3.3: Phytochemical Analysis Of Pyridine

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3.3.3: Phytochemical analysis
The leaves and bark extracts were tested for some secondary metabolites like-saponins, tannins, alkaloids, anthraquinones, phlobatannins, flavonoids, terpenoids, reducing sugar and poly phenols.
Test for reducing sugar: Mixture of 1ml or 1gm of plant sample is taken in a test tube and 10ml deionized water is added in it after this a few drops Fehling solution (1ml Fehling solution A and B) is also mixed and heated at 1000C in a water bath. The Brick red precipitate is produce and which shows a positive result.
Test for tannins: in test for tannins , 2gm of aqueous extract is mixed with 2 drops of 5% ferric chloride in a tube which produces brown color and shows positive result.
Test for phlobatannins: In Test
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About 5 mg of the sample was suspended in 50µl of methoxylamine hydrochloride solution in GC grade pyridine (20 mg ml-1). The mixture was shaken for 2 hr. at 370C before adding 70 µl of MSTFA. Shaking was continued for another 30 min. Analysis of lipid content by GC–MS was performed using Thermo Trace GC Ultra coupled with Thermo fisher DSQ II mass spectrometers. Chromatographic separations of metabolites were carried out on 30 m x 0.25 mm Thermo TR50 column (polysiloxane column coated with 50% methyl and 50% phenyl groups). Xcalibur software was used to process the chromatographic and mass spectrometric data. The GC oven temperature was maintained at 700C for 5 min, then gradually raised at the rate of 50C min-1 to 2900C and maintained for 5 min. The sample was injected in the split mode at a splitting ratio of 1:16. Helium was used as a carrier gas and set at a constant flow rate of 1 mL min-1. The mass selective detector was run in the electron impact (EI) mode, with electron energy of 70 eV. The resulting GC–MS profile was analyzed using Replib, WILLY and NIST mass spectral library by matching the chromatogram with commercially available standards. A freely available mass spectral deconvolution algorithm (Automated Mass Spectral Deconvolution and Identification System, AMDIS32) was used for processing multiple …show more content…
First agar media dissolve in water then keep for autoclave after autoclave it cool at room temperature and nutrient agar plates were prepare for all extracts, 20µl inoculums of each selected bacterium was uniformly dissolve in 25ml agar media then pour in Petri dish plates, after five minutes four well approximately 5mm diameter was bored with the help of borer.
The equal volumes (50µl) of plant extract (Drug) were poured in to the each well. The plates were incubated at 37ºC for 24hrs.

3.5.3: Determination of MIC

The minimum inhibitory concentration of plant metabolites against bacterial pathogens were calculated by broth micro dilution method in different test tubes. A series of test tubes (5 in number) were filled with 5 ml of nutrient broth, autoclaved and supplemented with varying concentration of plant extracts. 0.05 ml of bacterial pathogens was inoculated and the test tubes were incubated overnight. Next day, the growths of bacterial pathogens in presence of plant extracts were recorded by taking an un-inoculated nutrient broth as reference. The minimum concentration in the media which was able to check the growth of pathogens was considered as MIC of the compound against that

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