2.1 Collection of drugs
The fresh leaves of Bacopa monnieri were collected from the Herbal Garden Jamia Hamdard and three other different places of India (Kerala, Bangalore and Jammu) as given in Table 1. It was authenticated by comparing with the specimen available at Deptt. of Pharmacognosy and Phytochemistry, Jamia Hamdard, New Delhi. The leaves were dried at room temperature and powdered.
2.2 Preparation of Reagents [Harborne 1998; Khandelwal 2004]
2.2.1 Dragendorff’s reagent
It was arranged by blending two arrangements. Arrangement A (17 gm of bismuth subnitrate + 200 gm tartaric corrosive + 800 ml refined water) and arrangement B (160 gm potassium iodide + 4 ml refined water) were blended in 1:1 v/v extent. From this arrangement working standard was arranged by taking 50 ml of this arrangement and including 100 gm of tartaric corrosive and making up to 500 ml with refined water.
2.2.2 Mayer’s reagent
A mixture was created with Mercuric chloride (1.36 g) and 60 ml of refined water. An arrangement of 5 g of Potassium iodide (KI) in 20 ml of refined water was included to deliver to 100 ml and kept in a holder to utilize.
2.2.3 Wagner’s reagent
A mixture was created with iodine (1.27 g) and potassium iodide (2 g) with 5 ml of water and the arrangement was weakened to 100 ml with water.
2.2.4 Liebermann-Buchard’s reagent
Sulphuric acid (2.5 ml) was blended with acidic anhydride (2.5 ml), to it 45 ml of liquor was included, shaken well and put away for use.
2.2.5 Fehling’s arrangement A (Copper solution) Carefully chosen little precious stones of cupric sulfate (34.66 g) were dissolved appearing no follow of foam or following dampness in adequate water to create 500 ml, and put away in well closed container.
2.2.6 Fehling’s solution B (Alkaline tartarate solution)
Sodium potassium tartarate (176 g) and Sodium hydroxide (77 g) were broken down in adequate water to deliver 500 ml and put away in well stoppered container. These two arrangements (Fehling’s arrangement A and B) were blended in break even with volumes promptly some time recently use.
2.2.7 Ninhydrin’s reagent
Ninhydrin (30 g) was blended in 10 ml of n- butanol taken after by 0.3 ml of 98% v/v in acidic acid.
2.2.8 Millon’s reagent (Mercuric nitrate solution) It can be prepared by dissolving 3 g of mercury in 27 ml of raging nitric acid and cooled.
2.3 Qualitative chemical tests for active compounds
2.3.1 Tests for Alkaloids
Each of the extract buildups was taken independently in five ml of 1.5 % hydrochloric acid and sifted. The filtrate was at that point tried with taking after reagents:
2.3.1.1 Dragendorff's Test
Small quantity of Dragendorff's reagent were integrated in each of the extract and watched for arrangement of orange yellow precipitate.
2.3.1.2 Hager’s Test
A small number of drops of Hager's reagent were integrated in each of the extract and watched for arrangement of yellow precipitate.
2.3.1.3 Wagner’s Test
A small number of drops of Wagner's reagent were included in each of the extract and watched for arrangement of precipitate.
2.3.1.4 Mayer’s Test
Few drops of Mayer's reagent were included in each of the extract and watched for arrangement of white or cream colored precipitate.
2.3.2 Tests for Carbohydrates
2.3.2.1 Molisch Test: Small amounts of alcoholic and watery extricates were broken up independently in five ml of refined water and sifted. To this arrangement, 2-3 drops of α-naphthol were incorporated. To this, approximately one ml of concentrated sulphuric corrosive was included along the sides of slanted test tube so as to create two layers and watched for arrangement of violet colored layer at the border.
2.3.2.2 Fehling’s Test (Location of lessening sugar) : Few drops of Fehling's arrangements A and B in break even with volume were included in weaken extricates and warmed for 30 minutes and watched for the arrangement of brick ruddy coloured precipitate.
2.3.3 Tests for Glycosides
About two ml alcoholic extracts were utilized for the glycoside test.
2.3.3.1 Keller-Killiani Test: Dried extract was included with acetic acid containing ferric chloride and one ml of strong sulphuric acid and watched for the arrangement of ruddy brown colour at the intersection of two layers and the upper layer turnes somewhat blue green within the nearness of glycosides.
2.3.3.2 Borntrager's Test: Small amount of benzene and weak smelling salts arrangement were incorporated to the ethanolic extract and watched for the arrangement of ruddy pink colour.
2.3.3.3 Legal Test: concentrated ethanolic extracts were made antacid with few drops of 10% sodium hydroxide arrangement and after that naturally arranged sodium nitrosopruside arrangement was included to the arrangement and watched for arrangement of blue colour.
2.3.3.4 Baljet test: To the concentrated ethanolic extracts sodium picrate reagent was included and watched for arrangement of orange or yellow colour.
2.3.4 Tests for phenolics compounds
2.3.4.1 Test of Ferric chloride: The extracts were blended in water and heated. To this, two ml of ferric chloride arrangement was included and watched for arrangement of green or blue colour.
2.3.4.2 Lead Acetic acid test: To the extracts (2 ml) leads acetic acid arrangement was included and watched for arrangement of accelerate.
2.3.4.3 Gelatin solution: A few ml of gelatin arrangement was included to the watery extract and watched for arrangement of accelerate or turbidity.
2.3.5 Chemical tests for Flavonoids
2.3.5.1 Smelling salts test: Filter paper strips were plunged in alcoholic arrangement of the extracts, ammoniated and watched for colour alters from white to yellow.
2.3.5.2 Shinoda / seat test: A little amount of buildup was broken up in five ml of ethanol and treated with few of drops concentrated hydrochloric corrosive and 0.5 g of magnesium turnings and watched for arrangement of pink colour.
2.3.6 Tests for Proteins and Amino-acids
2.3.6.1 Millon’s test: To small amount of ethanol extract, five ml refined water was included and sifted. To two ml of this filtrate 5-6 drops of Millon's reagent (arrangement of mercuric nitrate and nitrous acid) were included and watched for arrangement of ruddy precipitate.
2.3.6.2 Xanthoprotein test: Small amount of extract was integrated with nitric acid and watched for arrangement of yellow colour.
2.3.6.3 Biuret Test: Soluble filtrate of the dried extracts was included with small amount of 0.02 % copper sulfate arrangement were included and watched for arrangement of ruddy or violet colour.
2.3.6.4 Ninhydrin Test: To the extract, lead acetate arrangement was included to accelerate tannins. The accelerate was spotted on a paper chromatogram, splashed with ninhydrin reagent and warmed at 110ºC for 5 minutes and watched for arrangement of ruddy or violet colour.
2.3.7 Tests for Saponins
2.3.7.1 Froth Test: Few mg of residue was taken in a test tube with little sum of water and shaken enthusiastically for one minute and watched for arrangement of wealthy foam which was steady for above ten minutes. To the alcoholic extracts, few drops of sodium bicarbonate arrangement were included, shaken well, and watched for the arrangement of honeycomb like frothing.
2.3.8 Tests for Acidic compounds: To the alcoholic extract sodium bicarbonate arrangement was included and watched for the generation of foam (Harborne, 1998).
2.3.9 Litmus paper test: A little sum of alcoholic extracts were taken in warm water and sifted. The filtrate was at that point tried by litmus paper and methyl orange and watched for the visibility of blue colour.
2.3.10 Tests for Mucilages: The extricate was treated with ruthenium red solution in lead acetate and watched for the arrangement of pink colour.
2.3.11 Tests for Resins: Distilled water was included to the extracts and watched for turbidity. A blend of extract in acetone (3 ml) and HCl (3 ml) was warmed on a water shower for half an hour and checked for a pink colour.
2.4 Assay for total phenolics and flavonoid content
2.4.1 Material and Reagent
Reference standard, for flavonoid Rutin and for phenol compound gallic acid were received from M/s Normal cures Pvt. Ltd. Bangalore, India. Each chemicals and reagents utilized were of explanatory rating and acquired from Merck Chemicals, India.
2.4.2 Preparation of methanol extracts of all samples: one gm of leaf and callus sample were dried and powdered then refluxed in 50 ml of methanol for two hours. Collected filtrate was filtered and heated till residue formation .The weight of residue was measured and reconstituted in methanol.
2.4.3 Reagents
- 10% Folin Ciocalteu reagent solution in distilled water
- 1M Na2CO3 (sodium carbonate) solution in distilled water
- Standard (Gallic acid) 1 mg/ml solution in methanol
- Dilution of standard Gallic acid: 50 µg/ml, 100 µg/ml and 150 µg/ml in methanol
2.4.4 Samples preparation: 10 mg / ml methanol leaf extract and 8 mg/methanol calli extracts were made. 0.5 ml sample was poured to mixture of 5 ml F.C compound and 4 ml volume of Na2CO3 then took absorbance on 765 nm after 15 minutes.
2.4.5 Preparation of Standard: 0.5 ml of each standard dilution was mixed to mixture of 5 ml volume F.C. reagent and 4 ml Na2CO3 solution, then took absorbance at 765 nm after 15 minutes.
2.4.6 Blank solution: Methanol 5 ml solvent and 5 ml F.C. reagent were taken and added to 4 ml Na2CO3 solution.
2.5 Evaluation of entire flavonoid content [Pourmorad et al. 2006]
2.5.1 Reagents
- 0.1 mg/ml AlCl3 (aluminium chloride) solution in distilled water
- 1M CH3COONa (sodium acetate) solution in distilled water
- Standard (Rutin) 1 mg/ml solution in methanol
- Dilution of standard Rutin: 10 µg/ml to 100 µg/ml in methanol
2.5.2 Samples preparation: 100 mg /ml solution for leaves extract and calli extract were included in methanol. Half ml of sample was added to 1.5 ml methanol. Combination of 0.1 ml volume of AlCl3 and 1/10 ml volume of CH3COONa reagents were poured to above solution. After that 2.8 ml volume distilled water mixed to solution and kept for half hour after that absorbance was maintained at 415 nm.
2.5.3 Preparation of Standard: 0.5 ml volume of standard dilution were taken and added to 1.5 ml methanol. After that 0.1 ml volume of AlCl3 and 0.1 ml of CH3COONa reagents were poured to above solution and then added 2.8 ml Distilled water and kept for half hour after that absorbance was taken at 415 nm.
2.5.4 Blank solutions: two ml methanol was added to mixture of 0.1 ml volume of aluminium chloride and 0.1 ml volume of CH3COONa and then added to 2.8 ml volume purified water.
2.6 Methods for antioxidant activity
2.6.1 DPPH liberated radical rummaging activity
0.1 mM DPPH was mixed in methanol and 1 ml mixture was included to 3 ml alcohol extract at one concentration (500 µg/mL). Butyrate hydroxytoluene (BHT) was utilized as a positive standard. Discoloration was calculated on 517 nm after hatching for 30 min. Estimations were taken at slightest in triplicate. The capacity to rummage the DPPH radical was calculated utilizing the taking after condition: DPPH rummaging impact (%) = [ADPPH - AS / ADPPH] x100 where, ADPPH is the UV absorbance of the DPPH arrangement and AS is the UV absorbance of the arrangement when the test extricate is included. The extract concentration giving 50% restraint of radical-scavenging movement (IC50) was calculated and communicated as mg/mL, d.w. (Blois, 1958).
2.6.2 Ferric diminishing control determination
2.5 ml phosphate buffer for pH maintaining (200 mM and pH 6.60) and 500 µg/mL alcohol extracts were blended with and 2.5 ml 1% potassium ferricyanide. At that point the blend was brooded at 50 °C with twenty minutes. 2.5 ml 10% dilute trichloroacetic acid was included to over blend and centrifuged at 10000 revolutions per minute for ten minutes. 5 ml of the top surface was poured with distill water (5 ml) and 0.1% FeCl3 (1 ml). The absorbance of the response blend was measured at 700 nm. The ultimate comes about were communicated as µg ascorbic acid counterparts / g based on dry weight of the extract (Zhao et al. 2008).
2.6.3 Hydrogen peroxide rummaging activity
Solution of H2O2 (43 mM) was made in buffer phosphate (0.10 M, pH 7.4). Extricates at concentration 50 µg/mL broken up in 3.4 mL phosphate buffer were included to a H2O2 arrangement (600µL). The absorbance of the response blend was recorded at 230 nm. The extricate concentration giving 50% of H2O2 rummaging movement (IC50) was calculated and communicated as µg/mL based on test dry weight. (Shon et al. 2007).
2.6.4 Statistical analysis
The tests were carried out utilizing Completely Randomized Design (CRD). Triple readings were attempted in each test. Data are maintained as means ± standard deviation (SD). Analysis of variance and significant differences among means were tried by one-way ANOVA using the COSTAT computer package (Cohort Software, 1989). The least significant difference (LSD) at P ≤ 0.05 level was included. Correlation coefficients (R2) from regression investigation between phenolic, flavonoid substance and antioxidant activities were moreover premeditated.
2.7 Methods for antibacterial activity
2.7.1 Microbial strains
The microbial strains were taken from Division of Biotechnology, University of Mewar, Chittorgarh, India. The reference strains of microbes ought to be refined in supplement broth (Hi-media,) at 37ºC, subcultured routinely (each 30 days) and put away in supplement agar inclines at 4ºC as well as at -20ºC by planning suspensions in 20% glycerol. Bacterial strains chosen for antimicrobial study are following
BS :Bacillus subtilis,
SA: Staphylococcus aureus,
EC: Escherichia coli,
PA: Pseudomonas aeruginosa
2.7.2 Formationand establishment of stock cultures
The microbial strains were inoculated on nutrient agar and maintainted during the night at 37°C. These cultures were set aside at 4°C. Fresh slope cultures were arranged every 3-4weeks.
2.7.3 Culture media:
2.7.3.1 Nutrient agar: Nutrient agar will be used for preparation of stock cultures on agar slopes. The formula was following
Peptone
|
5.00 gram
|
Sodium chloride quantity
|
5.00 gram
|
Beef extract quantity
|
1.50 gram
|
Yeast extract quantity
|
1.50 gram
|
Agar
|
1.50 gram
|
pH ( at room temprature )
|
7.40 ± 0.2
|
2.7.3.2 Nutrient broth
Beef extract quantity
|
3.00 g
|
Peptone quantity
|
5.0 g
|
pH
|
6.80 ± 0.2
|
2.7.3.3 YEPD medium will be used as the basic liquid culture medium for growing overnight fungal cultures. The formula was following
Yeast extract quantity
|
3.0 g
|
Peptone quantity
|
10.0 g
|
Dextrose quantity
|
20.0 g
|
pH
|
7.0
|
2.7.3.4 Mueller Hinton broth (MHB) medium
It is utilized for determining the antibacterial susceptibility testing.
Beef extract powder quantity
|
2.00 gram
|
Casein quantity
|
17.50 gram
|
Soluble starch quantity
|
1.50 gram
|
pH
|
7.3± 0.1
|
2.8 Water Extraction
25 gm of discuss dried powder was set in hot refined water and bubbled for 30 min and kept undisturbed for 24hr. It was sifted off utilizing sterile channel paper (Whatman no. 1) into a clean cone shaped carafe. Watery dissolvable was expelled beneath weight employing a rotatory vaccum evaporator at 60°C. The dried buildup of rough extricate was resuspended in 20% DMSO and after that put away in dim bottles at 4°C.
2.9 Solvent Extraction
Dried regenerated shoot powdered were extracted with solvent (Chloroform, acetone and 95% (v/v) ethanol) using soxhlet extractor. 25 gm powder was arranged in soxhlet thimble and 250 ml of solvent was poured to soxhlet flask then extracted at 40°C until the extract was apparent.
2.10 Preparation of bacterial inoculum:
Inoculums were prepared by of isolated bacterial colonies from 24 hr old culture and suspended in 5 ml of germ-free nutrient broth. The broth culture was maintainted at 37°C for 4hr.
2.11 Agar well dissemination strategy [Murray et al. 1995]
Agar well-diffusion strategy was taken after to decide the antimicrobial action. Supplement agar (NA) plates were cultured with 8 hour ancient - broth culture of individual microbes. Wells (10mm breadth and approximately 2 cm a portion) were made in each of these plates utilizing sterile plug borer. Stock arrangement of each plant extricate was arranged at a concentration of 1 mg/ml in several plant extricates viz. Ethanol, chloroform, Water. Almost 100 µl of distinctive concentrations of plant dissolvable extricates were included sterile syringe into the wells and permitted to diffuse at room temperature for 2hrs. Control tests including inoculums without plant extricate were set up. The plates were hatched at incubation temperature 37°C for 18 to 24 h for bacteria and incubation temperature 28°C for 48 hours parasitic pathogens. The breadth of the hindrance zone (mm) was calculated and the action record was too calculated. Triplicates were kept up and the test was rehashed thrice.
2.12 Assurance of Minimum inhibitory concentration [Baron and Fingold 1990].
Least amount inhibitory concentrations were decided by the tube weakening strategy. The MIC was decided utilizing the tube weakening strategy. A two- overlap serial weakening of the extract and divisions was carried out aseptically to grant changing concentrations extending from 30 mg/ml-0.23435 mg/ml. Each of the weakenings was immunized with 0.1 ml of the standardized inoculum and the tubes were brooded at 37 °C for 24 hours and microbial development watched as turbidity in tubes was looked out for. The most reduced concentration that appeared no development was considered the MIC. The complete strategy was replicated using Ampiclox and Zosyn at concentrations extending from 0.5 mg/ml-0.0004 mg/ml. A positive control tube containing broth with living beings without plant extricate and a negative control tube containing extricate without living beings were moreover hatched along. The MIC was taken as the slightest concentration that repressed the development of test organism.