PHYTOCHEMICAL ANALYSIS AND EVALUATION OF ANTIBACTERIAL ACTIVITY OF TERMINALIA CHEBULA, MOMORDICA CHARANTIA, DREGEA VOLUBILIS PLANT EXTRACTS

Thupurani Murali Krishna 1 , Urmila Bonkuri 2 , Racha Srikanth 3 , Challa Surekha 3 , Peddoju Pranay 3 and Venkalapally Thirupathaiah 2 . 1. Department of Biotechnology, Chaitanya Postgraduate College (Autonomous), Kishanpura, Hanamkonda506001 TS-India. 2. Natural Products Laboratory, Chaitanya Postgraduate College (Autonomous), Kishanpura, Hanamkonda506001-TS-India. 3. Department of Biochemistry and Bioinformatics, Gitam University,Rushikonda, Visakhapatnam, AP-India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 20 October 2018 Final Accepted: 22 November 2018 Published: December 2018


ISSN: 2320-5407
Int. J. Adv. Res. 6 (12), 1195-1201 1196 are ascertaining for plant based medications which are of affordable and safe. Around 50,000 plants have been identified for its attributed medicinal properties. Plants offer a variety of natural compounds related to different molecular families which exhibits several Pharmacological activities such as anti-microbial, anti-oxidant, anticancer, anti-diabetic, anti-inflammatory etc., in humans.
Dregea volubilis is a reference as a medicinal plant found in Ayurvedic, Siddha and Unani literature for the treatment of eye ailments including cataract Kumar and Totawar, 2015. The plant belongs to family Asclepiadaceae is extensively used in Indian traditional medicines. The leaf paste is used to treat cough, fever, severe cold and rheumatic pain Rajadurai, 2009. The preparations of leaf paste in combination with pepper and bark paste with hot milk is used to treat dyspepsia and primary urinary infections respectively (Pandikuma, 2007;Silija et al., 2008).
The preset study was framed out to evaluate the phytochemicals and the antibacterial activity of the fractions separated from crude extracts of the Terminalia chebula, Momordica charantia, Dregea volubilis. Phytochemical analysis:-Preparation of extracts:-All the extracts were dissolved separately using dilute HCL acid and filtered. The filtered extract is used for detection of phytochemicals according to the methods described by Harbone 1998; Kokate1997.

Material And
Test for detecting alkaloids:-Mayer's test:-Mayer's reagent was freshly prepared by dissolving the mixture of mercuric chloride (1.36 g) and of potassium iodide (5 g) in 100 ml of water Wagner's test:-Dissolve 1 g of picric acid in 100 ml of H 2 O. Wagner,s reagent and aqueous solution of iodine and potassium iodide.
Dragendorff test:-This is how to make dragenorff's reagent.our (0.5g) of bismuth nitrate in a empty beaker.add (10ml) of concentration hydrochloric acid.pour (4g) of potassium iodide into another beaker ,add a little after and stir unit.

Hager's Test :-
Dissolve 2g of picric acid in 100ml of H 2 O potassium antimonite. Boil 22g of potassium antimonite with 1liter of water until nearly all of the salt has dissolved, cool quickly and add 35ml of 10% potassium hydroxide.

Detection of Flavonoids:-Alkaline copper test:-
Prepared mixing (0.5ml) copper sulphate,(0.5ml) sodium carbonate .Foline phenol reagent diluted 1:1 with distilled water. Sandard protein solution (100mg%)bovine serum albumin in (0.1N) NaOH. 2 ] also known as lead acetate, lead diacetate, plum bous acetate, sugar of lead, lead sugar salt saturin or Goulard's powder, is a white (crystalline chemical compound with a sweetish taste it is made by treating lead (2) oxide with acetic acid like other lead compounds, it is toxic. lead acetate is soluble in water and glycerin with water it forms the trohydrate, Pb [CH3COO] 2 . 3H2O.

Detection of Tannins:-
Ferric chloride test use of ferric chloride (FeCl3) test for phenolics in general, alternatively, a portion at water extract is diluted with distilled H20 in ratio of 1:4 and few drops of 10% ferric chloride solution is added.

Inoculation preparation:-
The bacterial strains were inoculated into sterilized nutritive broth and incubated at 35 ± 2°C for 24 h. The turbidity of the resulting suspensions are diluted with same nutritive broth to obtain a transmittance of 25% at 580 nm, this percentage was calculated spectrophotometrically using Bausch & Lomb spectrophotometer comparable to McFarland turbidity standard. This level of turbidity is equivalent to approximately 3.0 × 108 CFU/ml (a stock standard from which a working standard was drawn with concentration of 1 × 108 CFU/ml).

Agar well diffusion assay:-
The antibacterial activity of selected medicinal plant extracts was performed based on the guidelines of Clinical and Laboratory Standard Institute.
The selective medium was inoculated with the test organism and once the agar was solidified, the wells were created using a six millimeters diameter bork corer. The wells were filled with 25 µL of the plants extracts of 25, 50, 75 µg/ml concentrations. ciproflaxacin (10µg/ml) is used as positive control. The test was carried out in triplicate. The plates were incubated at 35 ± 2°C for 24 h.

Minimum inhibitory concentration (MIC):-
The MIC of the fractions was determined by diluting the varied concentrations (0.0-1000 µg/ml) of Dregea volubilis, Terminalia chebula, Momordica charantia. Equal volume of the fractions and nutrient broth were mixed in the test tube. Specifically 0.1ml of standardized inoculums of 1to 2 X 10 7 cfu/ml was added to each tube. The tubes were incubated aerobically at 37 o C for 18-24hrs. Two control tubes were maintained for each test batch. This 1198 is as follows: tube containing extracts and the growth medium without inoculums (antibiotic control) and the tube containing the growth medium, physiological saline and the inoculums (organism control). MIC was determined as the lowest concentration of the extracts permitting no visible growth (no turbidity) when compared with the control tubes.

Discussion:-
The antibacterial activity of the fractions separated from crude extracts of Dregea volubilis, Terminalia chebula, Momordica charantia possessed significant inhibitory effects on all tested microorganisms. However, the sensitivity of the Gram positive and Gram negative strains is varied; perhaps this may be because of structural variation in the cell wall of bacteria. Based on the previous published reports, the high polar solvents which have good solubility of antimicrobial agents will easily penetrate through the lipopolysaccharide of bacterial outer membrane (primary permeability barrier of the cell) for the necessary action (Nikaido, 1996). Here we also agree with the above statement because the profound antimicrobial activity results especially on Gram negative organisms of the present work are obtained with only ethanol and aqueous solvents.
The secondary metabolites or phenolic compounds present in the fractions that are separated from the crude extracts of Dregea volubilis, Terminalia chebula, Momordica charantia may adopts several antibacterial mechanisms such as inhibition cell wall synthesis or causing energy depletion by accumulating in the cell wall or they increase the rate of permeability of cell membrane which leads to the loss of cellular constituents nor they disrupt membrane and change in the structure and function of cell organelles, consequently, resulting in the cell mutation and subsequent damage and death (Cowan, 1999;Marcucci et al., 2001;Conner, 1993;Kim et al., 1995).

Conclusion:-
According to the results obtained in the present work here conclude that the ethanol and aqueous fractions of separated from the Dregea volubilis, Terminalia chebula, Momordica charantiaedicinal plants (leaves) had significant phytochemicals which are responsible for antimicrobial activity. However, there should be an extension of this work for isolation of principle compound that responsible for the activity.

Conflict Of Interest:-
Authors here declare no conflict of interest.
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