4. MATERIALS AND METHODS 4.1 Chemicals and reagents …shodhganga.inflibnet.ac.in/bitstream/10603/8035/8/08_chapter 4.pdf · Determination of moisture content ... distilled water

Materials and methods

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4. MATERIALS AND METHODS

4.1 Chemicals and reagents used

Atomic absorption spectrometer (AAS) standard metal solutions of magnesium,

manganese, calcium, copper, zinc, silicon, lead, arsenic, mercury, cadmium, nickel,

chromium and aluminium were procured from Sigma Aldrich limited, Mumbai,

India. The metal solutions were diluted with deionised water to produce working

standards of 1000 ppm solutions and stored. Acrylamide, bisacrylamide, tris base,

ammonium persulphate, sodium dodecyl sulphate, mercaptoethanol, bromophenol

blue, glycine, coomassie brilliant blue G250, coomassie brilliant blue R250, bovine

serum albumin, TEMED, agarose, ethidium bromide, isoamyl alcohol, 2,4,6-

tripyridyl-s-triazine (TPTZ), dinitrophenyl hydrazine (DNPH), 2,6-dichlorophenol

indophenols were procured from Sigma Aldrich limited, Mumbai, India. Prestained

broad range SDS-PAGE standard and LA 393-5MT Dialysis membrane 70, flat

width 29.31mm, diameter 17.5mm, capacity approx. 2.41mL/cm were obtained

from Bio-Rad laboratories, Hercules, CA. Master mix, ITS1 forward primer, ITS4

reverse primer, pGEM-T easy vector, 1 kilobase pair (kb) DNA ladder were

procured from Bangalore Genei Pvt. Ltd., Bangalore, India. 3-(4, 5-dimethyl thiazol–

2–yl)–5–diphenyl tetrazolium bromide (MTT) was obtained from Acros Organics,

New Jersy, USA.

4.2 Instruments used

i. Rotary evaporator: Rota vapor R-205, Buchi Laboratory Equipments,

Switzerland.

ii. Muffle furnance: Scientec, Genuine equipment manufacturers, Coimbatore,

India

iii. Atomic absorption spectrometer: Shimadzu AA 6300 flame atomic

absorption spectrometer, Tokyo – Japan, equipped with a deuterium

background corrector and a hydride vapour generator for analysis of arsenic


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and mercury. Hollow cathode lamps of specific metals were used as a

radiation source.

iv. HPLC: Shimadzu, liquid chromatographic system equipped with LC20AT

solvent delivery system (pump), fluorescence detector and auto sampler

with 100µL loop volume was used. Class VP 6.01 data station was used for

data collection and processing.

v. Matrix Assisted Laser Desorption Ionisation: MALDI-ToF/ToF MS, Bruker

Daltonics, Ultraflex III, Germany.

vi. PCR: Eppendorf Master cycler, Germany

vii. DNA document analyser: Alpha Innotech Corporation, San Leandro,

California.

viii. Systronics pH meter

ix. Spectrophotometer: Shimadzu 160-A UV-VIS, Koyota, Japan.

x. Elisa reader : Thermo Multiskan, EX, USA.

4.3 Plant collection, authentication and extraction

The plant, Spergula arvensis Linn., was collected from in and around

Udhagamandalam in the month of August 2009 and was authenticated by

Dr.N.Selvaraj, Professor and Head, Horticulture Research Station (TNAU),

Udhagamandalam, The Nilgiris, Tamilnadu. A voucher specimen with number,

TIFAC 05, was deposited at the herbarium of JSS College of Pharmacy,

Udhagamandalam.

Processing of the sample

Approximately 5kg of the plant sample was collected and dried. Healthy plants

were removed and washed thoroughly with distilled water until no foreign

material remained. Some of the samples were dried using tray drier for 3h. The

dried samples were stored in a cupboard in capped bottles kept in dessicator and

used when required.


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Extraction

The dried plant material was powdered and successively extracted with petroleum

ether, chloroform, ethyl acetate and methanol in a Soxhlet extractor for 18–20h. The

extracts were concentrated to dryness in a rotavapor under reduced pressure and

controlled temperature (40-50°C). The nature and yields of the extracts were noted.

All the extracts were stored in a refrigerator at 4°C for further use.

4.4 Preliminary phytochemical screening of the sample

The dried plant sample obtained was subjected to preliminary phytochemical

screening. (Harborne, 1984; Kokate 2002; Raaman N., 2006).

4.5 Quantitative nutrient analysis

Proximate analysis

Proximate analysis is a technique that separates and identifies categories of

compounds in a mixture like moisture, ash content, starches, reducing sugars,

proteins, fats, esters, free acids, etc. This method of analysis helps to know about the

energy contained in the sample. The analysis was carried out as per the procedure

given by Sadasivam and Manickam, 2009.

Determination of total ash

About 3g of the powdered plant was accurately weighed in a silica crucible which

was previously ignited and weighed. The powder was spread as a fine even layer at

the bottom of the crucible. The crucible was incinerated at a temperature not

exceeding 450ºC until free from carbon. The crucible was cooled and weighed and

the procedure was repeated to get a constant weight.

Determination of moisture content

The sample material was taken in a flat-bottom dish and kept overnight in an air

oven at 100–110ºC and weighed. The loss in weight was regarded as a measure of

moisture content.


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Estimation of crude fat

The moisture free sample (2g) was extracted with petrol in a Soxhlet extractor. It

was then heated on a sand-bath for about 6h till a drop taken from the drippings

left no greasy stain on the filter paper. After boiling with petrol, the residual petrol

was filtered using Whatmann no. 40 filter paper and the filtrate was evaporated in a

pre weighed beaker. Increase in weight of the beaker gave the amount of crude fat.

Estimation of crude protein

The crude protein was determined using the micro Kjeldahl method. The oven-

dried material (2g) was taken in a Kjeldahl flask and 30mL of con H2SO4 was added

followed by 10g potassium sulphate and 1g of copper sulphate. The mixture was

then heated first gently and then strongly till the frothing ceased. When the solution

became colourless, it was heated for another hour, allowed to cool, diluted with

distilled water and transferred to a 800mL Kjeldahl flask by washing the digestion

flask. Three or four pieces of granulated zinc, and 100 mL of 40% caustic soda were

then added and the flask was connected with the splash heads of the distillation

apparatus. Dil H2SO4 (0.1N, 25mL) was taken in the receiving flask and distilled.

After two-thirds of the liquid distilled out, it was tested for the completion of the

reaction. The flask was removed and titrated against 0.1N caustic soda solution

using methyl red indicator for the determination of Kjeldahl nitrogen. The crude

protein content was calculated.

Estimation of crude fiber

The crude fiber, was determined by treating the moisture and fat-free material, first

with 1.25% dilute acid and then with 1.25% alkali, thus imitating the gastric and

intestinal action in the process of digestion. Then 2g of the moisture and fat-free

material was treated with 200mL of 1.25% H2SO4. After filtration and washing, the

residue was treated with 1.25% NaOH. It was then filtered, washed with hot water

and then 1% HNO3 and again with hot water. The residue was ignited and the ash

weighed. Loss in weight gave the weight of the crude fiber.


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Estimation of phenols

The plant sample (1g) was homogenized in 10mL of 80 per cent methanol with

pestle and mortar and agitated for 15 min at 70ºC. One mL of the methanolic extract

was added to a mixture of 5mL of distilled water and 250µL of Folin Cicalteau

reagent (1N) and the solution was kept at 25ºC. After 3 min 1mL of saturated

solution of sodium carbonate and one mL of distilled water was added and the

reaction mixture was incubated for 1h at 25ºC, when a blue colour developed. The

absorbance of the blue colour was measured using UV-Visible Spectrophotometer at

726 nm.

Estimation of carbohydrates

Carbohydrates are the important components of storage and structural materials in

plants. They exist as free sugars and polysaccharides. The basic units of

carbohydrates are the monosaccharides which cannot be split by hydrolysis into

simpler sugars. The carbohydrate content can be measured by hydrolyzing the

polysaccharidres into simple sugars by acid hydrolysis and estimating the resultant

monosaccharides. In the present study the carbohydrate was estimated by the

phenol sulphuric acid method.

Principle

In hot acidic medium, glucose gets dehydrated to hydromethyl furfural. This forms

a green coloured product with phenol and has an absorption maximum at 490nm.

Procedure

The sample (100mg) was taken in a boiling tube. It was then hydrolysed with 5mL

of 2.5N HCl, kept in a boiling water bath for 3h and cooled to room temperature.

The solution was neutralized with solid sodium carbonate until the effervescence

ceased. The volume was made up to 100mL and centrifuged. The supernatants

(0.5mL and 1mL) were collected and analysed. The glucose solutions (0.2, 0.4, 0.6,

0.8 and 1mL) were pipetted into a series of test tubes (working standards). Then the


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volume in all the tubes was made up to 1mL with distilled water. Phenol solution

(1mL) and 96% sulphuric acid (5mL) were then added to each tube. Water (1mL)

served as the blank. The contents in the tube were mixed well and after 10 min they

were placed in a water bath at 25-30ºC for 20 min and the absorbance was read at

490nm. The amount of the total carbohydrate present in the sample solution was

determined using the standard graph.

Estimation of cellulose

Principle

Cellulose undergoes acetolysis with acetic/nitric acid forming acetylated

cellodextrins which get dissolved and hydrolysed to form glucose molecules on

treatment with 67% H2SO4. This glucose molecule is dehydrated to form

hydroxymethyl furfural which forms a green coloured product with anthrone and

the colour intensity is measured at 630nm.

Procedure

Acetic acid (3mL) was added to a known amount (1g) of the sample in a test tube

and mixed in a vortex mixer. The tube was placed in a water bath at 100ºC for 30

min and cooled. The contents were then centrifuged for 15-20 min and the

supernatant was discarded. The residue was washed with distilled water and 10mL

of 67% H2SO4 was added to it and allowed to stand for 1h. The solution (1mL) was

diluted to 100mL. To 1mL of this diluted solution, 10mL of anthrone reagent was

added and mixed well. The tubes were heated in a boiling water bath for 10 min

and cooled. The colour was measured at 630nm. A blank was set with anthrone

reagent and distilled water. Cellulose (100mg) was taken in a test tube and the same

procedure was followed for the standard. A series of volumes (0.4-2mL,

corresponding to 40-200µg of cellulose) were taken and the colour developed was

read at 630nm.


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Estimation of free fatty acid

A small quantity of free fatty acid is usually present in oils along with triglycerides.

The free fatty acid content is known as acid number/acid value. It increases during

storage. The keeping quality of oil, therefore, relies upon the free fatty acid content.

Principle

The free fatty acid in oil is estimated by titrating it against KOH in the presence of

phenolphthalein indicator. The acid number is defined as the mg KOH required to

neutralize the free fatty acid present in 1g of sample. The free fatty acid content is

expressed as oleic acid equivalents.

Procedure

The oil (1g) was dissolved in 50mL of a neutral solvent in a 250mL conical flask and

a few drops of phenolphthalein was added to it. It was then titrated against 0.1N

potassium hydroxide. The contents were shaken vigorously until a pink colour

obtained persisted for fifteen seconds.

Titre value x Normality of KOH

Acid value (mg KOH/g) = X 56.1 Weight of the sample (g)

The free fatty acid was calculated as oleic acid using the equation,

1mL N/10 KOH = 0.028g oleic acid.

Note: To find out the exact strength of KOH, 0.1N oxalic acid solution (630mg in 100

mL water) was prepared and titrated against KOH with phenolphthalein as the

indicator. The strength of KOH was calculated using the formula V1N1 = V2N2.

Total free amino acid

The amino acids are colourless ionic compounds that form the basic building blocks

of proteins. Apart from being bound as proteins, amino acids also exist in free form

in many tissues and are known as free amino acids. They are mostly water soluble


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in nature. Very often in plants during disease conditions, the free amino acid

composition exhibits a change and hence, the measurement of total free amino acid

gives the physiological and health status of the plants.

Principle

Ninhydrin is a powerful oxidizing agent, decarboxylates the α-amino acids and yields an

intensely coloured bluish purple product which is colorimetrically measured at 570nm.

Procedure

Extraction of amino acids

The leaf sample (500mg) was ground with a small quantity of acid washed sand and

5mL of 80% ethanol was added to this homogenate and centrifuged. The supernatant

obtained was saved and the extraction was repeated twice and the supernatants were

pooled. The volume of the sample was reduced by evaporation and the extract was used

for the quantitative estimation of total free amino acid. To 0.1mL of the extract, 1mL of

ninhydrin solution was added and the volume was made up to 2mL with distilled

water. The test tube was heated in a boiling water bath for 20 min. To this 5mL of the

diluent mixture was added and the contents were mixed. After 15 min the intensity of

the purple colour formed was read against a reagent blank in a colorimeter at 570nm.

Standard

Leucine (50mg) was dissolved in 50mL of distilled water in a volumetric flask (stock

solution). Stock solution (10mL) was taken and diluted to 100mL in another

volumetric flask (working standard). A series of volume from 0.1-1mL of the

working standard solution gave a concentration range 10-100µg. The same

procedure as that of the sample was followed and read the colour at 570nm. The

total free amino acids in the sample was determined by a standard curve using

absorbance vs concentration expressed as percentage equivalent of leucine.


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Estimation of tannins

Tannin content in the leaf samples were estimated following the Folin-Dennis

method (Oberbacher and Vines ,1963).

Standard tannic acid solution

Tannic acid (100mg) was dissolved in 100mL of distilled water. The working

standard solution was prepared by diluting 5mL of the stock solution to 100mL

with distilled water.

Extraction of tannin

The powdered leaf material (250mg) was weighed and transferred to a 50mL

conical flask to which 10mL of water was added. The flask was heated gently and

boiled for 30 min. The flask was then cooled and the contents were centrifuged at

5000 rpm for 20 min, the supernatant was collected and the volume was made up to

10mL. The sample extract (0.5mL) was transferred into test tube and the volume

was made up to 7.5mL with water. To this, 0.5mL of Folin-Denis reagent and 1.0mL

of sodium carbonate solution were added and shaken well. The absorbance was

read at 700 nm after 30 min. A blank was run with water instead of the sample. The

tannin content was calculated as tannic acid equivalents from the standard graph.

Estimation of lycopene

Lycopene is responsible for the red colour of tomato and the fleshy part of water

melon. It is a carotene having the formula C40H56. Though, it has no nutritional

value, its contribution to the colour of tomato has a great role in consumer

acceptability.

Principle

The carotenoids in the sample are extracted in acetone and then taken in petroleum

ether. Lycopene has absorption maxima at 473nm and 503nm. One mole of

lycopene when dissolved in one litre light petroleum (40 - 60ºC) and measured in a


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spectrophotometer at 503nm in 1cm light path gives an absorbance of 17.2 x 104. A

concentration of 3.1206µg lycopene/mL, therefore, gives unit absorbance.

Procedure

The plant sample (2g) was taken and grinded well to a smooth consistency in a

blender. About 1g of the ground sampled was washed accurately. The sample was

then repeatedly extracted with acetone using pestle and mortar until the residue

became colourless. The acetone extracts were pooled and transferred to a separating

funnel containing about 20mL petroleum ether and mixed gently. About 20mL of

5% sodium sulphate solution was then added. The separating funnel was gently

shaked. The volume of petroleum ether reduced during this process because of

evaporation. More of petroleum ether (20mL) was then added to the separating

funnel for a clear separation of two layers. Most of the colour was noticed in the

upper petroleum ether layer. The two phases were separated (the lower aqueous

phase was colourless). The petroleum ether extracts were pooled and washed once

with a little distilled water. The washed petroleum ether extract containing

carotenoids was poured into a brown bottle containing anhydrous sodium sulphate

(10g) and kept aside for 30 min. It was then poured into a 100mL volumetric flask

through a funnel containing cotton wool. The sodium sulphate slurry was washed

with petroleum ether until it was colourless and the washings transferred into the

volumetric flask. The volume was made up and the absorbance was measured in a

spectrophotometer at 503nm using petroleum ether as blank.

Calculation

Absorbance (1 unit) = 3.1206 µg lycopene/ mL

31.206 x absorbance Lycopene (mg) in 100g sample = Weight of sample (g)


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Estimation of starch

Starch is an important polysaccharide. It is the storage form of carbohydrate in

plants and abundantly found in roots, tubers, stems, fruits and cereals. Starch,

which is composed of several glucose molecules, is a mixture of two types of

components, namely amylase and amylopectin. Starch is hydrolysed into simple

sugars by dilute acids and the quantity of simple sugars is measured

colorimetrically.

Principle

The sample is treated with 80% alcohol to remove sugars. Starch is then extracted

with perchloric acid. In hot acidic medium, starch is hydrolysed to glucose and

dehydrated to hydroxymethyl furfural. This compound forms a green coloured

product with anthrone.

Procedure

The sample (0.1g) was homogenised in hot 80% ethanol to remove sugars,

centrifuged and the residue was retained. The residue was washed repeatedly with

hot 80% ethanol till the washings did not give colour with anthrone reagent. The

residue was dried well over a water bath. To the residue, 5mL of water and 6.5mL

of 52% perchloric acid were added and extracted at 0ºC for 20 min, centrifuged and

the supernatant was saved. The extraction was repeated using fresh perchloric acid

and centrifuged, again the supernatants were pooled. The supernatant (0.1mL) was

pipetted out and made up the volume to 1mL with distilled water. Standards were

prepared by taking 0.2, 0.4, 0.6, 0.8 and 1mL of the working standard and the

volume made up to 1mL in each tube with water. Anthrone reagent (4mL) was then

added to each tube and heated for 8min in a boiling water bath. The solution was

rapidly cooled and the intensity of the green to dark green colour formed was read

at 630nm.


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Calculation

Glucose content in the sample was obtained using the standard graph and

multiplying the value by a factor 0.9 to arrive at the starch content.

Estimation of total antioxidants

Total antioxidant activity is measured by ferric reducing antioxidant power (FRAP)

assay (Benzie and Strain, 1999). FRAP assay uses antioxidants as reductants in a

redox linked colorimetric method.

Principle

At low pH, reduction of ferric tripyridyl triazine (Fe 3+ TPTZ) complex to ferrous

form (which has an intense blue colour) can be monitored by measuring the change

in absorption at 593nm. The reaction is non-specific, in that any half reaction that

has a lower redox potential, under reaction conditions, than that of ferric-ferrous

half reaction, will drive the ferric to ferrous (Fe 3+ to Fe 2+) ion formation. The

change in absorbance is therefore, directly related to the combined or total reducing

power of the electron donating antioxidants present in the reaction mixture.

Reagent preparation

Acetate buffer 300mM (pH 3.6)

i. Sodium acetate trihydrate (3.1g) was weighed and 16mL of glacial acetic acid

was added and made upto 1L with distilled water.

ii. 10mM TPTZ (2,4,6-tripyridyl-s-triazine) was dissolved in 40mM HCl

iii. 20mM FeCl3.6H2O were taken

The working FRAP reagent was prepared by mixing solution (i), (ii) and (iii) in the

ratio 10:1:1 at the time of use.

Standard : 1000µM Ascorbic acid


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Procedure

The sample (100µL) was mixed with 3mL of working FRAP reagent and the

absorbance was read at 593nm at 0 min after vortexing. The samples were placed at

37ºC in water bath and the absorption was again measured after 4 min. Ascorbic

acid standards (100µM-1000µM) were processed in the same way.

Calculation

Change in absorbance of sample from 0-4 min FRAP value of = X FRAP value of the Sample (µM) Change in absorbance of standard from 0-4 min std (1000µM)

Estimation of vitamin C

Ascorbic acid, otherwise known as vitamin C, is an antiscorbutic. It is present in

gooseberry, bittergourd, etc., in high amounts. Generally it is present in all fresh

vegetables and fruits. Vitamin C is a water soluble and heat liable vitamin. Ascorbic

acid was determined colorimetrically. Dehydroascorbic acid alone reacts

quantitatively and not the other reducing substances present in the sample. This

method thus gives an accurate analysis of ascorbic acid content than the dye (2,6-

dichlorophenol indophenol) method.

Principle

Ascorbic acid is first dehydrogenated by bromination. The dehydroascorbic acid is

then reacted with 2,4 dinitrophenyl hydrazine to form osazone and dissolved in

sulphuric acid to give an orange-red colour solution which is measured at 540nm.

Extraction

The sample (1g) was grinded in a mortar and pestle in 25-50mL of 4% oxalic acid

solution. This solution was filtered and the filtrate was collected. The collected

filtrate (10mL) was transferred to a conical flask and bromine water was added

dropwise with constant stirring. The enolic hydrogen atoms in ascorbic acid were


55

removed by bromine. The extract turned orange yellow due to excess bromine. The

bromine was expelled by blowing in air. This was made upto 25mL with 4% oxalic

acid solution. Similarly the ascorbic acid stock solution was converted into dehydro

form by bromination.

Procedure

The standard dehydroascorbic solution (10-100µg) was pipetted out into a series of

test tubes. The brominated sample (0.1-2mL) was also pipetted out. The volume

was made upto 3mL in each tube by adding distilled water. Dinitrophenyl

hydrazine (DNPH) reagent (1mL) was added to which 2 drops of thiourea was also

added. Distilled water (3mL), 1mL DNPH reagent and 2 drops of thiourea were

taken as blank. The contents in the tubes were mixed thoroughly and incubated at

37ºC for 3h. The orange red osazone crystal formed was dissolved by adding 7mL

of 80% sulphuric acid. The absorbance was read at 540nm. For calculating the

ascorbic acid content in the sample, a graph was plotted with ascorbic acid

concentration against absorbance.

Estimation of vitamin A

Vitamin A (retinol) is a fat-soluble vitamin. It is important for proper vision.

Vitamin A is supplied to the body in the form of its precursor, β-carotene, which is

present in fruits, vegetables, greens, etc. Vitamin A is not stored in the body when

consumed abundantly.

Principle

Vitamin A and its palmitate forms blue colour with trichloroacetic acid (TCA)

which is proportional to its concentration measured at 620nm.

Extraction

The sample (1g) was grinded to a fine paste and 1mL of saponification mixture (2N

KOH in 90% alcohol) was added to it. The tube was refluxed gently for 20 min at

60ºC. The tube was cooled at room temperature and 20mL water was added and


56

mixed well. Vitamin A was extracted with petroleum ether in a separating funnel,

twice. The organic layers were pooled and anhydrous sodium sulphate was added

to remove the moisture for 30-60 min. The ether extract (5mL) was evaporated to

dryness at 60ºC. The dried residue was dissolved in 1mL of chloroform.

Procedure

Aliquots of the standard vitamin A concentrations ranging from 1.5-7.5µg, acetate

were pipetted out into a series of test tubes. The volumes were made upto 10mL

with chloroform. Trichloroacetic (2mL) solution was added by rapidly mixing the

contents of the tube. The absorbance was read immediately at 620nm with a

spectrophotometer. The absorbance of the sample was also determined in the same

manner. The amount of vitamin A was calculated using a standard graph plotting

absorbance against vitamin A concentration.

Estimation of β-carotenes

Carotenoids, the tetraterpenoid (C40) compounds, are ubiquitous in plants. These

terpenoids existing as hydrocarbons (carotenes) or oxygenated derivatives, are

accessory pigments in photosynthetic systems and give characteristic colour to

plant parts, particularly flowers and fruits. Carotenes occurring in different

chemical forms have characteristics features and functions. Their levels are altered

during physiological and pathological conditions.

Principle

The total carotenoids are extracted and partitioned in organic solvents on the basis

of their solubility.

Procedure

The plant was finely cut with scissors. The weighed sample (2g) was placed in a

high speed blender. Acetone (40mL), hexane (60mL) and MgCO3 (0.1g) were added

and blended for 5 min. The residue was allowed to settle and decanted into a

separator. The residue was washed twice with 25mL portions acetone then with


57

25mL hexane and the extracts were combined. The acetone, present in the extracts,

was removed by washing the extract with five 100mL portions water. The upper

layer was transferred to a 100mL volumetric flask containing 9mL acetone and the

volume diluted with hexane. In a chromatographic tube a pack of magnesia-

diatomaceous earth (1:1) was activated. The column was prepared by placing a

small glass plug inside the tube and the adsorbent was added to 15cm depth, the

tube was attached to the suction flask and vacuum was applied. Sodium sulphate

(anhydrous) (1cm layer) was placed. Vacuum was continuously applied to the flask

and the extract was poured. Acetone-hexane mixture (1:9) (50mL) was used and the

chromatogram was developed. Visible carotenes were washed through the

adsorbent. The column at the top was covered with the solvent during the process.

The entire eluate was collected and transferred to a 100mL volumetric flask to

which acetone-hexane mixture was added for dilution. The instrument was

calibrated with different concentrations of high purity β-carotene. The absorbance

of the sample solution was determined with a spectrophotometer. Carotene content

in the sample was calculated using the calibration curve.

Elemental analysis by Atomic Absorption Spectrophotometric (AAS) method

(atomic adsorption cook book).

All the reagents used for the study were of analytical grade. Deionised water was

used for all the dilutions. Nitric acid, perchloric acid and hydrogen peroxide were

of supra pure quality. All the plastic materials and glasswares were cleaned by

soaking in dilute nitric acid for 24h and rinsed with distilled water, followed by

deionised water prior to use.

The calibration curves for the analyte ions were drawn after setting the various

parameters of flame atomic absorption spectrometer including the wavelength, slit

width, lamp current, flame type, fuel gas flow rate, support gas flow rate and the

burner height at optimum levels. These instrumental parameters were tabulated.


58

Linearity

The standard solutions of the analytes, namely sodium, potassium, magnesium,

manganese, calcium, copper, zinc, silicon, lead, arsenic, mercury, cadmium, nickel,

chromium, iron, palladium and aluminium. The solutions were diluted to 1000 ppm

solutions and stored in light resistant containers. The standard solutions for

calibration curve were prepared by diluting the stock solution with deionised

water.

Limit of detection and Limit of quantification

The limit of detection and the limit of quantitation of the developed method were

determined by using the formula LOD = 3Sbl/slope and LOQ = 10Sbl/slope where

Sbl is the standard deviation of blank measurements and slope of the calibration

curve.

Accuracy

The accuracy of the method was determined by recovery experiments. The recovery

of the optimised extraction procedures for the estimation of major trace minerals

and heavy metals in the plant sample were determined at single level by adding a

known quantity of the standard mineral and metal to the plant sample of pre

analysed sample and the mixtures were analysed.

Preparation of samples

Dry ashing technique was used for the digestion of the plant materials in order to

remove the organic matters (Barbara, 2003). The experiment was performed in

triplicate.

The shade dried plant material (5g) was ground into coarse powder. The ground

powder (1g) was accurately weighed and placed in clean and dry quartz beaker.

The quartz beaker was placed in a muffle furnace and the temperature was raised at

a rate of 50ºC/h until it reached 500ºC. The samples were heated at 500ºC for 24h to

conduct ashing. After the ashing was complete the samples were cooled to room


59

temperature in a dessicator. After cooling 2 drops of con HNO3 were added to the

ashes for dissolving. The samples were then diluted with deionised water, filtered

using Whatmann filter paper No.42 impregnated with HNO3 and the volume of the

clear solution was made to 100mL with deionised water. The blanks were also

prepared. The above solutions were appropriately diluted prior to analysis for the

estimation of minerals and heavy metals. The recovery of the minerals and heavy

metals were carried out, percentage recovery was established and tabulated.

4.6 Amino acid analysis

Sample preparation

The dried plant sample was analyzed in duplicate for amino acids. The sample

(5mg) was weighed and placed in a 2mL ampoule, to which 0.45mL of 6N HCl were

added. The ampoules were evacuated, sealed and kept for 24h digestion.

Preparation of reagents

Mobile Phase A [pH : 3.2 (The pH was adjusted with perchloric acid)]

Sodium Citrate : 19.6g (the crystals were dissolved completely in

500mL water)

Ethanol : 70mL

Perchloric acid : 16.6mL

The total volume was made up to 1L

Mobile Phase B [pH : 10 (The pH was adjusted with 4M Sodium hydroxide)]

Sodium Citrate : 39.2g (dissolved completely)

Boric acid : 8.26g (dissolved completely)

4N Sodium hydroxide :20mL

The total volume was made upto to 666mL.


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Mobile Phase C

Sodium hydroxide : 1.3g

The total volume was made upto 166mL.

The prepared solution A, B and C were filtered through 0.45µm membrane filter, to

avoid contamination with ammonia.

Preparation of Reaction Reagent

Carbonic Acid Buffer pH: 10

Sodium carbonate : 40.7g

Boric Acid : 13.56g

Potassium Sulphate : 18.18g

The volume was made upto 1L with water

Reagent A: O-Phthaladehyde (OPA)

O-Phthaladehyde : 160mg dissolved in 2.8mL of ethanol.

50mL of carbonic acid buffer was added

Mercaptoethanol : 0.4mL

Brij : 0.8mL (5g of Brij was dissolved in 25mL of water

and the bottle immersed in warm water. After cooling, the volume was made

up to 50mL)

Finally the volume was made up to 200mL with carbonic acid buffer

Reagent B: Sodium Hypochlorite

80µL of sodium hypochlorite in 200mL of carbonic acid buffer.


61

Sample Diluent pH 2.2 (The pH was adjusted with perchloric acid)

Trisodium citrate : 17.4g

Caprylic acid : 0.1mL

Perchloric acid : 11mL

The volume was made up to 1L

Procedure

The standard and sample solutions were analyzed by reverse phase high pressure

liquid chromatography (RP-HPLC) method using fluorescence detector for the

quantification of amino acids. A shimadzu LC20AT system equipped with a

quarternary pump and a fluorescence detector was used. A gradient elution for 70

min was performed using a stationary phase of Hibar C18 column (250 x 4.6mm i.d.,

5µ) and a mobile phase A consisting of sodium citrate buffer (pH adjusted with

perchloric acid) and ethanol (30:70) : mobile phase B consisting of sodium citrate

buffer (pH 10 adjusted with 4M sodium hydroxide : mobile phase C consisting of

2M sodium hydroxide). The mobile phase was pumped at a flow rate of

0.5mL/min.

Precolumn derivatisation of the sample and standard solutions were performed

using o-phthalaldehyde reagent. The retention times of the various amino acids

were determined using an amino acid calibration mixture. Quantitation of

individual amino acids was achieved by monitoring the absorption of the column

eluate at 348 and 450nm and comparing the areas under the individual peaks with

those of the corresponding amino acid standards. The essential acids were then

calculated using the formula,

Essential amino acid score= g of essential amino acid in 100g of test protein/g of

essential amino acid in 100g of FAO/WHO (1991) reference pattern x 100 (Vadivel

and Janarthanan, 2001).


62

4.7 Molecular authentication

Genomic DNA extraction

Principle

In prokaryotes, the DNA is double stranded and circular and is found throughout

the cytoplasm. The cell membranes must be disrupted in order to release the DNA

in the extraction buffer. Nucleic acids are generally precipitated using ethanol,

isopropanol or PEG. Ethanol precipitation is simple, rapid and quantitative. It

precipitates even nanogram quantities of DNA and RNA.

Cetyltrimethylammonium bromide (CTAB) is a cationic detergent. The positively

charged detergent has a high affinity for the negatively charged phosphate

backbone on nucleic acid molecules and thus binds to DNA strongly and forms an

insoluble complex. CTAB solubilizes membranes, lysis the cells and precipitates cell

wall polysaccharides in presence of high salt concentration and at high

temperature. It precipitates DNA at low salt concentration and at low temperature.

Sodium dodecyl sulphate (SDS) is used to disrupt the cell membrane. DNA can be

protected from endogenous nucleases by chelating with Mg2++ ions using EDTA.

Mg2++ ions are considered as a necessary cofactor for most nucleases. Proteinase

enzyme is used to degrade the proteins in the disrupted cell soup. Phenol–

chloroform is used to denature and separate the protein from the DNA. Chloroform

is also a protein denaturant, which stabilizes the rather unstable boundary between

an aqueous phase and a pure phenol layer at the interface between the aqueous and

organic phases which are removed by centrifugation. DNA released from the

disrupted cells is precipitated by cold absolute ethanol or isopropanol.


63

Preparation of the reagent

Cetyltrimethylammonium bromide (CTAB) solution

CTAB - 2g

0.1M Tris HCl (pH 8.0) – 10mL

1.4M NaCl – 8.18g

0.5M EDTA (pH 8.0) – 4mL

PVP – 1g

Mercaptoethanol – 1mL

Sodium sulphite – 1g

Procedure

The plant was ground into a fine powder using liquid nitrogen and incubated in

2mL of CTAB extraction buffer (0.1M Tris–HCl, pH 7.5, 0.7M NaCl, 10mM EDTA,

1% CTAB) at 65°C for 10 min. The mixture was treated with 750µL of chloroform :

isoamyl alcohol (24:1) and the solution was gently rotated for 10 min at room

temperature and centrifuged at 10,000 rpm for 10 min. The upper aqueous layer

(300µL) was mixed with 0.5 volume of 5M sodium chloride and 2 volumes of ice

cold ethanol, incubated at -20°C for 1h and then centrifuged at 13,000 rpm at 4°C for

10 min. The precipitated DNA was then washed with 70% ethanol and resuspended

in 300µL of TE buffer (10mM Tris HCl, pH 8.0 and 1mM EDTA). RNA was digested

with RNase (1mg/mL) at 37°C for 1h. The chloroform extraction was repeated one

more time. The DNA was precipitated by adding 100µL of 5M sodium acetate (pH

5.2) and 725µL of ice cold 100% ethanol. The pellet was washed with 500µL of ice

cold 70% ethanol, air dried and dissolved in 50µL of Tris- EDTA (TE) buffer

depending on the pellet size. The DNA concentration was determined by 1%

agarose gel.


64

Polymerase Chain Reaction (PCR)

Polymerase chain reaction is a process where the two synthetic oligonucleotide

primers, which are complementary to two regions of the target DNA, are added in

the presence of excess deoxynucleotides and Taq polymerase.

Principle

The polymerase chain reaction (PCR) is an in vitro DNA amplification of target

DNA with a pair of primers and a DNA polymerase, resulting in several million

fold amplification of the target sequence within a few hours. In this way, the sample

(target DNA) is allowed to react with a pair of primers (specific for each microbe),

deoxynucleotide triphosphates, buffer and Taq DNA polymerase (heat stable DNA

polymerase). Of the primers that are complementary to the target DNA, a new

DNA strand is synthesized. During each cycle, the DNA strand is doubled. Each

cycle consists of three segments, namely denaturation step (during which the two

strands of target DNA is separated), annealing step (during which the primers

attaches to the complementary target sequences) and synthesis or extension step

(during which a new strand is synthesized with a help of dNTPs and enzyme).

Procedure

Amplification of DNA was performed in a total volume of 20µL. The reaction

mixture contained 10µL of 10X reaction buffer [500mM KCl, 15mM MgCl2, 100mM

tris HCl (pH 8.3), 0.1% w/v gelatin] Template DNA–DNA sample was added at a

concentration of 50ng/µL. Each of the forward (ITS1 5'-

TCCGTAGGTGAACCTGCGGAAGGATCATTG-3') and reverse primer (ITS4 5'-

TCCTCCGCTTATTGATATGC-3') (20pmol) was then added. dNTP mix (3µL of

5mM) was then added. The four deoxyribonucleotide triphoshate (dATP, dTTP,

dGTP, dCTP) were used at a concentration of 100mM each. 1µL of Taq polymerase

(0.5U/µL) was added to each sample. The remaining volume was made up by

sterile water. Amplification was carried out in an Eppendorf Master Cycler. Initial

denaturation was carried out at 94ºC for 4 min, followed by 35 cycles, each cycle


65

consisting of denaturation of DNA for 1 min at 94ºC, annealing of the primers for 1

min at 52ºC, elongation at 72ºC for 2 min and final elongation for 10 min. The PCR

products were analysed by gel electrophoresis in a 1.2% agarose in Tris – acetate

buffer (pH 8.3) – EDTA (TAE) buffer (0.04M Tris – acetate, 0.001M EDTA, pH 8.0).

A 1000 base pair (1Kbp) ladder was used as a size standard. To visualize DNA, gels

were stained with ethidium bromide (0.1mg/L) and then photographed under

transmitted ultraviolet light using an Alpha Imager 2000 (Alpha Innotech, San

Leandro, CA, USA). For primer and plant isolate combination, the amplification

reactions were performed three times to determine the reliability and

reproducibility of the method.

DNA cloning

Competent cell preparation

Principle

E.coli cells are more likely to incorporate foreign DNA if their cell walls are altered

so that DNA can pass through more easily. Such cells are said to be competent.

Cells that are undergoing very rapid growth are made competent more easily than

cells in other stages of growth. The cells can be made competent artificially by

treating the cells with calcium chloride prior to adding DNA. The calcium

destabilizes the cell membrane and adheres to the cell surface favouring the

formation of the pores for the entry of DNA. The DNA is taken during the heat

shock step when the cells are exposed briefly at the temperature of 42ºC. Immediate

chilling on ice ensures closure of pores.

Selection of cells containing transformed DNA is enhanced by selection markers

carried by DNA. PUC series and PBR322 plasmids have ampicillin resistance factor

which enables only the transformed cells to grow on Luria Bertani – ampicillin

plate.


66

The PUC plasmid also has the genes for β – galactosidase enzyme. The lacZ is a

gene that has a series of unique restrictions site that has a series of unique

restrictions site engineered into it such that the plasmid be cut within lacZ gene. If

the plasmids are the host or the plasmid encoded fragments are themselves active,

they can associate to form an enzymatically active protein. This type of

complementation is known as α – complementation.

LacZ positive bacteria, result from α–complementation can produce active β–

galactosidase enzyme which hydrolyse X–gal (5 – bromo, 4–chloro, 3–indolyl β–D–

galactoside) into a blue coloured compound and thereby appears as a blue coloured

colony in presence of X–gal and IPTG. Any plasmid which has been inserted with

DNA fragment in LacZ gene will not have a functional LacZ gene and thus will

produce white colonies which are unable to cleave X–gal.

Reagent Preparation

i. Calcium chloride (100mM)

Calcium chloride (1.47g) was dissolved in 100mL of distilled water, sterilized

and stored at 4˚C.

ii. Isopropyl thiogalactoside (IPTG) stock

IPTG (20mg) was dissolved in 1mL of distilled water.

iii. X–gal stock

X–gal (20mg) was dissolved in 1mL dimethyl formamide and stored at –

20˚C.

iv. IPTG / X – gal

IPTG stock (800µL) was added to 3.8mL of sterile water and swirled well to

mix. X–gal (400µL) stock was added and mixed till the solution was

colourless. 250µL per plate was used and the solution was stored at – 20˚C.


67

Procedure

Competent preparation

The isolated colonies of E.coli was plated and inoculated into the LB broth and

incubated for overnight at 37ºC. From the overnight culture, a fresh culture was

prepared. The broth (2mL) was taken in the eppendorf tube and centrifuged at 6000

rpm for 10 min at 4ºC. The supernatant was discarded and the pellet was

suspended in 1mL of 0.1M calcium chloride. The tubes were kept in ice for 20 min.

It was then centrifuged at 6000 rpm for 10 min at 4ºC. The supernatant was again

discarded and the pellet was resuspended gently in 100µL of 0.1M calcium chloride

and 16µL of 40% glycerol. The competent cell prepared was stored at 4ºC.

Transformation

The competent cell (100µL) was taken and 3µL of plasmid pGEM-T easy vector was

added and mixed gently. The tubes were kept in ice for 30 min. It was then

subjected to heat shock at 42ºC for 1 min by keeping in the water bath. This was

kept in ice for 5 min and 600µL of LB broth was added and incubated at 37ºC for 1h.

LB ampicillin plate along with IPTG and X – gal was prepared with the antibiotic

concentration of 100µg/ml and IPTG X–gal concentration of 200µg/mL. The

inoculums was plated and incubated at 37ºC for overnight and the results were

observed.

Isolation of Plasmid DNA

Plasmids are extrachromosomal, self replicating double stranded, circular DNA

molecule present in most prokaryotes. Plasmids are responsible for antibiotic

resistant gene transfer between individual cells. The plasmids can be transferred

from one cell to another and therefore function as vectors or carriers in genetic

engineering techniques.


68

Principle

Glucose provides an iso osmotic condition to prevent physical shock. The

resuspended solution‟s pH is raised to basic level with Tris to help denature DNA.

EDTA is added to protect DNA from endogenous nucleases. It is a chelating agent

that binds Mg2++ ions, which is considered a necessary cofactor for most nucleases.

The sodium dodecyl sulfate (SDS) is an ionic detergent, which dissolves the

phospholipids and protein components of cell membrane. Sodium hydroxide in the

solution denatures the plasmid and the genomic DNA into single strands.

Potassium acetate forms an insoluble precipitate of SDS / lipid / protein complex.

At this pH, the circular DNA renatures. The chromosomal DNA is trapped in the

SDS / lipid / protein precipitate. The plasmid DNA renatures into its double

stranded form and escape being trapped in the precipitate and remains in the

supernatant. Phenol chloroform is used to denature and separate the proteins from

DNA. Phenol efficiently denatures proteins and probably dissolves the denaturized

protein. Chloroform, also a protein denaturant, stabilizes the rather unstable

boundary between an aqueous phase and a pure phenol layer. The use of this

mixture also reduces the amount of aqueous solution remained in the organic phase

in order to maximize the yield. The enzyme RNAse will completely degrade the

contaminating RNA available in the DNA solution.

Preparation of reagents and medium

i. Luria Bertani broth

Tryptone - 10mg /L

Yeast extract - 5g /L

NaCl - 10g /L

Ampicillin - 10µg /mL

(pH adjusted to 7.5 adding NaOH dropwise)


69

ii. Solution I

50mM glucose

25mM Tris HCl (pH 8.0)

10mM EDTA (pH 8.0)

iii. Solution II

0.2N NaOH (freshly diluted from a 10N stock)

1% SDS

iv. Solution III

5M potassium acetate - 60mL

Glacial acetic acid - 11.5mL

H2O - 28.5mL

(The resulting solution is 3M with respect to potassium and 5M with

respect to acetate)

v. Phenol chloroform mixture

Equal volumes of phenol and chloroform were mixed. The mixture

was kept on ice and added 20mL of TE buffer. The mixture was mixed

for 15 min to remove the dust on the surface layer using a pipette.

This was repeated 5 times. TE buffer (30-40mL) was added and it was

stored on ice.

vi. 95% ethanol

vii. 70% ethanol

viii. TE buffer containing DNase free RNase

10mM Tris HCl (pH 8.0)

1mM EDTA (pH 8.0)

RNase

Steps

Growing the bacteria and amplifying the plasmid – preparation of

preculture


70

Harvesting and lysing the bacteria

Purifying the plasmid away from the bacterial host

Procedure

The bacterial culture was streaked on LB agar containing ampicillin (100µg/mL)

and incubated at 37ºC overnight. A single colony was picked using autoclaved

tooth pick and the inoculums were transferred into 5mL of LB broth containing

100µg/mL ampicillin and incubated at 37˚C with shaking for 8–12h (150 rpm). The

overnight culture (1mL) was transferred into 1.5mL eppendorf tube and centrifuged

for 5 min at 10000 rpm. The supernatant was discarded. Solution I (100µL) was

added to the cell pellet and vortexed in a vortex mixture so that the cells dissolved

and dispersed uniformly. It was incubated at room temperature for 5 min. Solution

II (200µL) was added and gently mixed by inverting the tube 5 times and incubated

at ice for 10 min. Ice cold solution III (150µL) was then added and gently mixed by

inverting the tube 5 times and incubated at ice for 10 min.

The tube containing the lysate was centrifuged at 10000 rpm for 10 min at 4ºC. The

supernatant was transferred into a fresh tube without disturbing the pellets. Phenol

chloroform mixture (400µL) was added and gently mixed by inverting the tube 5

times and incubated at room temperature for 5 min. The tube was centrifuged at

10000 rpm for 10 min. The supernatant was transferred into a fresh tube without

disturbing the precipitate. Absolute ethanol (1mL) was added and mixed by

inverting the tube 5 times and incubated at 20ºC for 1h for DNA to precipitate. It

was centrifuged again at 10000 rpm for 5 min and the supernatant were discarded.

The precipitate obtained was washed with 1mL of 70% ethanol. The alcohol was

completely drained off. When the pellet turned transparent, 20µL of 1X TE buffer

containing RNase enzyme was added and incubated for 1h. The presence of

plasmid DNA was checked on agarose gel electrophoresis.


71

Agarose Gel Electrophoresis

Agarose gel electrophoresis is employed to quickly determine the yield and purity

of DNA isolated or check the product of PCR reaction, check progression of a

restriction enzyme digestion and to size fractionate DNA molecules which then

could be eluted from the gel. Agarose is a natural product purified from red

seaweed (Rhodophta). It is a polysaccharide of alternate 1,4 linked α-D-

galactopyranose and 1,4 linked 3,6-anhydro-α-L-galactopyranose residue and

arranged into double helix. It dissolves in water on boiling and forms a gel when

cooled to about 40ºC.

Agarose gel is the first choice for nucleic acid analysis. The gel is easily prepared,

nontoxic, optically clear (preferred for densitometric scanning and photography),

chemically inert having pores for a wide range of molecules to pass through,

available with least or minimum electroendosmosis (EEO) possessing good gel

strength.

Agarose gels have large pore size and can be used to separate macromolecules like

nucleic acids. Agarose gels are hydrocolloids, held together by hydrogen and

hydrophobic bonds. They are somewhat brittle and they break when bent. Hence,

agarose gels should be handled carefully with some form of support for the entire

gel, such as gel tray or wide spatula. The pore size and sieving characteristics of

agarose gel to a certain extent is determined by its concentration. The higher the

concentration the smaller is the pore size. Agarose gel is generally used in the

concentration range of 0.4- 4% (w/v).

Principle

The separation of DNA on the gel is carried out under an electric field applied to

the gel matrix. DNA molecules migrate towards the anode due to the negatively

charged phosphates along the backbone of the DNA. Fragments of linear DNA

migrate through agarose gels with the mobility that is inversely proportional to the


72

log10 of their molecular weight. Thus the larger molecules travel at a slower speed

than the smaller ones.

Circular forms of DNA migrate in agarose distinctly from linear DNAs of the same

mass. Typically uncut plasmids will appear to migrate more rapidly than the same

plasmid when linearised. Additionally, most preparations of uncut plasmid contain

at least two topologically different forms of DNA, corresponding to supercoiled

forms and nicked circles. Several parameters like agarose concentration, voltage

applied and molecular size of DNA affect the migration of DNA.

Reagent preparation

i) Tris acetate (TAE) buffer (50X) was stocked.

Tris base (1.6M) – 242g

Glacial acetic acid – 57.1mL

0.5M EDTA (pH 8.0) – 100mL

ii) 1X TAE buffer

1mL of stock TAE buffer was diluted to 50mL with autoclaved distilled

water.

iii) Gel loading dye (1X)

Bromophenol blue – 0.25g

Xylene cyanol – 0.25g

Sucrose – 40g

The above were dissolved in 100mL of distilled water and stored at -20˚C

iv) Ethidium bromide stock

10mg of ethidium bromide was dissolved in 1mL of distilled water.


73

Procedure

Agarose gel electrophoresis was performed based on the method described by

Sambrook et al. (1989) to check the quality of DNA and also to separate the products

amplified through polymerase chain reaction. 1X TAE tank buffer in 500mL

quantity was prepared to fill the electrophoresis tank and for gel preparation. In a

separate conical flask, agarose (0.8 per cent for genomic DNA and 1.5% for PCR

product) was added to 1X TAE buffer, boiled till the agarose dissolved completely

and cooled to lukewarm temperature. Ethidium bromide was added at the rate of

5L/100mL to agarose solution and was allowed to mix completely. It was then

poured into the gel mould, the comb was placed properly and allowed to solidify

for half an hour at room temperature.

After solidification, the comb was removed carefully. The casted gel was placed in

the electrophoresis tank containing 1X TAE buffer with the well near the cathode

and submerged to a depth of 1cm. Fifteen microlitre of the PCR product was mixed with

3L of 6X tracking dye and mixed well by pipetting in and out 3 times. The mixture was

loaded into the wells with the help of the micropipette. Two microlitre of 1 kbp DNA

ladder was loaded in one of the wells as a standard marker. The cathode and anode

were connected to power pack using power cord and the gel was run at a constant

voltage of 60 volts. The negatively charged DNA molecules moved towards the

anode and got separated according to their molecular weight. The power was

turned off when the marker reached the anode end and the gel was viewed in an

UV transilluminator and the banding pattern was analyzed. The sizes of the PCR

products were determined by comparison with standard 1 kbp molecular marker

(Genei, Pvt. Ltd., Bangalore, India).

Gel documentation

After the separation of the PCR products with 1.0% agarose gel, it was viewed and

photographed using Alpha imager TM1200 documentation and analysis system. The

PCR products were resolved on 2% agarose at 50V stained with ethidium bromide


74

(0.5µg/mL), photographed and analyzed using gel documentation system (Alpha

Innotech Corporation, San Leandro, California).

Sequencing of ITS region

ITS region was amplified with respective ITS primers. Amplified 16S rDNA was

purified from each reaction mixture by agarose (1.2%, w/v) gel electrophoresis in

TAE buffer containing 0.5µg of ethidium bromide per mL. Colony PCR was

performed for the cloned DNA. The plasmid was isolated by conventional method.

The isolated plasmid was then sequenced.

ITS sequence analysis

To obtain the genomic sequence of the plant, vector sequence was removed using

the software Vecscreen. Vecscreen is a system for quickly identifying segments of a

nucleic sequence that may be of vector origin. This software helps researchers to

identify and remove segments of vector origin before sequence analysis or

submission. This helps us to deduce the vector contamination from the cloning

history of the sequenced DNA.

Sequence submission

The rDNA homology searches were performed using the BLAST program (Altschul et

al., 1990) through the internet server at the National Center for Biotechnology

Information (National Institutes of Health, Bethesda, USA). Sequences were compared

with the GenBank database. Sequin software was used to prepare the sequence format

for submission. The newly obtained sequences were submitted to GenBank database,

New York, USA.

4.8 Protein Extraction

The plant was homogenized with a mortar and a pestle in liquid nitrogen. The

frozen plant was placed in 500mL Scott Duran bottle and about (1:4 ratio) 0.1M

potassium phosphate buffer (pH 7.0) was added and vortexed. The homogenates

were mixed thoroughly and incubated at 4°C overnight. The homogenates were


75

taken in a separate centrifuge tube and centrifuged at 7500 g for 15 min in a

refrigerated centrifuge at 4°C and the supernatant solutions were collected.

Electrophoresis

Polyacrylamide Gel (PAG) is a synthetic gel. This can be prepared from pure

acrylamide monomers and cross linkers and hence free from any contaminant. PAG

is thermo stable, transparent, strong and relatively inert chemically. PAG can be

prepared with a wide range of average pore sizes as well as gradient gels. PAG is

non-ionic and hence exhibits least Electroendosmosis. PAG can withstand high

voltage gradients, buffers with extreme pH, detergents and other chemicals and

reagents used in electrophoresis. PAG is feasible to various staining and destaining

procedures and can be digested to extract separated fractions or dried for

autoradiography and permanent recording.

Acrylamide is a white crystalline powder that forms the major ingredient in

polyacrylamide gel. As it dissolves in water “autopolymerization” takes place. It is

a slow spontaneous process by which acrylamide molecules join together by head

on tail. But in the presence of a free radical generating system, acyrlamide

monomers are activated in to a “free radical” state. These activated monomers

polymerize quickly and form long chain polymers. A solution of these polymer

chains becomes viscous but does not form gel because, these polymers slide over

one another. Gel formation requires hooking various chains together. This is done

by polymerizing acrylamide in the presence of a cross linker. N, N‟-

methylenebisacrylamide, commonly called as „bis‟ is the most frequently used cross

linking agent for polyacrylamide gels. Acrylamide and bis when activated by free

radicals form a mesh like gel.

Two proteins of different sizes, but identical charge densities, could be separated by

Polyacrylamide gel electrophoresis (PAGE) since the molecular sieving effect would

slow down the migration rate of the larger protein relative to that of the smaller

protein.


76

PAGE is carried out as Native PAGE and Sodium dodecyl sulphate (SDS) PAGE. In

native PAGE, separation takes place according to both size and charge difference of

molecules. In SDS–PAGE, proteins are denatured by heating in the presence of

excess SDS and a thiol reagent (usually 2-mercaptoethanol). During this treatment

most polypeptides bind SDS in a constant weight ratio such that they have

essentially identical charge densities and migrate in polyacrylamide gels of the

correct porosity according to polypeptide size.

Principle

Acrylamide gels are formed by polymerizing acrylamide with a ‘bis’ in the

presence of a catalyst TEMED. Polymerization is accelerated by addition of

ammonium per sulphate with free radical source. The rate at which the gels

polymerise can be controlled by varying the concentrations of TEMED and

ammonium persulphate. The relative proportions of acrylamide monomer and ‘bis’

can control the porosity of the gel.

Sodium dodecyl sulphate is the common dissociating agent used to denature native

proteins to individual polypeptides. When a protein mixture is heated to 100˚C in

presence of excess SDS, the detergent wraps around the polypeptide backbone. It

binds to polypeptides in a constant ratio of 1.4g/g of polypeptide. SDS binding also

imparts a large negative charge and shadows any other charge previously present

on the polypeptide. Thus, polypeptides after treatment become a rod like structure

possessing a uniform charge density, namely same net negative charge per unit.

The mobilities of the proteins are a linear function of the logarithms of their

molecular weights.

Preparation of solutions

i) Acrylamide- bisacrylamide solution

Acrylamide - 30g

Bisacrylamide - 0.8g


77

Both these were dissolved in 80mL distilled water and made upto 100mL,

filtered and stored at 4ºC in a brown bottle.

ii) 1.5M Tris-HCl, pH 8.8

Tris base -18.15g

It was dissolved in 80mL distilled water, pH adjusted to 8.8 with 1N HCl and

made up to 100mL and stored at 4ºC.

iii) 1.5M Tris-HCl, pH 6.8

Tris base -6.05g

It was dissolved in 60mL distilled water, pH adjusted to 6.8 with 1N HCl and

made up to 100mL and stored at 4ºC.

iv) 1N HCl solution

10mL concentrated HCl was mixed with 120mL distilled water.

v) 10% Ammonium persulphate (APS)

APS (100mg) was dissolved in 1mL distilled water (freshly prepared).

vi) 10% Sodium dodecyl sulphate (SDS)

SDS (1g) was dissolved in little distilled water, and made up to 10mL with

distilled water and stored at room temperature.

vii) 0.5% Bromophenol blue - 0.05g of bromophenol blue in 10mL distilled water

viii) 5X Sample Buffer

Distilled water – 2.6mL

0.5M Tris Hcl pH 6.8 – 1mL

Mercaptoethanol – 0.8mL

Glycerol – 1.6mL

10% SDS – 1.6mL

0.5% bromophenol blue – 0.4mL


78

Total volume – 8mL and it was stored at room temperature.

ix) Electrode Buffer

Glycine - 4.32g

Tris base - 0.9g

SDS - 0.3g

Distilled water - 300mL

x) Staining solution

Coomassie brilliant blue R-250 - 0.1g

Methanol - 40mL

Acetic acid - 10mL


xi) Destaining solution

Methanol - 40mL

Acetic acid - 10mL


xii) Separating gel composition

Acrylamide - 3mL

Distilled water - 2.5mL

Tris Hcl pH 8.8 - 1.875mL

10% SDS - 75µL

10% APS - 40µL

TEMED - 5µL

Total - 7.5mL

xiii) Stacking gel composition

Acrylamide - 0.65mL


79

Distilled water - 3.25mL

Tris Hcl pH 8.8 - 1.25mL

10% SDS - 50µL

10% APS - 25µL

TEMED - 5µL

Total - 5mL

Procedure

SDS-PAGE was performed following the method of Laemmli (1970) using a

separating gel of 12% and a stacking gel of 4% to separate the plant protein. The

samples containing equal amount of proteins were loaded into the wells of

polyacrylamide gels (Sigma-Aldrich Techware system; Sigma). Broad range, pre

stained molecular weight marker (Bio-Rad Laboratories, Hercules, CA) mixed with

sample buffer was also loaded in one of the wells. Electrophoresis was carried out

at constant voltage of 75 volts. Gels were stained with 0.2% Coomassie brilliant blue

R250 solution overnight and then destained. Based on the Rf value of each protein

band stained, the molecular weight was calculated.

Ammonium Sulphate Fractionation of Proteins

The solubility of proteins is markedly affected by the ionic strength of the medium.

As the ionic strength is increased, protein solubility at first increases. This is

referred to as salting in. However, beyond a certain point the solubility begins to

decrease and this is known as salting out.

At low ionic strengths the activity coefficients of the ionizable groups of the

proteins decreases so that their effective concentration decreases. This is because the

ionisable groups become surrounded by counter ions which prevent interaction

between the ionisable groups. Thus protein – protein interactions decreases and the

solubility increases.


80

At high ionic strengths much water becomes bound by the added ions and remains

to properly hydrate the proteins. As a result, protein–protein interactions exceed

protein–water interactions and the solubility decreases.

Because of the differences in structure and amino acid sequence, proteins differ in

their salting in and salting out behavior. This forms the basis for the fractional

precipitation of proteins by means of a salt.

Ammonium sulphate is a particularly useful salt for the fractional precipitation of

proteins. It is available in highly purified form, has great solubility allowing for

significant changes in the ionic strength and is inexpensive. Changes in the

ammonium sulphate concentration of a solution can be brought about either by

adding solid substance or by adding a solution of known saturation, generally, a

fully saturated (100%) solution. Final concentration of ammonium sulphate used in

the present study is given in the Table A .

During 20% of protein precipitation from the sample, the initial concentration of the

sample was 0%, to precipitate the proteins from the sample, 10.7g of ammonium

sulphate was added. During 40% of protein precipitation, the initial concentration

of the sample was 20% to precipitate the proteins from the sample 11.5g of

ammonium sulphate was added. For precipitating 60% of proteins from 40%

concentrate sample 12.2g of ammonium sulphate was added. Similarly for

precipitating 80% proteins from 60% and 100% proteins from 80%, 13.1g and 14.1g

of ammonium sulphate were added respectively. For precipitation of each

concentration, the ammonium sulphate was added in small amounts by placing the

sample beaker in a magnetic stirrer by constant stirring and the sample was left for

overnight stirring in cold condition.


81

Table A: Final concentration of ammonium sulphate, % saturation at 0˚C

20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

10.7

8.0

5.4

2.6

0

13.6

10.9

8.2

5.5

2.7

0

16.6

13.9

11.1

8.3

5.6

2.7

0

19.7

16.8

14.1

11.3

8.4

5.7

2.8

0

22.9

20.0

17.1

14.3

11.5

8.5

5.7

2.8

0

26.2

23.2

20.3

17.4

14.5

11.7

8.7

5.8

2.9

0

29.5

26.6

23.6

20.7

17.7

14.8

11.9

8.8

5.9

2.9

0

33.1

30.0

27.0

24.0

21.0

18.2

15.0

12.0

9.0

6.0

3.0

0

36.6

33.6

30.5

27.5

24.4

21.4

18.4

15.3

12.2

9.1

6.1

3.0

0

40.4

37.3

34.2

31.0

28.0

24.8

21.7

18.7

15.5

12.5

9.3

6.2

3.1

0

44.2

41.1

37.9

34.8

31.6

28.4

25.3

22.1

19.0

15.8

12.7

9.4

6.3

3.1

0

48.3

45.0

41.8

38.6

35.4

32.1

28.9

25.8

22.5

19.3

16.1

12.9

9.6

6.4

3.2

0

52.3

49.1

45.8

42.6

39.2

36.0

32.8

29.5

26.2

22.9

19.7

16.3

13.1

9.8

6.6

3.2

0

56.7

53.3

50.0

46.6

43.3

40.1

36.7

33.4

30.0

26.7

23.3

20.0

16.6

13.4

10.0

6.7

3.3

0

61.1

57.8

54.5

51.0

47.6

44.2

40.8

37.4

34.0

30.6

27.2

23.8

20.4

17.0

13.6

10.2

6.8

3.4

0

65.9

62.4

58.9

55.5

51.9

48.5

45.1

41.6

38.1

34.7

31.2

27.7

24.2

20.8

17.3

13.9

10.4

6.9

3.4

0

70.7

67.1

63.6

60.0

56.5

52.9

49.5

45.9

42.4

38.8

35.3

31.7

28.3

24.7

21.2

17.6

14.1

10.6

7.1

3.5

0


82

Dialysis

Dialysis is a process by which small molecules are selectively removed from a

sample containing mixture of both small and large molecules. Dialysis is effectively

accomplished using a special type of membrane known as semipermeable

membrane. The semipermeable membrane allows small molecules to pass freely

through, holding the large molecules inside. These membranes are essentially made

up of cellulose derivatives.

Pretreatment of dialysis membrane

For biological work, the membranes are pretreated to remove some undesirable

impurities such as glycerol, heavy metals, sulphides, etc., that are associated during

manufacturing process. To remove glycerol, heavy metals, sulphur and also to

inactivate any enzyme that may be present in the dialysis tube, the tube is cut into

pieces of about 5-10cm, placed inside a beaker containing 500mL of 2% sodium

bicarbonate, 10mM EDTA and boiled for 5 min. The membrane is boiled in distilled

water for 5 min., the procedure is repeated again with sodium bicarbonate solution

and then with water and stored in 20% methanol at 4ºC.

Dialyzing the protein sample

The protein sample was dialyzed in the membrane by tying one end with a thread

and the samples were filled in through the other end. The tubing was checked for

leakage. The dialysis bag was placed in the phosphate buffer solution and dialyzed

overnight on a magnetic stirrer. The temperature was maintained at 4ºC throughout

the process. The proteins were collected in a centrifuge tube and stored at 4ºC the

next day.

Estimation of proteins

The protein in a solution can be measured quantitatively by different methods. The

method described by Bradford uses the concept, namely the protein‟s capacity to


83

bind a dye, quantitatively. This method is simple, rapid and inexpensive (Jagadish

et al., 2010; Salekdeh et al., 2002).

Principle

The assay is based on the ability of proteins to bind coomassie brilliant blue G250

and form a complex whose extinction coefficient is much greater than that of the

free dye.

Reagent preparation

Dye concentrate

Coomassie brilliant blue G250 (100g) was dissolved in 50mL of 95% ethanol.

Concentrated ortho phosphoric acid (100mL) was added followed by

distilled water to a final volume of 200mL. The solution was stored in amber

bottles and refrigerated.

One volume of the concentrated dye solution was mixed with 4 volumes of

distilled water.

Procedure

A series of protein samples in the test tubes in the concentration 0.2, 0.4, 0.6, 0.8 and

1mL was prepared in phosphate saline buffer. The experimental samples were

prepared in 100µL of phosphate buffer saline. Diluted dye binding solution of 5mL

was added to each tube. The tubes were mixed well and left aside for 5 min for the

colour to develop but not more than 30 min. The red dye turned blue when it binds

with protein. The absorbance was read at 595nm. A standard curve was plotted

using the standard protein absorbance Vs concentration. The protein concentration

was calculated using the standard curve.


84

Protein purification

Ultrafiltration

This is a simple method of purification. Various molecular cut off units like 3kDa,

10kDa and 30kDa were used and the proteins above 30kDa molecular weight were

collected and stored at 4ºC.

Reverse phase high pressure liquid chromatography (RP-HPLC)

The protein sample of more than 30kDa were analysed using RP-HPLC for

separating the protein as fractions. A shimadzu LC20AT system equipped with a

quarternary pump and a fluorescence detector was used. An isocratic elution was

performed for 70 min using the stationary phase of Hibar C18 column (250x4.6mm

i.d., 5µ) and a mobile phase consisting of buffer A- 0.1% trifluoroacetic acid (TFA) in

water and buffer B-0.1% TFA in acetonitrile (ACN). Fractions were collected in

every 5 min. the 1st fraction was collected at 0-5 min, 2nd at 6-10 min, 3rd at 11-15

min, 4th at 16-20 min, 5th at 21-25 min, 6th at 26-30 min, 7th at 31-35 min, 8th at 36-40

min, 9th at 41-45 min, 10th at 46-50 min, 11th at 51-55 min, 12th at 56-60 min, 13th at 61-

65 min and 14th fraction at 65-70 min. All the 14 fraction collected were concentrated

in a concentrator and then screened for in vitro anticancer activity.

4.9 In vitro anticancer studies

In vitro short term toxicity studies

The short term toxicity studies were carried out against Dalton‟s Lymphoma

Ascities (DLA) cells using standard procedures (Unnikrishnan and Ramadasan

Kuttan., 1988). This test relies on a breakdown of membrane integrity determined

by the uptake of a dye such as (Tryphan blue, erythorisin and nigrosin) to which

the cell is normally impermeable.

Procedure

DLA cells were cultured in peritoneal cavity of mice by injecting intraperitoneally a

suspension of DLA cells (1.0 x 105 cells/mL). The peritoneal fluid containing cells


85

were withdrawn from the peritoneal cavity of the mice between 12- 15 days with

the help of a sterile syringe. The cells were washed with Hanks balanced salt

solution (HBSS) and centrifuged for 10-15 min at 1,200 rpm. The procedure was

repeated thrice. The cells were then suspended in known quantity of HBSS and the

cell count was adjusted to 2x106 cells/mL. The diluted cell suspension was

distributed into Eppendorf tubes (0.1 mL containing 2x106 cells). The cells were

exposed to the protein sample and incubated at 37ºC for 3h. After 3h, an equal

quantity of the protein treated cells and tryphan blue (0.4%) were mixed and left for

a min and loaded into a haemocytometer. The viable and non-viable count were

recorded within two min. Viable cells do not take up colour, whereas dead cells

take up colour. The percentage growth inhibition was calculated and CTC50 value

was generated from the dose-response curves for the cell line.

Total Cells – Dead Cells % Growth Inhibition = 100 – X 100

Total Cells

In vitro cytotoxicity studies

Drug development programmes involve pre–clinical screening of a vast number of

chemicals for their specific and non–specific cytotoxicity against many types of

cells. Use of in vitro assay systems for the screening of potential anticancer agents

has been a common practice almost since the beginning of cancer chemotherapy in

1946. The National Cancer Institute now routinely measures the growth inhibitory

properties of every compound under test against a panel of 60 human tumor cell

lines which are representative of major human tumor types. There are a number of

advantages in in vitro testing using cell cultures which include analysis of species

specificity, feasibility of using only small amounts of test substances, and facility to

do mechanistic studies. A novel anticancer drug should possess cytotoxicity at low

concentration against cancerous cell lines and should be safe against normal cell


86

lines even at higher concentrations (Masters, 2000). All the proteins isolated were

tested for cytotoxicity by MTT assay.

Chemicals and reagents

Chemicals such as sodium bicarbonate, sodium pyruate, L-glutamine, glucose,

HEPES, penicillin and streptomycin were purchased from Sigma, USA. Likewise,

media ingredients such as Dulbecco‟s MEM, Eagle‟s MEM Medium (Sigma, USA),

and Fetal Bovine serum were procured from GIBCO USA.

Preparation of test solutions

The protein was weighed, dissolved in distilled dimethyl sulphoxide (DMSO) and

the volume was made up to 10mL with MEM/DMEM, at pH 7.4 and supplemented

with 2% inactivated FBS/NBCS (maintenance medium) to obtain a stock solution of

1mg/mL concentration, sterilized by filtration and stored at -20˚C till use. Serial

two fold dilution of the extract was prepared from the stock solution to obtain

lower concentrations.

Cell culture maintenance

Cell lines used in this study such as HBL 100 (Normal Cell line), HeLa (Cervical

Cancer), A549 (Lung carcinoma), Hep2 (Larynx Carcinoma) and OAW42 (Ovary

cancer Cells) were purchased from the National Centre for Cell Sciences (NCCS),

Pune, India. The above cell lines were maintained in Dulbecco's Modified Eagles

medium supplemented with 2mM L-glutamine and Earle‟s BSS adjusted to contain

1.5g/L sodium bicarbonate, 0.1mM non-essential amino acids and 1.0mM of

sodium pyruvate. Penicillin and streptomycin (100 IU/100µg) were adjusted to

1mL /L. Cells were maintained at 370C with 5% CO2 atmosphere.

Cell viability

Cell viability was assessed by trypan blue dye exclusion test as reported by

Chakraborty et al., (2004). The numbers of stained and unstained cells were counted

using a haemocytometer (Improved Neubauer Brightline, USA).


87

Determination of mitochondrial synthesis by MTT assay

The ability of the cells to survive a toxic insult has been the basis of most

cytotoxicity assays. This assay is based on the assumption that dead cells or their

products do not reduce tetrazolium salt. The assay depends both on the number of

cells present and on the mitochondrial activity per cell. The cleavage of tetrazolium

salt, 3-(4, 5 dimethyl thiazole-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), to a

blue formazan derivative by living cells is clearly a very effective principle on

which the assay is based. The principle involved is the cleavage of MTT into a blue

coloured product (formazan) by mitochondrial enzyme succinate dehydrogenase.

The number of cells is known to be proportional to the extent of formazan

production by the cells used (Francis and Rita, 1986).

Procedure

The cell suspensions were dispensed (100µL) in triplicate into 96-well culture plates

at optimized concentrations of 1.5 x 105 cells/mL for all the cancer cells, after a 48h

recovery period, to form formazone crystal reaction. To each well of the 96 well

microtitre plate, 0.1mL of the diluted cell suspension (approximately 10,000 cells)

was added. After 24h, when a partial monolayer was formed, the supernatant was

flicked off, and the monolayer washed once with medium. Different protein

samples (100μL each) were added to the cells in microtitre plates. The plates were

then incubated at 37ºC for 3 days in 5% CO2 atmosphere, microscopic examination

was carried out and observations were noted every 24h. After 72h, the sample

solutions in the wells were discarded and 50µL of MTT in MEM – PR (Minimum

essential medium without phenol red) was added to each well. The plates were

gently shaken and incubated for 3h at 37ºC in 5% CO2 atmosphere. The supernatant

was removed, 50µL of propanol was added and the plates were gently shaken to

solubilize the formed formazan. The absorbance was measured using a ELISA

multiwell plate reader (Thermo Multiskan EX, USA) at the wavelength of 620 nm.

The percentage growth inhibition was calculated using the following formula and


88

IC50 (concentration of drug or test sample needed to inhibit cell growth by 50%)

values were generated from the dose-response curves for each cell line. The relative

viability of the treated cells as compared to the control cells was expressed as the %

cytoviability, using the following formula (Sukirtha et al., 2011);

OD value of experimental samples Percentage of viability = X 100

OD value of experimental control (untreated)

IC50 was then determined by the corresponding dose response curve.

4.9 Protein characterisation

Protein characterisation is a broad term which involves creating a profile or

fingerprint of the protein molecule‟s physical, chemical and biological properties.

Mass Spectrometry

The protein fraction was run on the SDS-PAGE gel. The protein spot was excised

from preparative gel (stained with Coomassie brilliant blue) (Salekdeh et al., 2002)

and the mass spectrometry and peptide mass finger printing and MS-MS of the

protein was done. The data from matrix-assisted laser desorption/ionization- time

of flight (MALDI-TOF) MS were searched against the data bases with MASCOT

(www.matrixscience.com) and Profound software

(http://bioinformatics.genomicsolutions.com) to identify the annotated functions

of the proteins sequenced.

Protein digestion (In gel trypsin digestion)

The excised stained gel piece (minced into 1mm3 pieces) was transferred into a

sterile microcentrifuge tube. The gel was washed with 500µL of wash solution (50%

acetonitrile, 50mM ammonium bicarbonate) and incubated at room temperature for

15 min with gentle agitation (vortex mixer on lowest setting). The solution was

removed with a pipette. The gel was washed several times with 500µL of wash


89

solution (15 min each) until the Coomassie dye was completely removed. The gel

was dehydrated in 100% acetonitrile for 5 min. When dehydrated, the gel pieces

had an opaque white color and were significantly smaller in size. Acetonitrile was

removed with a pipette and the gel was dried completely at room temperature for

10-20 min in a centrifugal evaporator. The gel piece was rehydrated in 150µL

reduction solution (10mM DTT, 100mM ammonium bicarbonate) for 30 min at 56ºC.

The reduction solution was discarded with a pipette and added 100µL alkylation

solution (50mM iodoacetamide, 100mM ammonium bicarbonate) and incubated for

30 min in dark at room temperature. Again the alkylation solution was discarded

with a pipette and added 500µL of wash solution and incubated at room

temperature for 15 min with gentle agitation. The wash solution was removed and

the gel was dehydrated using 100µL 100% acetonitrile for 5 min. Acetonitrile was

completely discarded and the gel was dried at room temperature in a centrifugal

evaporator. While the gel was drying protease digestion solution was prepared.

Typically, this is a modified sequencing grade trypsin (Product number V5111,

Promega, Madison, WI). The lyophilized trypsin (20µg/vial) was resuspended in a

solution containing 50mM ammonium bicarbonate (1mL), sample (50µL) and stored

at -70ºC. The gel was rehydrated with a minimal volume of protease digestion

solution. Small gel plugs (20µL) were used. The gel piece was hydrated throughout

the digest. The gel was digested overnight at 37ºC. The samples were centrifuged

and the supernatant was transferred into a sterile tube containing tryptic peptides.

The extracted solution 50µL (60% acetonitrile, 0.1% TFA) was then added to the gel

piece and sonicated in ultrasonic waterbath for 10 min. The pooled extracted

peptides were dried by centrifugal evaporation to near dryness. Resuspension

solution 5µL (50% acetonitrile, 0.1% TFA) was added to each tube and sonicated in

water bath and gently agitated on a vortex at the lowest setting. The samples were

spinned down and spotted 0.5µL on MALDI plate followed by 0.5µL of alpha-

cyano-4-hydroxycinnamic acid matrix (10mg/mL in 50% acetonitrile, 0.1%


90

TFA).The spots were dried completely and the plate was loaded into Voyager and

calibrated using internal tryptic peaks of 842.5 and 2211.1 Daltons (Da).

Protein identification

The protein identification was performed automatically by searching (NCBInr)

using MASCOT search engine (Matrix Science, UK). The following are the

paramaters used;

Type of search

Enzyme used

Fixed modification

Variable modification

Peptide and fragment mass tolerance

Mass values

Protein mass

4.10 In silico analysis

Target identification

A number of methods for the computational prediction of protein structure from its

sequence have been developed. The first requirement in the construction of the

protein structure model is the multiple sequence alignment between the templates

and the target sequence. The sequence alignment is based on identifying

structurally conserved regions (SCR) common to template and target sequences.

The multiple sequence alignment was performed using the software Discovery

Studio 3.5. Query and the template sequences were given as input.

Homology modeling

Homology modeling relies on the identification of one or more known protein

structures likely to resemble the structure of the query sequence, and on the

production of an alignment that maps residues in the query sequence to residues in

the template sequence. Homology modeling can produce high-quality structural


91

models when the target and template are closely related, which has inspired the

formation of a structural genomics consortium dedicated to the production of

representative experimental structures for all classes of protein folds. Homology

modeling, also known as comparative modeling of protein, refers to constructing an

atomic-resolution model of the target protein from its amino acid sequence and an

experimental three-dimensional structure of a related homologous protein. Using

the output of the multiple sequence alignment homology modeling was carried out.

The homology modeling of the protein identified by MASCOT search was done

using the software Discovery Studio 3.5

Model validation

Model validation was carried out using SAVS server. To verify the protein model,

the co-ordinates of the protein model were submitted to PROCHECK. The stereo

chemical quality of the protein structures was examined by Ramachandran plot.

The number of residues that are in the allowed and disallowed regions of

Ramachandran plot determines the quality of the model, which indicates the region

of possible angle formation.

Gene identification of the active protein

From the obtained protein sequence, the gene was identified by BLAST search in

NCBI website, derived by automated computational analysis using gene prediction

method: GNOMON. The whole genomic sequence for this reference sequence

record is from whole-genome assembly released by the US DOE Joint Genome

Institute (JGI-PGF) and the International Brachypodium Initiative as v1.0 in

December 2009 (http://www.brachypodium.org/).

http://www.brachypodium.org/

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