1 and shade dried. Dried stem leaves, and

1 Collection of plant material

 Two plants Flacourtia jangomas and Leucas aspera were selected for present work after extensive literature survey. The plants were collected from Munger, Bihar, India.

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 Flacourtia jangomas- Fresh Flacourtia jangomas leaves stem and fruits were collected from Munger, Bihar with latitude 25.3748° N and 86.4735° E longitude. The plant part was authenticated by Dr. S.B.Padal, Department of Botany, Andhra University. Voucher specimen number-22229 and was deposited in Botany Department Herbarium, Andhra University, India.

Fig- Flacourtia jangomas stem

Leucas aspera – Fresh Leucas aspera leaves stem and flowers were collected from Munger, Bihar with latitude 25.3748° N and 86.4735° E longitude. The plant part was authenticated by Dr. S.B.Padal, Department of Botany, Andhra University. Voucher specimen number-22230 and was deposited in Botany Department Herbarium, Andhra University, India.

 

3.1.1 Preparation of solvent extract (HARBORNE, 1984)

Fresh Flacourtia jangomas stem, fruits, and leaves were collected washed intensely with distilled water 2-3 times and shade dried. Dried stem, fruits, and leaves of Flacourtia jangomas were powdered using electric pulverizers. The shade dried Flacourtia jangomas stem, fruits and leaves were filled in the thimble of Whatman No 1 filter paper and extracted individually with different organic solvents that are Ethyl acetate, Hexane and Diethyl-ether in Soxhlet extractor for 48h. The solvent was concentrated under reduced in Rotary evaporator and stored at 4° C in the aseptic airtight bottle for further use.

 

Fresh Leucas aspera stem leaves, and flowers were collected washed intensely with distilled water 2-3 times and shade dried. Dried stem leaves, and flowers of Leucas aspera were powdered using electric pulverizers. The shade dried Leucas aspera stems, leaves and flowers was filled in the thimble of Whatman No 1 filter paper and extracted individually with organic solvent Ethyl acetate, Hexane and Diethyl-ether in Soxhlet extractor for 48h. The solvent was concentrated under reduced pressure in Rotary evaporator and stored at 4° C in the aseptic airtight bottle for further use.

3.1.2 Test Microorganism

The cultures were procurred from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh

Test Bacteria- Bacteria used are Staphylococcus aureus MTCC 3160, Bacillus polymyxa(local isolate), Bacillus megaterium MTCC2444,Bacillus pumilis MTCC2466 Escherichia coli MTCC 723, Salmolella typhi MTCC 3216, Vibrio cholerae MTCC3906, Pseudomonas aeruginosa MTCC 7837.

Test Fungi- Fungi used for research are Aspergillus niger MTCC 1881, Aspergillus flavus MTCC 1883,Neurospora crassa MTCC 1855.Trichoderma viridae MTCC 2417.

 

 

 

3.1.3 Antibacterial activity

 

Antibacterial activity was implemented by Agar diffusion method. The test bacterial cultures were revived by inoculating in Nutrient broth and incubated at 37ºC for 18 hrs. The Nutrient Agar plates were prepared. Each plate was inoculated with 18-hour old cultures (100 ?l, 10-4 CFU) and spread evenly on the plates. After 20 min, on the plates the wells of size 6mm was punctured by sterile cork borer and was filled with 25 ?l, 75 ?l and 100?l of Flacourtia jangomas stem fruits and leaves extracts and 25 ?l, 75 ?l ,100 ?l  of Leucas aspera leaves, stem and flowers extracts. Ciprofloxacin antibiotic was used as positive control. All the plates were incubated at 37ºC for 24 h and the diameter of inhibition zone was noted in mm

 

 

 

3.1.4 Antifungal activity

Antifungal activity was performed by Agar diffusion method. Czapek Dox Agar media 19. was used for the antifungal activity. The test fungi cultures were revived by inoculating in Czapek Dox broth media and incubated at 270C for 48 hrs. The agar plates of the Czapek dox media were prepared. Each plate was inoculated with 48 hour old cultures (100 ?l 10-4 CFU) and spread evenly on the plate. After 20 min, the well was punctured by sterile cork borer of 6mm size and was filled with filled with 25?l, 75?l and 100?l of Flacourtia jangomas stem fruits and leaves extracts and 25?l, 75?l ,100 ?l of Leucas aspera leaves, stem and flowers extracts. Amphotericin was used as a positive control. All the plates were incubated at 270C for 96 hours and the diameter of inhibition zone was noted in mm.

 

 

 

 

 

3.2 Phytochemical analysis

 

3.2.1 Qualitative analysis-  Leucas aspera flower, Leucas aspera leaves, Flacourtia jangomas stem, Flacourtia jangomas leaves and Flacourtia jangomas fruits were carried on further for bioassays. The plant extracts were screened for Phytochemical analysis.

 

3.2.2 Preparation of solvent extracts-

Fresh Flacourtia jangomas stem, fruits, and leaves were collected washed intensely with distilled water 2-3 times and shade dried. Dried stem, fruits, and leaves of Flacourtia jangomas were powdered using electric pulverizers. The shade dried Flacourtia jangomas stem, fruits and leaves were filled in the thimble of Whatman No 1 filter paper and extracted with Ethyl acetate in Soxhlet extractor for 48h. The solvent was concentrated under reduced in Rotary evaporator and stored at 4° C in the aseptic airtight bottle for further use.

Fresh Leucas aspera leaves, and flowers were collected washed intensely with distilled water 2-3 times and shade dried. Dried leaves and flowers of Leucas aspera were powdered using electric pulverizers. The shade dried Leucas aspera leaves and flowers was filled in the thimble of Whatman No 1 filter paper and extracted with Ethyl acetate for 48h. The solvent was concentrated under reduced pressure in Rotary evaporator and stored at 4° C in the aseptic airtight bottle for further use.

 

Invitro- Antioxidant activity

Nitric oxide scavenging assay

 

Nitric oxide scavenging assay was done by method described by Marcocci 20.The Nitric oxide scavenging assay is done by Griess reagent 21. Antioxidant activity of Flacourtia jangomas stem leaves and Leucas aspera flowers leaves and stem was evaluated by Nitric oxide scavenging activity. Different concentrations (10?l, 50?l and 100?l) of Flacourtia jangomas stem extract and Butylated hydroxy anisole (BHA) were taken in different test tubes and made up to 3ml with 0.1M phosphate buffer (pH 7.2).Sodium Nitroprusside (5mM) prepared in buffered saline (pH7.2) was added (1ml) to each tube. The reaction mixture was incubated for 30 min at room temperature.

                A control without the test sample, but with an equivalent amount of methanol was maintained. After 30 min, 1.5 ml of above solution was mixed with 1.5 ml of Griess reagent

(1% Sulphanilamide, 2% phosphoric acid and 0.1% N-1-Naphthylethylenediamine dihydrochloride).The absorbance of the Flacourtia jangomas stem extract was measured at 546 nm. Nitric oxide radical scavenging activity was calculated using the following formula:

 

% Nitric oxide radical scavenging activity = (control OD – sample OD) ×100

                                                                ______________________

                                                                         Control OD

 

 Free radical scavenging activity (Braca A, De Tommasi N, Di Bari L, Pizza C, Politi M, Morelli I, et al. Antioxidant principles from Bauhinia tarapotensis. J Nat Prod 2001;64:892-5)

Concentration (10?l, 50?l and 100?l) of plant extracts in Dimethyl sulfoxide (DMSO), was added in a series of test tubes. The volume was adjusted to 500?l by adding Methanol. Five milliliters of a 0.1 mM methanolic solution of 1,1-diphenyl-2-picryl hydrazyl (DPPH; from Sigma –Aldrich, Bangalore) was added  and shaken earnestly. A control without the test compound, but with an equivalent amount of methanol was maintained. The tubes were allowed to stand at room temperature for 20 min. The absorbance of the samples was measured at 517 nm. Butylated Hydroxy Anisole (BHA) was used as reference standard. Free Radical scavenging activity was calculated using the following formula:

 

% radical scavenging activity = (control OD – sample OD) ×100.

                                                  ————————————

                                                                 Control OD

 

Hydroxyl radical scavenging activity

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