CHAPTER 2 MATERIAL AND METHODS

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CHAPTER 2

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MATERIAL AND METHODS

PLANT TISSUE CULTURE

TISSUE CULTURE ROOM STERILIZATION

Tissue culture room was fumigated over night and sterilized with potassium permanganate and formaldehyde (2:3) at the interval of 24 hrs for two times before every inoculation.

GLASSWARES

The glassware used for culture work comprised of 6"x1" Borosil test tubes, glass and plastic jars (Hi media), 100 ml, 250 ml, 500 ml, and 1000 ml corning and Borosil flasks, pipettes, and measuring cylinders (100 ml, 500 ml). Before use, glassware were thoroughly brushed with alkaline detergent teepol and then washed in running water and reprocessed with chromic acid (mixture of K2Cr2O7 + H2SO4

+ H2O) for about 4 hours followed by through rinse washing in tap

water and DDH2O to avoid the adverse effect on the pH of the

medium. The glass-wares were then inverted in a clean tray and left to dry in the oven. Plugs for the tubes and flasks were made out of non-absorbent cotton wrapped in muslin. The glass-wares were then steam sterilized in an autoclave at a pressure of 15lb/in2_at

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PLANT MATERIAL

Argyreia nervosa was collected from Bapalal vaidya botanical research centre, VNSGU, Surat, Gujarat, India.

The explants like node, leaf, internode and shoot apex were excised and were placed in different bottles and covered with net and washed for 30 minutes under running tap water to remove all the adhering dust particles from the surface. The explants were then washed with 1.0% liquid detergent (teepol) for another 15 minutes and then washed properly to remove the detergent. The explants were then treated with bavistin (fungicide) for another 10-15 minutes to remove the fungus and then washed properly to remove the fungicide.

MEDIA

A basal MS (1962) media plus sucrose (2.5%) was used for plant tissue culture. Composition of the media is given in Table No.3.

Table No.3

Composition of Murashige and Skoog’s Culture Medium Constituent Concentration

(mg/liter) Macro Elements

Potassium Nitrate (KNO3) 1900

Ammonium Nitrate (NH4NO3) 1650

Magnesium Sulphate (MgSO4·7H2O) 370

Calcium Chloride (CaCl2·2H2O) 440

Potassium dihydrogen phosphate (KH2PO4) 170

Manganese Sulphate (MnSO4·4H2O) 22.3

Potassium Iodide (KI) 0.83 Phosphoric acid (H3BO3) 6.2

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Micro Elements

Zinc Sulphate (ZnSO4·7H2O) 8.6

Copper Sulphate (CuSO4·5H2O) 0.025

Sodium Molybdate (Na2MoO4·2H2O) 0.25

Iron Sulphate (FeSO4·7H2O) 27.8

Na2EDTA 37.3 Organics in mg/ml Nicotinic acid 0.5 Pyridoxine-HCl 0.5 Thiamine-HCl 0.1 myo-Inositol 100 Glycine 2.0 Sucrose 25000-30000 STOCK PREPARATION:

Stock solution of organic and inorganic nutrients (Major, Minor, Iron and Vitamins) were prepared in double distilled water and stored in refrigerator. Macro nutrients stock was prepared at twenty times (X20) of their final concentration while Micro nutrients were prepared at 100 times (X100) of their final strength.

PLANT GROWTH REGULATORS:

All plant growth regulators were added in medium before autoclave the medium. Main growth hormones used was as followed: Auxins:

IAA, 2, 4-D and NAA dissolved in few drops of 0.1N NaOH and final volume made by sterile double distilled water. 50 mg 2, 4-D was

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dissolved in few drops of 0.1N NaOH and final volume (10 ml) made up by adding DDH2O.

Cytokinins:

Kinetin, ADS and BAP (Dissolved in few drops of 0.1N NaOH solution and final volume made by sterile double distilled water). 25

mg BAP, KN and ADS were separately dissolved in few drops of 0.1N NaOH and final volume (10 ml) made up by adding DDH2O.

Gibberellins:

GA3 (Dissolved in few drops of ethanol and final volume made by

sterile double distilled water). 20 mg GA3 was dissolved in few drops

of ethanol and final volume (10 ml) made up by adding DDH2O.

Carbon source: Sucrose (25-30 g)

Gelling Agent: Agar-Agar Powder (0.65-0.9%)

MEDIA PREPARATION: Callus:

The media contained MS (Murashige and Skoog, 1962) basal mineral nutrients plus, vitamin and sucrose (2.5-30%) as the carbon source. Plant Growth Regulators were added and mixed well with help of glass rod. The pH of the medium was adjusted 5.75 with the help of 0.1 N NaOH and 0.1 N HCl. 0.65-0.7% agar-agar powder was used to solidify the media. Then medium was heated and continuously stirred with the help of glass rod. It was cooled and in each culture tube 20-25 ml medium and each jar 30-40 ml medium was

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dispensed. All the tubes were plugged with non-absorbent cotton plugs and mouth of tube was wrapped with aluminium foil or non-absorbent glossy paper. All the jars were wrapped with brown paper and aluminium foil. These tubes and jars with medium containing growth regulators were sterilized by autoclaving at 121°C and 15 lbs/sq inch pressure for 20 - 25 minutes.

The media were supplemented with different auxins and cytokinins viz., BAP, IAA, NAA, and KN (Kinetin), and in different concentrations and combinations from 0.20 mg/l to 20.00 mg/l has been used for all experiments.

Micropropagation:

The media contained MS (Murashige and Skoog, 1962) basal mineral nutrients plus, vitamin and sucrose (2.5-30%) as the carbon source. Plant Growth Regulators were added and mixed well with help of glass rod. The pH of the medium was adjusted 5.75 with the help of 0.1 N NaOH and 0.1 N HCl. 0.7-0.9% agar-agar powder was used to solidify the media. Then medium was heated and continuously stirred with the help of glass rod. It was cooled and in each culture tube 20-25 ml medium and each jar 30-40 ml medium was dispensed. All the tubes were plugged with non-absorbent cotton plugs and mouth of tube was wrapped with aluminium foil or non-absorbent glossy paper. All the jars were wrapped with brown paper and aluminium foil. These tubes and jars with medium containing growth regulators were sterilized by autoclaving at 121°C and 15 lbs/sq inch pressure for 20 - 25 minutes.

The media were supplemented with different auxins, cytokinins and gibberellic acid viz., BAP, IAA, KN (Kinetin) and GA3 in different

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concentrations and combinations from 1.0 mg/l to 5.0 mg/l has been used for all experiments.

Cell Suspension Culture:

The media contained MS (Murashige and Skoog, 1962) basal mineral nutrients plus, vitamin and sucrose (3.0-4.0%) as the carbon source. Plant Growth Regulators were added and mixed well with help of glass rod. The pH of the medium was adjusted 5.75 with the help of 0.1 N NaOH and 0.1 N HCl. in each 250 ml flask 50 ml medium was dispensed. All the flasks were plugged with non-absorbent cotton plugs and mouth of tube was wrapped with aluminium foil or non-absorbent glossy paper. These flasks with medium containing growth regulators were sterilized by autoclaving at 121°C and 15 lbs/sq inch pressure for 20 - 25 minutes.

The media were supplemented with BAP and IAA in different concentrations from 1.0 mg/l to 7.00 mg/l has been used for all experiments.

INOCULATION and CULTURE MAINTENANCE

The maintenance of sterile conditions was inevitable for successful tissue culture experiments. Hands and arms were washed with alcohol before inoculation. The rims of the test tubes and the sides of the plugs were flame sterilized. Instruments (forceps, scalpels, spatula etc.) were all sterilized by dipping in the alcohol and flaming a number of times. Care was taken to cool the instruments before putting into operation. Primary washing of the explants was done with tap water and DDH2O for all the explants. The explants then treated

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treated with 8% Hydrogen peroxide solutions for 5-10 minutes respectively depending upon the explants. The explants were then thoroughly washed (4 - 5 washings) with sterilized distilled water to remove the traces of Hydrogen peroxide. Fresh cuts were given to the stem, node and leaf explants after sterilization to remove undesirable or dead portions. 1cm2_{segments of leaves were made}

with sterilized blade. The explants were then planted on MS medium. 1 cm of node and internodal explants were inoculated in all the media with various growth regulators.

Before starting the inoculation work, the slab of the laminar flow was swabbed with rectified spirit and culture tubes containing autoclaved media, pertidishes and spirit lamps, cotton and other things required were kept on the slab. The forceps, scalpels, needles, scissors etc. were kept in a glass tube column containing rectified spirit. There after chamber was sterilized by UV light for an hour. The sterile explants (Leaf, node, internodes etc.) were aseptically transferred into culture tubes containing 20 ml medium or in culture jars containing 30ml medium.

Callus Culture:

The explants were placed on culture tubes (150 x 25 mm) containing 20-25 ml of medium with respective growth regulators. The cultures were incubated at 25±2°C under 24 hours with white fluorescent light intensity of 1800-2000 lux. Sub-culturing was carried out at an interval of 30-40 days. The cultures were observed and examined in every day and data was recorded. The resulting callus of all the explants was maintained by frequent subcultured at every 8 weeks in culture tubes containing fresh medium.

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The initiation of callus first started from the cut margin of explant (leaf, stem and node) ultimately unorganized mass proliferates establishing a callus culture. After every 8 weeks of culture initiation the primary cultures thus obtained having undifferentiated mass of callus were sectioned into small pieces and then transferred to fresh medium for proliferation or regeneration under air laminar flow cabinet. The callus was harvested regularly at the transfer age of 8-9 weeks for analysis.

Micropropagation:

The explants were placed on culture tubes and jars containing medium with respective growth regulators. The cultures were incubated at 25±2°C under 16/8 hours (light/dark) photoperiod with white fluorescent light intensity of 1800-2000 lux. The cultures were observed and examined in every day and data was recorded. Shoot buds were initiated from explants after 20-25 days of inoculation. Leaves appeared after 10-12 days.

Regenerated shoots were excised and 1-2 shoots were transferred on the fresh shooting medium for the proliferation and elongation of the sprouted shoots. The elongated shoots were separated from the clump and further transferred on rooting medium. Acclimatization/Hardening of Plantlets:

Elongated shoots were excised and further sub-cultured on the rooting media for the rhizogenesis. Rooted plantlets were removed from the culture vessels and transferred into polybegs containing sterilized coco peat and sand mixture (2:1 ratio). In other method the elongated shoots were excised and directly transferred into polybegs

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containing sterilized coco peat and sand mixture (2:1 ratio) and MS medium contained auxin. The potted plants were maintained inside a culture room at 25 ± 2°C for 16 hr/day illumination with cool white fluorescent light. The polythene bags were gradually removed over a period of 6 days. The plants were kept in the culture room for 2 weeks before transferring into the field.

Cell Suspension Culture:

Liquid MS Basal sterilized medium without agar was used for cell suspension culture. 50 ml MS medium containing 3% sucrose with respective growth regulators was placed in 250 ml flask. 1 g friable callus was used for cell suspension culture. Cell Piece of callus transferred to a sterilized pertidish in laminar air flow. Gently broke the callus of ~1cm diameter with forceps into 10-20 small pieces. These small pieces of callus were transferred to the liquid media containing different concentration of IAA and BAP (1.0 BAP+1.0 IAA; 1.0 BAP+3.0 IAA; 1.0 BAP+5.0 IAA; 1.0 BAP+7.0 IAA). Opening of the flask was flame sterilized and place the sterilized cap on the flask. Replicated samples were prepared. Sucrose level was increased to check cell growth.

The cultures incubated on an incubating shaker at 120 rpm and 25±2°C. After 10days 5ml liquid media with single cells and small cell clumps was transferred to other flaks containing 50 ml media.

The growth of cells was measure by haemocytometer. 1ml media pipette out from each flask containing different concentration of IAA and BAP in sterile condition and transferred to both chambers of a haemocytometer and a cover-slip was used to allow each chamber to be filled with capillary action.

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Cell counting started with one chamber of the haemocytometer, all the cells in the four 1-mm corner squares are counted in each chamber. Each 1-mm square of the haemocytometer represented total volume of 10-4_cm3_{. Since cm}3_{is equivalent to 1ml, the cell}

concentration per ml determined as followed:

Cells per ml= the average count per square X dilution factor X 104

Total cells= cells per ml X original volume of culture medium from which cell suspension was taken.

1ml media pipetted out from flasks at short intervals of time (3-5 days) and counted the cell numbers. Plotted the cell count data of a passage on a graph and the curve indicated the growth pattern of suspension culture.

Evans blue stain was used to check cell viability. Evans blue stain is excluded by living, functional membranes. Thus it is taken up by dead cells and excluded by living cells. This is easily seen under the light microscope.

PHARMACOGNOSTIC CHATACTERS (ANATOMY)

Temporary mounts of free hand sections were employed for the anatomical studies. The sections were stained with phenolphthalein in which ratio of water and glycerine is 60:40. The sections were observed and photographs were taken by Trinocular Research Fluorescence Microscope (Model Axio Scope A1; Carl Zeiss, Germany).

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PHYTOCHEMICAL SCREENING

QUALITATIVE ANALYSIS:

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY Extraction:

Alkaloids: (Wagner and Bladt, 1996)

100 mg powdered plant material was mixed thoroughly with 0.1ml 10% ammonia solution and then extracted for 30 minutes with 5ml methanol under reflux on water bath at 600_{C. It was then}

centrifuged at 2500 rpm for 10 minutes and concentrated to 1ml. different parts of plants and their cultured cells were extracted from above mentioned method.

Flavonoids: (Wagner and Bladt, 1996)

100 mg powdered plant material was extracted for 15 minutes with 5ml methanol under reflux on water bath at 600_{C. It was then}

Tannin: (Harborne, 1973)

100mg powdered plant material refluxed with 5 ml methanol for 30 minutes on a water bath at 600_{C/heating mental at 40}0_C

consecutively 3 times. Filtered and removed solvent under reduced pressure. Concentrated and made up the volume 1ml.

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Glycoside: (Wagner and Bladt, 1996)

100 mg powdered plant material was extracted for 15 minutes with 5ml methanol under reflux on water bath at 600_{C. It was then}

Stationary Phase: TLC Plate Silica gel 60 F 254 (Merck) 20cm x 20cm was cut in to 20cm x 10cm size with the help of cutter and used as stationary phase.

Sample Application: Samples of in vivo plant parts and in vitro produced cells was loaded on the separate pre-activated silica gel TLC plate. Material was applied by using Camag Linomat-5 sample applicator. Linomat-5 was programmed by Win CATS Planar Chromatography Manager for loading the samples. Samples were loaded on each track. Total 9 tracks were loaded on TLC plates. On each track 20µl sample was applied. Band length was kept 0.8 cm Chromatogram Development: Sample applicated HPTLC Plates were allowed to dry first afterwards they were placed in twin trough glass chamber of size 20 x 10 cm. for the development of chromatogram. Chromatogram was allowed to develop up to 8 - 10 cm. HPTLC plate was removed from the chamber and immediately marked the total migration of solvent through pencil and then it was dried with hair dryer to remove the traces of solvent by evaporation.

Solvent system: Combination of various organic solvent was used for the better resolution of the secondary metabolites

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Flavonoids : Ethyl acetate: Formic acid: Glacial acetic acid: Water (20:2:2:5.2)

Tannin : Chloroform: Acetic acid (18:2)

Glycoside : Ethyl acetate: Methanol: Water (20:2.6:2)

Chromatogram Scanning : The developed chromatogram was scanned through· Camag TLC Scanner 3 at 200 nm and 254 nm wavelength selecting deuterium lamp, 366 nm wavelength using Mercury lamp and 520 nm wavelength using white lamp. Scanning speed was adjusted 20 mm/s and data resolution was 100 µm/step. HPTLC Data was generated by using Win CATS Planar Chromatography Manager.

Detection:

Alkaloid : Dragendroff’s reagent, 5% methanolic H2SO4

The plate was heated at 1000_{C. Orange to brown}

coloured

zone appeared. (Wagner and Bladt, 1996)

Flavonoids : sprayed plate with 1% ethanolic solution of AlCl3.

Yellow fluorescence on long wavelength UV light (360nm)

Yellow coloured zone appeared when heated at 1000_C

for 5-10

minutes. (Wagner and Bladt, 1996)

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Blue coloured bands appeared. (Goldstein and Swain, 1965)

Glycoside : 10% ethanolic H2SO4

The plate was heated at 1000_{Cfor 3-5 min, coloured}

zones

appeared. (Wagner and Bladt, 1996)

QUANTITATIVE ESTIMATION:

QUANTITATIVE ESTIMATION OF CARBOHYDRATE:

Determination of total carbohydrate by Anthrone method: (Sadasivam and Manickam, 1996)

Materials:

 2.5N HCl

 Anthrone Reagent: dissolve 200mg anthrone in 100ml of ice

cold 95% H2SO4.Prepare fresh before use.

 Standard Stock: Dissolve 100 mg in 100 ml water.

 Working standard: 10 ml of reagent diluted to 100 ml with

distilled water. Store refrigerated after adding a few drops of toluene.

Procedure:

100 mg of the sample took into a boiling tube to hydrolyze by keeping it in a boiling water bath for three hours with 5 ml of 2.5N HCl and cool to room temperature. Solid sodium carbonate used to neutralize it until the effervescence ceased. Made up the volume to

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100 ml and centrifuge. The supernatant was collected and took 0.5 and 1ml aliquots for analysis. The standard was prepared by taking 0, 0.2, 0.4, 0.6, 0.8 and 1.0 ml of the working standards. ‘0’ served as blank. Made up the volume 1 ml in all the tubes including the sample tubes by adding distilled water. Then 4 ml of anthrone reagent added. Heated for eight minutes in a boiling water bath. Cooled rapidly and read the green to dark green colour at 630nm Drew a standard graph by plotting concentration of the standard on the x-axis versus absorbance on the y-axis. From the graph calculated the amount of carbohydrate present in the sample.

 Calculation:

Mg of glucose

Amount of carbohydrate in 100mg of the sample =_____________________ X 100

Volume of test sample

QUANTITATIVE ESTIMATION OF PROTEIN

:

Protein in plant material was estimated by the method given by Lowry et al, (1951).

Reagents:

 80% ethanol

 5% perchloric acid (HClO4) or trichloro acetic acid (C2HCl3O2)

 Reagent A: 2% sodium carbonate (Na2CO3) in 0.1N sodium

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 Reagent B: 0.5g copper sulphate (CuSo4) and 1g potassium

sodium tartarate (KnaC4H4O6, 4 H2O) 100 ml double distilled

water.

 Reagent C: 98 ml of Reagent A+ 2 ml of Reagent B

 Reagent D: Folin phenol diluted with distilled water(1:2)

Sample preparation:

Powder (dried) 0.1g was crushed, homogenized and extracted in 10 ml of 80% ethanol. The resulting slurry was centrifuged and residue obtained, to the residue, 10 ml 5% perchloric acid was added and incubated at 800_{C for 20 min. It was brought to room}

temperature and centrifuged. The supernatant was discarded and precipitates were washed with 10 ml distilled water. Centrifuged and discarded the supernatant. Then to the residue, 10 ml of reagent A was added and incubated at 300_{C for 1 hour. The protein was}

extracted in supernatant by centrifugation and residues were discarded. This supernatant was used as the sample for protein estimation and the total volume of sample was recorded. To 1 ml or 0.1 ml sample solution 5 ml of reagent C was added and incubated for 10 mins. To this add 1 ml of reagent D and mixed thoroughly. The optical density was measured at 660nm before 30 mins.

Standard graph:

Protein stock solution was prepared by dissolving 50 mg of bovine serum albumin (Fraction) in 50 ml double distilled water, thus making a solution of concentration 1mg/ml. Freshly prepared stock must be used earliest. Double distilled water added to the aliquots of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 ml of the stock

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solution to adjust final volume 1 ml. Then to this 5 ml of reagent C was added and incubated for 10 mins. 1 ml of reagent D was added and optical density was measured at 660nm. Blank was prepared using distilled water. The standard graph was obtained by plotting optical density on y-axis and concentration on x-axis.

QUANTITATIVE ESTIMATION OF LIPID (OIL CONTENT):

Agrawal et al., (1987) method was followed for the determination of lipid.

Procedure:

The plant samples were dried for 12-17 hours at 60-700_{C and}

ground to coarse powder. 5 gm weighed sample is taken in a cellulose thimble. The thimble was fixed in the Soxhlet funnel and about 150-200ml of petroleum ether was taken in the flat bottom flask. The funnel over the flask was fixed and attached to the water condenser. The samples refluxed for at least 4 hours. The heater was switched off to let the apparatus cool by maintaining the water flow as such. The condenser and funnel were detached. Petroleum ether was evaporated in flat bottom flask over hot lat at 800_{C. When a}

small quantity about 10 ml of ether was left in the flask, it is transferred in weighed butter paper (W1) and kept in oven at 70±100C

till ether evaporates (presence can be detected by its smell). The oil quantity (%) was calculated on the basis on the weight of plant material.

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QUANTITATIVE ESTIMATION OF SECONDARYMETABOLITES: PHENOLIC COMPOUNDS:

Folin method of Swain and Hills (1959) was used to estimate total phenolics compounds in plant and cultured cells.

Reagents:

 80% ethanol (chilled)  Folin phenol reagent (1:1)

 Saturated sodium carbonate (20%): (dissolve 1g sodium

carbonate in 3 ml of distilled water.)

 Tannic acid for standard graph. Sample Preparation:

50 mg powder sample was ground in chilled mortar and pestle with minimal amount of 80% ethanol. Then this pulp was centrifuged at 10,000 rpm for 15 minutes and supernatant was collected to the residual pellet. Repeated extraction with 80% ethanol twice, till supernatant volume reaches 25 ml. From 25 ml supernatant prepared suitable aliquots (0.2 to 1.0 ml). Make the final volume of aliquots 8.5 ml with distilled water. Then to this 0.5 ml Folin phenol reagent was added and incubated at room temperature for 3 minutes. After incubation, 1 ml of saturated sodium carbonate was added and mixed thoroughly. The optical density was measured at 650 nm, not later than 60 minutes.

Standard solution:

Phenol stock solution was prepared by dissolving 100 mg tannic acid in 100 ml of double distilled water in a volumetric flask.

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Thus, concentration of solution was 1 mg/ml or 1000µg/ml. The prepared stock must be used at the earliest.

To the aliquots of 0.2, 0.4, 0.6, 0.8 and 1.0 ml of stock solution add double distilled water to make the final volume 8.5 ml. Then 0.5 ml Folin phenol reagent added and incubated for 3 minutes. To above preparation 1 ml saturated sodium carbonate solution added. This solution was incubated 60 minutes and read optical density at 700 nm. Blank was plotted taking optical density on X-axis and concentration of phenol on y-axis. The concentration of unknown sample was read from the graph.

TANNINS:

Folin Denis method by Schanderl (1970) was used to estimate tannin compounds in plant.

Reagents:

 Folin Denis Reagent: 100 g sodium tungestate dissolved with 20

g phosphomolybdic acids in 750 ml of distilled water and then 50 ml of phosphoric acid added. Refluxed the mixture for 2 hours and made volume up to 1 litre with water. The reagent was protected from exposure to light.

 Sodium Carbonate Solution: 350 g Sodium carbonate dissolved in 1 litre of water at 70-800_{C. Filtered through glass wool and}

allowed it to stand overnight.

 Standard tannic acid solution: 100 mg tannic acid was dissolved in 100 ml of distilled water.

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 Working solution: 5 ml of standard tannic acid solution was

diluted to 100 ml with distilled water. Thus, concentration of working solution was 50 µg/ml.

Sample preparation: Extraction of tannin:

Weighed 0.5 g of the test sample and transferred to 250 ml conical flasks. 75 ml water added. Then the flasks were gently boiled for 30 minutes and centrifuged at 2000 rpm for 20 min. The supernatant was collected in 100 ml volumetric flask and made up the volume. 1 ml of the sample extract transferred to a 100 ml volumetric flask containing 75 ml of double distilled water. 5 ml Folin Denis reagent and 10 ml of sodium carbonate solution were added. Entire solution diluted to 100 ml with water and mixed well. Read the absorbance at 700 nm after 30 minutes. The standard graph was plotted taking optical density on X- axis and concentration of tannin on Y-axis. The concentration of unknown sample was read from the graph.

FLAVONOIDS:

Flavonoids estimation in test samples (Hongbin et al., 2009). Extraction of flavonoids:

Powders of the samples (0.5 g) were accurately weighed and placed in a sealed vessel by adding 25 ml of the ethanol-water (70:30, v/v) solvent, followed by the extraction for 48 hours at room temperature. The refluxed it at 450_{C for 15-20 minutes.}

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Determination of flavonoids:

Firstly, 2ml of the sample solution was accurately removed in a volumetric flask (10 ml) by adding 0.6 ml of NaNO2 (5%) solution,

shaken up and then standing for 6 min. secondly, 0.5 ml of the Al(NO3) (10% solution was added to the volumetric flask, shaken, and

was left to stand for 6 min. Finally 3.0 ml of the NaOH (4.3%) solution was added to the volumetric flask, followed by addition of water to the scale, shaken and left to stand for 15 min before determination. Using the sample solution without coloration as reference solution and 500 nm as determination wavelength, the coloration method was used to determine the content of flavonoids in the sample by ultraviolet-visible detector.

Preparation of standard solution:

A standard solution (0.16 mg/ml) of phloroglucinol was prepared as follows: phloroglucinol (200 mg) was accurately weighed and dissolved in ethanol-water (70:30, v/v) solution and then the solution was diluted to 100 ml in a volumetric flask (100 ml) by ethanol- water (70:30, v/v) solvent. Two millilitres of this solution was removed and diluted to 25 ml in a volumetric flask by ethanol-water (70:30, v/v) solvent.

Standard curve:

Five portions of the phloroglucinol solution were accurately removed (0.4, 0.8, 1.2, 1.6, and 2.0 ml) in five volumetric flasks (10 ml), and then the following work was done (as the description in determination of flavonoids). Using the concentration of phloroglucinol standard solution as X-axis and the absorbency as Y-axis.

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Gas Chromatography – Mass Spectrometry/ NMR

The methanolic samples were used into a GC-MS system Perkinelmer- CLAURAS 500 to identify compounds present in the leaf sample. For the detection of the peaks found in the graph, The Organic Compounds database of Colby University, Chemistry

department was used.

ANTIFUNGAL ACTIVITY

To check antifungal activity two types of extracts were used. 1) Aqueous extract

2) Methanolic extract

Aqueous extract: Aqueous extract was prepared by Soxhlet extraction. The extract was evaporated at 50-600_{C and the residue collected and}

stored in refrigerated till used (Raman, 2005).

Methanolic extract: Methanolic extraction was done by cold maceration. 5 g powder of plant material was mixed with 100 ml distilled water, and kept on rotatory shaker for 24 hours. Then it was filtered with Whatman filter paper No.1. Filtrate was evaporated at 500_{C and dried. Residue was used to check antifungal activity.}

Culture of Fungi:

For culturing of fungi glucose peptone (Sabouraud’s) agar was used. Nutrient agar medium was also used for maintaining the cultures. The most optimum growth temperature for pathogenic fungi was 300_C.

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Culture media:

Normally nutrient agar medium was used for growing fungi. But for testing plant antifungal activity, we used Sabouraud’s Agar (SDA) medium. The composition of the media was as follow:

1. Nutrient Agar Medium (pH 7) Peptone 1.5g

Beef extract 0.9g NaCl 1.5g Agar 9g

Distilled water 300ml

2. Sabouraud’s Agar (SDA) medium(pH 5.6) Peptone 10g

Dextrose 40g Agar 15g Distilled water 1000ml

Determination of Inhibition Concentration: (MIC)

Plates with SDA medium with different concentration of plant extract (aqueous/methanolic) with a difference of 100µg/ml were prepared for initial screening on Rhizopus spp. (MTCC No.2591), Fusarium spp. (MTCC No.6084), and Aspergillus niger (MTCC No.2587). Actively growing fungi from the margin of the plates (before spore formation) were inoculated on to the test plates with cork-borer and incubated at 250_{C in the dark for 2 days (Meyer and Afolayan, 1995).}

A negative blank and the positive control were also inoculated simultaneously. The negative blank containing only SDA medium and in positive control, solvent added in SDA medium. Growth zones were observed up to 72 hrs.

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In first stage of screening for MIC, plates which had 100ug/ml difference of drug concentration in each plate were used. The concentration of drug in which there was complete inhibition and concentration one step lower to that where there was slow growth were selected and second stage screening was done with drug concentration difference of 10 µg/ml. final concentration of the drug which was completely inhibiting the fungal growth was identified as Minimum Concentration of Inhibition(MIC).

ANTIBACTERIAL ACTIVITY

MATERIALS AND METHODS

Collection and Identification of Plant Material:

Fresh plant material was washed under running tap water, air dried and then homogenized to fine powder and stored in airtight containers.

Extraction of Plant Material Aqueous extraction:

10 g of air-dried powder was added to distilled water and boiled on Soxhlet extractor for 3 hrs. It was then filtered through vacuum filtration method. The extract was evaporated at 40-500_{C and}

the residue collected and stored in refrigerated till used (Raman, 2005)

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Methanol extraction

10 g of air-dried powder was taken in 100 ml of methanol in a conical flask, plugged with cotton wool and then kept on a rotary shaker at 190-220 rpm for 24 h. After 24 hours the supernatant was collected and the solvent was evaporated.

Bacterial Strains In vitro antimicrobial activity was examined for aqueous and methanol extracts from different parts of Argyreia nervosa used. Amongst 3 microorganisms investigated, two positive bacteria were Streptococcus spp. (MTCC No.1928) and Gram-negative Proteus vulgaris (MTCC No.1771) and Salmonella spp. (MTCC No.733).

All the microorganisms were maintained at 40_{C on nutrient agar}

slants.

Media Preparation and Antibacterial Activity

The antimicrobial assay was performed by agar well diffusion method for aqueous extract and methanolic extract. The molten Mueller Hinton agar was inoculated with 100 µl of the inoculum (1 x 108 cfu/ml) and poured into the Petri plate (Hi-media). For agar well diffusion method, a well was prepared in the plates with the help of a cork-borer (0.85 cm). 100 µl of the test compound was introduced into the well. The plates were incubated overnight at 370_{C. Microbial}

growth was determined by measuring the diameter of zone of inhibition. For each bacterial strain, controls were maintained where pure solvents were used instead of the extract. The result was obtained by measuring the zone diameter. The experiment was done three times and the mean values are presented.

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GENETIC STABILITY ANALYSIS

In this study, sample of Argyreia nervosa collected from the south Gujarat area was analyzed for variation using RAPD markers to determine the level of genetic diversity between the original plant and the cultured cells.

(A) Total Genomic DNA Extraction and Isolation:

Total Genomic DNA was extracted according to the CTAB method of Doyle and Doyle (1990), with a few modifications. Initially about 0.5gm of the frozen material of the test samples was taken. It was then mixed with the 2.5ml of the preheated extraction buffer (1.5M NaCl, 100mM Tris-Hcl pH-8, 40mM EDTA, 1% PVP, 3% C-Tab and 1% β-mercaptoethanol). The mixture was grinded and crushed

into the fine paste with the help of pestle and mortal and transferred to the sterile tubes and then incubated at 65°C for 1hour with occasional shaking.

The suspension was then mixed with 1/2 volume of chloroform: isoamyl alcohol (24:1), content was mixed by inversion to form an emulsion and then the tubes were centrifuged at 13,000 rpm for 15min. The upper phase (supernatant) was transferred to a new sterilized tube. Extraction was repeated with an equal volume of chloroform: isoamyl alcohol (24:1) again. The aqueous layer was transferred to a new tube, 0.6 volume of ice-cold isopropanol was added and tubes were incubated at -20°C for overnight period for precipitation and then centrifuged at 10,000rpm for 25 min at 4°C.

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The pellet was sedimented by centrifugation and washed carefully twice with 70% ethanol, briefly dried at room temperature and resuspended in 100µl of TE buffer (10mM Tris-HCl, 1mM EDTA pH8). The mixture is kept at 20°C for approximately 1015min at -20°C. To the above mixture, 0.1µl of BSA (Bovine albumin serum) is then added. The final mixture is then centrifuged at 10,000 rpm for 5min, and the supernatant is taken. The presence and quality of the extracted DNA is checked by the electrophoresis.

(B) Quality check and quantification of DNA:

Quantification of DNA was determined by UV-Spectrophotometer and the quality of the DNA was checked on agarose minigel electrophoresis according to Sambrook et al., (1989). For quantification, the stock of genomic DNA was diluted to 1:50 in millipore water and OD was measured at 260nm. The concentration

of DNA was calculated as; DNA (µg/ml) = O.D.260 x 50 x 50

The quality of DNA was checked on 0.8% agarose containing 5 µl of ethidium bromide (50µg /ml) per 100 ml of gel. Genomic DNA (5 µl) from stock was mixed with 1 µl of 6X BPB (Bromo Phenol Blue) gel loading dye and loaded on the gel. The gel was run at 80 mA. The bands were visualized under UV light and scanned by gel documentation system. The DNA sample with the ratio of 1.8-2.0 at OD 260/280 was retained for DNA fingerprinting. The stock solution was diluted to the concentration of 50-60ng/µl and used for further analysis.

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Figure: 24 shows the bands of DNA extracted from the samples and named according to that. The table: 4 show the sample names used for identifying the different samples during the procedure.

Table: 4

Samples for RAPD Analysis

SR NO

SAMPLE NO

NAME

1. 1A Cultured Leaf cell 2. 1B Cultured Leaf cell 3. 2A Cultured Seed cell 4. 2B Cultured Seed cell 5. 2C Cultured Seed cell 6. 2D Cultured Seed cell 7. 3 Cultured Stem cell 8. 4 Original Plant Stem

Amplification Procedure:

After the DNA extraction procedure, two DNA samples viz. 1A and 2B (Table: 4) were selected for the screening procedure for Polymerize Chain Reaction. The reason for considering these two samples is because of their quantity and quality (figure: 24) during the extraction and electrophoresis (figure: 24). The primers (Integrated DNA Technologies, Inc. Coralville, IA) taken for the PCR and RAPD analysis were given the specific codes for the analysis purpose. The details of each primer are been given in the table: 5. The cycler

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selected for the procedure was of Biometra, and the complete information of the system is as follows;

Cycler Type: T Professional Company: Biometra

Serial No: 2203303 Rev. No: 32090200d Block type: Gradient 96 Serial No: 20071379 Software version: 1.22 – 1.22 – 1.10 tpr Protocol version: 1.0.0.0 – 0.4.1.0 – 0.4.1.0 Table: 5 Primer Details Sr No

Code Sequence Tm GC% Mol Wt 1. OPG1 5’ – CTA CGG AGG A – 3’ 31.5°C 60.0% 3077.1 2. OPG2 5’ – GGC ACT GAG G – 3’ 36.6°C 70.0% 3093.1 3. OPG3 5’ – GAG CCC TCC A – 3’ 37.8°C 70.0% 2973.0 4. OPG4 5’ – AGC GTG TCT G – 3’ 35.3°C 60.0% 3059.0 5. OPG5 5’ – CTG AGA CGG A – 3’ 32.8°C 60.0% 3077.1 6. OPG6 5’ – GTG CCT AAC C – 3’ 31.8°C 60.0% 2988.0 7. OPG7 5’ – GAA CCT GCG G – 3’ 38.0°C 70.0% 3053.0 8. OPG8 5’ – TCA CGT CCA C – 3’ 34.5°C 60.0% 2948.0

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9. OPG9 5’ – CTG ACG TCA C – 3’ 32.4°C 60.0% 2988.0 10. OPG10 5’ – AGG GCC GTC T – 3’ 41.2°C 70.0% 3044.0 11. OPH1 5’ – GGT CGG AGA A – 3’ 33.2°C 60.0% 3117.1 12. OPH2 5’ – TCG GAC GTG A – 3’ 36.7°C 60.0% 3068.0 13. OPH3 5’ – AGA CGT CCA C – 3’ 33.9°C 60.0% 2997.0 14. OPH4 5’ – GGA AGT CGC C – 3’ 37.5°C 70.0% 3053.0 15. OPH5 5’ – AGT CGT CCC C – 3’ 38.0°C 70.0% 2964.0 16. OPH6 5’ – ACG CAT CGC A – 3’ 39.9°C 60.0% 2997.0 17. OPH7 5’ – CTG CAT CGT G – 3’ 33.7°C 60.0% 3019.0 18. OPH8 5’ – GAA ACA CCC C – 3’ 32.1°C 60.0% 2966.0 19. OPH9 5’ – TGT AGC TGG G – 3’ 33.0°C 60.0% 3099.1 20. OPH10 5’ – CCT ACG TCA G – 3’ 30.5°C 60.0% 2988.0 21. OPH11 5’ – CTT CCG CAG T – 3’ 35.1°C 60.0% 2979.0 22. OPH12 5’ – ACG CGC ATG T – 3’ 40.4°C 60.0% 3028.0 23. OPH13 5’ – GAC GCC ACA C – 3’ 38.7°C 70.0% 2982.0 24. OPH14 5’ – ACC AGG TGG G – 3’ 33.8°C 60.0% 3068.0 25. OPH15 5’ – AAT GGC GCA G – 3’ 37.1°C 60.0% 3077.1 26. OPH17 5’ – CAC TCT CCT C – 3’ 29.5°C 60.0% 2898.0 27. OPH18 5’ – GAA TCG GCC A – 3’ 35.2°C 60.0% 3037.0 28. OPH19 5’ – CTG ACC AGC C – 3’ 36.6°C 70.0% 2973.0 29. OPH20 5’ – GGG AGA CAT C – 3’ 30.0°C 60.0% 3077.0 30. OPL1 5’ – GGC ATG ACC T – 3’ 34.2°C 60.0% 3028.0 31. OPL2 5’ – TGG GCG TCA A – 3’ 38.1°C 60.0% 3068.0 32. OPL3 5’ – CCA GCA GCT T – 3’ 35.5°C 60.0% 2988.0 33. OPL4 5’ – GAC TGC ACA C – 3’ 33.3°C 60.0% 2997.0 34. OPL5 5’ – ACG CAG GCA C – 3’ 41.7°C 70.0% 3022.0 35. OPL6 5’ – GAG GGA AGA G – 3’ 29.2°C 60.0% 3166.1 36. OPL7 5’ – AGG CGG GAA C – 3’ 39.8°C 70.0% 3102.1

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37. OPL8 5’ – AGC AGG TGG A – 3’ 36.0°C 60.0% 3117.1 38. OPL9 5’ – TGC GAG AGT C – 3’ 34.2°C 60.0% 3068.0 39. OPL10 5’ – TGG GAG ATG G – 3’ 32.2°C 60.0% 3148.1

1st_Screening:

As stated above, the first screening for the primers was carried out on two samples viz. 1A and 2B with two series of primers viz. OPG1 to OPG10 and OPH1 to OPH10 respectively. After the successful run of 38 cycles, the amplified products were separated on agarose gel electrophoresis.

a) Preparation of reaction mixture:

 Buffer = 2 X 10 = 20µl  dNTPs = 0.5 X 10 = 5µl  MgCl2 = 1.2 X 10 = 12µl  Primer = 1.5 X 1 = 1.5µl  Enzyme = 0.4 X 10 = 4µl  DNA = 0.8 X 10 = 8µl  Sterile Water = 15 X 10 = 150µl

The reaction mixture is referred to as the master mixture and is transferred to 10 different sterile eppendorf tubes at the volume of 19µl with addition of 1µl of primer to each tube making the final volume at 20µl. These tubes having same DNA with different primers are then amplified.

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2nd_Screening:

Second screening for the primers was carried out on two samples viz. 1A and 2B with two series of primers viz. OPL1 to OPL9 and OPH11 to OPH20 (except OPH16) respectively. After the successful run of 38 cycles, the amplified products were separated on agarose gel electrophoresis.

The mixture is prepared as per the first screening. b) PCR Cycle:

The cycle is kept as per the first screening (figure: 25). 3rd_Screening:

Third screening for the primers was carried out on two samples viz. 1A and 2B with two series of primers viz. OPS1 to OPS10 and OPL1 to OPL10 respectively. After the successful run of 38 cycles, the amplified products were separated on agarose gel electrophoresis.

The mixture is prepared as per the first screening. b) PCR Cycle:

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RAPD Analysis:

With above selected primers final RAPD is carried out for analyzing the genetic diversity. The samples selected for the procedure are 1A, 2B, 3 and 4 (Table: 4) as they show the clearer band during separation (figure: 24).

1st_Analysis:

The first analysis was carried out with primers OPH13 (Table: 5). In the amplification process, the system was set to same as that of screening procedure (figure: 24). Also while electrophoresis, the known 500 base pair DNA ladder is used to compare the banding pattern.

Preparation of reaction mixture:

 Buffer = 2 X 5 = 10µl  dNTPs = 0.5 X 5 = 2.5µl  MgCl2 = 1.2 X 5 = 6.0µl  Primer = 1.5 X 5 = 7.5µl  Enzyme = 0.3 X 5 = 1.5µl  DNA = 1 X 1 = 1µl (each)  Sterile Water = 13.5 X 5 = 67.5µl

The reaction mixture is referred to as the master mixture and is transferred to 4 different sterile eppendorf tubes at the volume of 19µl with addition of 1µl of DNA to each tube making the final

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volume at 20µl. These tubes having same primer with different DNA are then amplified.

2nd_Analysis:

The second analysis was carried out with primer OPH14 (Table: 5). In the amplification process, the system was set to same as that of screening procedure (figure: 24). Also while electrophoresis, the known 500 base pair DNA ladder is used to compare the banding pattern.

The reaction mixture is referred to as the master mixture and is transferred to 4 different sterile eppendorf tubes at the volume of 19µl with addition of 1µl of DNA to each tube making the final volume at 20µl. These tubes having same primer with different DNA are then amplified.

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3rd_Analysis:

The third analysis was carried out with primer OPH15 (Table: 5). In the amplification process, the system was set to same as that of screening procedure (figure: 24). Also while electrophoresis, the known 500 base pair DNA ladder is used to compare the banding pattern.

4th_Analysis:

The fourth analysis was carried out with primer OPL3 (Table: 5). In the amplification process, the system was set to same as that of screening procedure (figure: 24). Also while electrophoresis, the

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known 500 base pair DNA ladder is used to compare the banding pattern.