dwivedi et al., 2008), and as an antipoison (Shinwari and Khan, 2000). V. sativa is used against asthma, bronchitis, in urinary diseases and skin infections. In the present research, antibacterial activity of n-hexane extract of V. sativa was determined against four bacteria and their zones of inhibition and MIC were observed. It has been observed that n-hexane extract showed the maximum antibacterial activity at 100 mg/mL concentration. Previous phytochemical studies showed that V. sativa contains various constituents e.g, apigenin (Boulos, 1995), kaempferol (Tschiersch and Hanelt, 1966), Luteolin (Seabra et al., 2001), quercetin (Roy et al., 1996), these compounds are identified by NMR-spectroscopy (Agrawal, 1989). Lectin also has been isolated from the plant (Gebauer et al., 1979).