2.5 Phytochemical screening
To confirm the presence or absence of the following plant secondary metabolites phytochemical screening were carried out: phenols, alkaloids, steroids, cardiac glycosides, flavonoids, saponins, phlobatannins, volatile oil and glycosides.
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2.5.1 Test for Phenols
Equal volumes of each extract and ferric chloride solution (which is prepared by dissolving 1352g of FeCl3.6H2O in distilled water containing 20 ml of concentrated HCl dilute to 1 liter) are added together. A deep bluish green precipitate indicates the presence of phenol.
2.5.2 Test for Alkaloids
To each extract was added to 1% aqueous HCl over water bath and filtered. The filtrate was treated with (2g of Iodine in 6g of Potassium iodide in 100 ml of distilled water). Formation brown or reddish brown precipitate indicates presence of alkaloids.
2.5.3 Test for Steroids
Into each extract was added to 2ml acetic anhydride and 2ml H2SO4. Color change from violet to blue or green indicates the presence of steroids.
1g each extract was boiled with 5ml distilled water and filtered. 3ml distilled water was added to the filtrate and shaken vigorously for 5 minutes. Persistent frothing on warming indicates the presence of Saponins.
2.5.5 Cardiac glycosides
The extract was treated with 2ml glacial acetic acid with a drop of Ferric Chloride solution and underplayed with 1ml H2SO4. A browning at the interface indicates the presence of cardiac glycosides.
A few drops of 1% HCl were added to 1ml of test extract and was boiled. A reddish precipitates indicate the presence of phlobatannins.
5ml Ammonium solution was added to the aqueous filtrate of extract and then a few drops of concentrated H2SO4. Yellow coloration indicates the presence of Flavonoids.
2.5.8 Volatile oil
A small quantity of the test extract was shaken with dilute NaOH and 0.1ml HCl. The formation of a white precipitate indicates a positive result.
A small amount of the alcoholic extract was taken in 1mlof water in a test tube and a few drops of aqueous NaOH were added. A yellow coloration indicates the presence of glycoside.
2.6 Maintenance of pure culture
2.6.1 Test organism
Test organisms were collected from MTCC and an antibacterial activity of various extracts of A. sativum and gomutra was tested against Pseudomonas sp and S. aureus.
2.6.2 Standardization of broth culture
To the test tube containing 10ml of nutrient broth, 0.1ml of the bacterial inoculum was added and mixed for homogeneity and was incubated for 3 hrs. Then the broth cultures were estimated for the presence of l08 cfu/ml of bacterial inoculums. Standardization of broth culture was done by comparing the turbidity to that of 0.5 McFarland turbidity standards.
2.7 Agar well diffusion method
The antibacterial activity of the aqueous, ethanol and methanol extracts of A. sativum and the synergistic effect of aqueous, ethanol and methanol extracts of A. sativum with gomutra was evaluated by agar well diffusion method. The Muller-Hinton agar was prepared and poured into sterile petri dishes and allowed to solidify. The test organism was inoculated onto the sterile agar plates. Four wells of 6mm in diameter each were aseptically bored using a sterile cork borer on each agar plates. 100µl of the different extracts of A. sativum and gomutra were added to each well. Also 100 µl of A. sativum with gomutra (50 µl + 50 µl) was added to a well. The effect of solvent on the bacteria was also checked by adding the respective solvent on one well. The plates were then incubated at 37°C for 18-24 hrs and room temperature for 3-4 days for bacteria and fungi respectively. Effects of the extracts were assessed by measuring the diameters of zones of inhibition.
2.8 FTIR spectral analysis
The FTIR spectra were performed and recorded with a Fourier transform infrared spectroscopy. The infrared radiation is propagated through the sample to obtain the corresponding spectrum, which was averaged from several data acquisitions. FTIR spectra were acquired in the wave number range of 500-4000 cm-1.
2.9 HPLC analysis
The HPLC analysis was done with aqueous and ethanolic extracts of A. sativum both with and without skin. The extracts were sent to VIT University, Vellore for HPLC analysis with HPLC model 1525. The flow rate was 0.5 ml/min. The injected volume was 20µl and the UV detector was set at 254 nm.
2.10 GCMS analysis
The bioactive compound was identified in ethanolic extracts of A. sativum. The extracts were sent to VIT University, Vellore for GC-MS analysis and results with identity of compound were collected.
2.11 Calculation of minimal inhibitory concentration (MIC) by broth dilution method
Serial dilutions of aqueous and ethanolic extract of A. sativum with and without skin (GJA, GJWSA, GJE and GJWSE) were prepared in Mueller-Hinton broth with 1ml of standard inoculums of the microorganisms. To the first tube 1mg/ml of A. sativum juice was added and serially diluted. A tube of the growth medium without A. sativum juice, served as a growth control. An uninoculated tube of the medium was incubated to serve as a negative growth control. After 24 hrs of incubation, the tubes were examined for turbidity, indicating growth of the microorganisms. The lowest concentration of the garlic extracts that inhibits growth of the organism was designated the minimum inhibitory concentration (MIC) (Forbes et al., 1998).