APPENDIX-1 ESTIMATION OF TOTAL CARBOHYDRATE The total

APPENDIX-1

ESTIMATION OF TOTAL CARBOHYDRATE

The total carbohydrate content was estimated by the method of Hedge and

Hofreiter, 1962.

Principle

Carbohydrate is first hydrolysed into simple sugars using dilute hydrochloric

acid. In hot acidic medium glucose is dehydrated to hydroxmethyl furfural. This

compound forms with anthrone a green coloured product with absorption maximum at

630 nm.

Reagents

1. Glucose stock standard: 100 mg of glucose was dissolved in 100 ml of water in

a standard flask.

2. Working standard: 10 ml of the stock was diluted to 100 ml. 1.0 ml of this

solution contains 100µg of glucose.

3. Anthrone reagent: 0.2% anthrone was dissolved in ice cold concentrated

sulphuric acid. Prepared fresh before use

4. 2.5 N HCl.

Procedure

weighed 100mg of the sample into a boiling tube, hydrolysed by keeping it in a

boiling water bath for three hours with 5.0 ml of 2.5 N HCl and cooled to room

temperature. Neutralized it with solid sodium carbonate until the effervescence ceas

made up the volume to 100 ml and centrifuged, collected the supernatant and take 0.2

to 1.0 ml for analysis. Prepared the standards by taking 0.2-1.0 ml of the working

standards. 1.0 ml of water serves as a blank made up the volume to 1.0 ml in all the

tubes with distilled water, then added 4.0 ml of anthrone reagent, heated for eight

minutes in a boiling water bath, cooled rapidly and read the green to dark green colour

at 630 nm.

Calculation

A standard graph was drawn by taking the concentration of glucose on X axis

and spectrophotometer reading on Y axis. From the graph the concentration of glucose

in the sample was calculated.

APPENDIX-2

ESTIMATION OF PROTEIN BY LOWRY’S METHOD

Principle:

The blue colour developed by the reduction of the phosphomolybdic-

phosphotungstic components in the Folin –ciocalteau reagent by the amino acids

tyrosine and tryptophan present in the protein plus the colour developed by the biuret

reaction of the protein with the alkaline cupric tartrate are measured in the Lowry’s

method.

Reagents:

i. Folin –ciocalteau reagent (reagent D)-reflux gently for 10 hours a mixture

consisting of 100g Sodium tungstate (Na2WoO4.2H2O), 25g Sodium

molybdate (Na2WoO4.2H2O), 700ml water, 50ml of 80% phosphoric acid, and

100ml of concentrated hydrochloric acid in a 1.5L flask. Add 150g lithium

sulfate, 50ml water and a few drops of bromine water. Boil the mixture for 15

min without condenser to remove excess bromine. Cool, dilute to 1L and filter.

The reagent should have no greenish 20% Sodium carbonate in 0.1N sodium

hydroxide (Reagent A).

ii. 0.5% Copper Sulphate (CuSO4.5H2O) IN 1% potassium sodium tartrate

(Reagent B).

iii. Alkaline copper solution.: Mix 50ml of A and 1ml of B prior to use (Reagent C)

iv. Protein Solution (Stock Standard):

Weigh accurately 50mg of bovine serum albumin (fraction V) and dissolve in

distilled water and make up to 50ml in a standard flask.

v. Working Standard Solution:

Dilute 10ml of the stock solution to 50ml with distilled water in a standard

flask. 1.0ml of this solution contains 200µg protein.

Procedure

Extraction of protein from Sample:

Extraction is usually carried out with buffers used for the enzyme assay. Weigh

500mg of the sample and grind well with a pestle and mortar in 5-10mL of the buffer.

Centrifuge and use the supernatant for protein estimation.

Estimation of Protein:

1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1.0ml of the working standard into a series

of test tubes.

2. Pipette out 0.1 ml and 0.2 ml of the sample extract in two other test tubes.

3. Make up the volume to 1.0 ml in all the test tubes. A tube with 1.0ml of

water serves as the blank.

4. Add 5.0 ml of reagent C to each tube including the blank. Mix well and

allowed to standing for 10mins.

5. Then add 0.5 ml of reagent D, Mix well and incubate at room temperature in

the dark for 30min, blue colour is developed.

Take the reading at 660nm.Draw a standard graph and calculate the amount of protein

in the sample.

APPENDIX-3

ESTIMATION OF AMINO ACIDS (Ninhydrin method)

Principle

Ninhydrin, a powerful oxidizing agent , decarboxylates the alpha-amino acids and yields

an intencsly coloured bluish purple product which is colormetrically measured at 570 nm.

Reagents

i. Dissolve 50mg leucine in 50ml of water in a volumetric flask. Take 10ml of this

stock standard aand dilute to 100ml in another volumetric flask for working

standard solution. A series of volumefrom 0.1-1 ml of this standard solution

gives a concentration range 10 µg-100µg. Proceed as that of the sample and

read the colour.

ii. Ninhydrin: Dissolve 0.8 stanous chloride in 500 ml of 0.2 M citrate buffer (pH

5.0). Add this solution to 20g of ninhydrin in 500ml of methylcellosolve (2

methoxyethanol)

iii. 0.2M Citrate buffer pH 0.5

iv. Diluent solvent: Mix equal volumes of water and n-propanol and use.

Procedure

1. To 0.1 ml of extract , add 1ml of ninhydrin solution

2. Make up the volume to 2ml with distilled water

3. Heat the tube in a boiling water bath for 20min.

4. Add 5ml of the diluents and mix the contents.

5. After 15min reat the intensity of the purple colour against a reagent blank in a

colorimenter at 570 nm. The colour is stable for 1h.

6. Prepare the reagent blank as above by taking 0.1ml of 80% ethanol instead of the

extract.

APPENDIX-4

ESTIMATION OF STEROIDS

The amount of steroid was determined by Zak’s method (Zak ,1954)

Principle

Steroids react with ferric chloride in the presence of concentrated sulphuric acid

to give a pink colour. The intensity of colour developed is directly proportional to the

amount of steroids present and it is read at 540 nm in a calorimeter.

Reagents

1. Stock ferric chloride

840 mg of pure dry ferric chloride was weighed and dissolved in 100 ml glacial

acetic acid.

2. Ferric chloride precipitating reagent

10 ml of stock ferric chloride reagent was taken in a 100 ml of standard flask

and made up to the mark with pure glacial acetic acid.

3. Ferric chloride diluting reagent

8.5 ml of stock ferric chloride was diluted to 100 ml with pure glacial acetic acid.

4. Concentrated Sulphuric acid.

5. Cholesterol Solution.

(i) Stock Standard – 100 mg of cholesterol was dissolved in 100 ml of glacial

acetic acid.

(ii) Working standard – 10 ml of stock was dissolved in 0.85 ml of stock ferric

chloride reagent and made up to 100 ml with glacial acetic acid. The concentration of

working standard is 100µg / ml.

Procedure

0.1 ml and 0.2 ml of triple acid extract is taken and a set of standards (0.5 to 2.5

ml) were taken and made up to 5 ml with ferric chloride diluting reagent. A blank was

prepared simultaneously by taking 5.0 ml diluting reagent. Then add 4.0 ml of

concentrated sulphuric acid to each tube. After 30 minutes incubation, intensity of the

colour developed was read at 540 nm.

APPENDIX-5

ESTIMATION OF TOTAL PHENOLS

The amount of total phenols in the plant tissues was estimated by the method

proposed by Mallick and Singh (1980).

Principle

Phenols react with phosphomolybdic acid in Folin-Ciocalteau reagent to

produce a blue-coloured complex in alkaline medium, which can be estimated

spectrophotometrically at 650nm

Reagents

1. Ethanol (80%)

2. Folin-Ciocalteau reagent (1N)

3. Sodium carbonate (20%)

4. Standard gallic acid solution (100μg/ml in water)

Procedure

The sample (0.5g) was homogenized in 10X volume of 80% ethanol. The

homogenate was centrifuged at 10,000rpm for 20 minutes. The extraction was repeated

with 80% ethanol. The supernatants were pooled and evaporated to dryness. The

residue was then dissolved in a known volume of distilled water. Different aliquots

were pipette out and the volume in each tube was made up to 3.0 ml with distilled

water. Folin- Ciocalteau reagent (0.5ml) was added and the tubes were placed in a

boiling water bath for exactly one minute. The tubes were cooled and the absorbance

was read at 650nm in a spectrophotometer against a reagent blank. Standard gallic acid

solutions (0.2-1ml) corresponding to 2.0-10μg concentrations were also treated as

above. The concentration of phenols is expressed as mg/g tissue.

APPENDIX-6

ESTIMATION OF TOTAL ALKALOIDS

Total alkaloids was measured by the method of Harborne, 1973

Procedure:

10mg of plant material was homogenized in a mortar and pestle, added around

20 ml of methanol : ammonia (68:2) decanted the ammonical solution after 24 hrs and

added fresh methanolic ammonia, repeated the procedure thrice and pooled the extracts,

evaporated the extracts using a flash evaporator, treated the residue with 1N HCl and

kept it overnight, extracted the acidic solution with 20 ml of CHCl3 thrice, pooled the

organic layers and evaporated to dryness, basic fraction basified the acidic layer with

conc. NaOH to pH-12 and extracted with CHCl3 (20 ml) thrice, pooled the CHCl3

layers, dry over absorbent cotton and evaporated to dryness, weighed the fraction that

contains ajmalicine and serpentine expressed as mg/100g.

APPENDIX-7

EXTRACTION AND ESTIMATION OF FLAVONOIDS

Flavonoid was extracted and estimated by the method of Cameron et al., 1993

Extraction:

A portion of the plant material was weighed out and extraction was carried out

in two steps, firstly with MeOH: H2O (1:1). at each step, sufficient solvent was added

to make liquid slurry and the mixture was left for 6-12 hrs, filtration to separate the

extract from the plant material was carried out rapidly by using a glass wool or cotton

wool plugged in the neck of a filter funnel. The two extracts were then combined and

evaporated to about one third the original volume or until most of the MeOH has been

removed, the resultant aqueous extract was cleared of low polarity contaminants such

as fats, terpenes, chlorophylls and xanthophylls by extraction (in a separating funnel)

with hexane or chloroform, this was repeated several times and the extracts obtained.

The solvent extracted aqueous layer containing the bulk of the flavonoids was then

concentrated.

Reagents

1. Vanillin reagent -1% vanillin in 70% conc.H2SO4

2. Catechin standard 110 µg/ml

Procedure

An aliquot of the extract was pipette into a test tube and evaporated to dryness.

Then added 4 ml of vanillin reagent and heated for 15 min in a boiling waterbath. A

standard was also treated in the same manner. Then the optical density was read at 340

or 360 nm.

APPENDIX-8

ESTIMATION OF GLYCOSIDES

Principle

Cardiac glycosides develop an orange red colour complex with Baljet’s reagent

(Picric acid in alkaline medium). The intensity (absorbance) of colour produced is

proportional to the concentration of glycosides.

Reagents

Standard digitoxin: 0.02% digitoxin is prepared in chloroform: methanol (1:1).

Baljet’s reagent: Freshly prepared 95ml 1% picric acid + 5ml 10% NaOH are mixed

immediately before use and filtered through a sintered glass funnel.

Procedure

1. 10ml of the extract and 10ml of Baljet’s reagent are taken and allowed to stand

for one hour. Then dilute the solution with 20ml distilled water and mix. Read

the intensity of the colour obtained against blank at 495nm using a

spectrophotometer. The difference between test and control is taken for

calculation.

2. Standard graph can be prepared using standard digitoxin.

Calculation

Concentration (%) = Absorbance×100

g %17

APPENDIX-9

ESTIMATION OF SAPONINS

20 g of crude taken from each plant were put into a conical flask and 100 cm3 of

20% aqueous ethanol were added. The samples were heated over a hot water bath for 4

h with continuous stirring at about 55 ºC. The mixture was filtered and the residue re-

extracted with another 200 ml of 20% ethanol. The combined extracts were reduced to

40 ml over water bath at about 90 ºC. The concentrate was transferred into 250 ml

separatory funnel and 20 ml of diethyl ether was added and shaken vigorously. The

aqueous layer was recovered while the ether layer was discarded. The purification

process was repeated.

60 ml of n-butanol was added. The combined n-butanol extracts were washed twice

with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a

water bath. After evaporation, the samples were dried in the oven to constant weight

and the saponin content was calculated.

APPENDIX-10

ESTIMATION OF TANNINS

Estimation of Tannins by Folins- Denis method (1970)

Principle

Tannins like compounds reduce posphotungsto molybdic acid in alkaline

solution to produce a blue colour complex and the colour intensity is proportional to the

concentration of Tannin and measured at 700nm.

Reagents

1. Folin-Denis reagent: Dissolve 100g of sodium tungstate and 20 g phosphomolybdic

acid in 750ml distilled water in suitable flask and add 50ml phosphoric acid. Reflux

with mixture for 2 hours and make up to one litre with distilled water, protect the

reagent from exposure to light.

2. Sodium carbonate solution: Dissolve 350g sodium carbonate in one litre of water at

70ºC-80ºC. Filter through glass wool after allowing it to stand overnight.

3. Tannic acid solution:

stock standard: Dissolve 100mg tannic acid in 100ml of distilled water.

Working standard: Dissolve 5ml of stock solution in 100ml with distilled water

(concentration 50µg/ml)

Procedure:

1. Extraction of Tannin: Weigh 0.5g of the powdered sample and transfer to 250ml

conical flask. Add 75ml of water. Heat the flask gently and boil for 30mins.

Centrifuge at 2000rpm for 20mins and collect the supernatant in 100ml

volumetric flask and make up the volume.

2. Transfer 1ml of the sample extract to 100ml volumetric flask containing 75ml

water.

3. Add 5ml of Folin-Denis reagent, 10ml of sodium carbonate solution and dilute

to 100ml with water.

4. Shake well. Read the absorbance at 700nm after 30mins.

5. Prepare a standard graph using 0-100µg tannic acid.

APPENDIX-11

ASSAY OF SUPEROXIDE DISMUTASE (SOD)

Superoxide dismutase activity was determined by the method of Kakkar et al.,

1984

Principle

Superoxide dismutase uses the photochemical reduction of riboflavin as oxygen

generating system and catalyses the inhibition of NBT reduction, the extent of which

can be assayed spectrophotometrically.

REAGENTS

1. Potassium phosphate buffer, (500 mM pH 7.8)

2. Methionine (450 M)

3. Riboflavin (53 mM)

4. Nitro Blue Tetrazolium (NBT) (840 M)

5. Potassium cyanide (200 M)

Procedure

Samples (0.5g) were ground with 3.0 ml of potassium phosphate buffer,

centrifuged at 2000 rpm for 10 minutes and the supernatant was used for the assay. The

incubation medium contained, a final volume of 3.0 ml, 50 mM potassium phosphate

buffer (pH 7.8), 45 M methionine, 5.3 mM riboflavin, 84 M NBT and 20 M

potassium cyanide. The amount of homogenate added to this medium was kept below

one unit of enzyme to ensure sufficient accuracy.

The tubes were placed in an aluminium foil-lined box maintained at 25°C and

equipped with 15W fluorescent lamps. Reduced NBT was measured

spectrophotometrically at 600 nm after exposure to light for 10 minutes. The maximum

reduction was evaluated in the absence of the enzyme. One unit of enzyme activity was

defined as the amount of enzyme giving a 50% inhibition of the reduction of NBT. The

values were calculated as units/mg protein.

APPENDIX-12

ASSAY OF CATALASE

Catalase activity was assayed spectrophotometrically following the method of

Luck (1974) in the fresh leaves of the plant.

Principle

The UV light absorption of hydrogen peroxide can be easily measured between

230 – 250 nm. On decomposition of hydrogen peroxide by catalase, the absorption

decreases with time. The enzyme activity can be arrived at from this decrease

Reagents

1. Phosphate buffer : 0.067 M (pH 7.0)

2. Hydrogen peroxide in phosphate buffer (2mM)

Procedure

A 20% homogenate of the leaves was prepared in phosphate buffer (0.067M,

pH 7.0) and the homogenate was employed for the assay. The samples were read

against a control without homogenate, but containing the H2O2-phosphate buffer.

To the experimental cuvette, 3.0 ml of H2O2-phosphate buffer was added, followed

by the rapid addition of 40µl enzyme extract and mixed thoroughly. The time interval

required for a decrease in absorbance by 0.05 units was recorded at 240nm. The enzyme

solution containing H2O2-free phosphate buffer served as control.

One enzyme unit was calculated as the amount of enzyme required to decrease

the absorbance at 240nm by 0.05 units.

APPENDIX-13

ESTIMATION OF GLUTATHIONE PEROXIDASE (Ellman, 1959)

Principle

Glutathione peroxidase catalyses the following reaction:

Se-GPx

2GSH + H2O2 GSSH + 2H2O

glutathione was measured by its reaction with DTNB to give a compound that

absorbs at 412 nm.

Reagents

1. 0.4 M Phosphate buffer, pH 7.0

2. 10 mM Sodium azide

3. 2.5 mM Hydrogenperoxide

4. 4 mM Reduced glutathione

5. 10% TCA

6. 0.3 M phosphate solution

7. 4mM EDTA: 14.88 mg/10ml water

8. Ellman’s reagent: 19.8 mg DTNB in 1%sodium citrate

Procedure

To 0.4 ml of buffer, 0.2 ml of EDTA, 0.1 ml of sodium azide and 0.2 ml of

reduced glutathione, 0.1ml of H2O2 were added to two test tubes labelled as test and

control. To the test added 0.2 ml of sample and to the control added 0.2 ml of water.

The contents were mixed well and incubated at 37°C for 10 min, the reaction was

arrested with the addition of 0.5 ml of 10% TCA. To determine the glutathione content,

1.0 ml of supernatant was removed by centrifugation, to that 3.0 ml of buffer and 0.5

ml of Ellman’s reagent. The colour developed was read at 412nm.

Standards in the range of 40-200 µg was taken and treated in the similar

manner. the activity was expressed in term of µg of glutathione consumed/min/mg

protein.

APPENDIX-14

ASSAY OF GLUTATHIONE S-TRANSFERASE

The method of Habig et al. (1974) was employed for the assessment of

glutathione S-transferase in the leaves of the selected plant.

The enzyme was assayed by its ability to conjugate GSH and CDNB, the extent

of conjugation causing a proportionate change in the absorption at 340 nm.

Reagents

1. Chloro-2,4-dinitrobenzene (CDNB) (1mM in ethanol)

2. Reduced glutathione (1mM)

3. Phosphate buffer (0.1M, pH 6.5)

Procedure

Sample (0.5g) was homogenized with 5.0 ml of phosphate buffer. The

homogenate was centrifuged at 5000rpm for 10 mins and the supernatant was used for

the assay. The enzyme activity was determined by monitoring the change in absorbance

at 340 nm in a spectrophotometer. The assay mixture contained 0.1ml of GSH, 0.1 ml

of CDNB and phosphate buffer in a total volume of 2.9 ml. The reaction was started by

the addition of 0.1 ml of enzyme extract to this mixture and the readings were recorded

against distilled water blank for a minimum of three minutes. The complete assay

mixture without the enzyme served as the control to monitor non-specific binding of

the substrates.

One unit of GST activity is defined as the nmoles of CDNB conjugated per

minute.

APPENDIX-15

ASSAY OF GLUTATHIONE REDUCTASE

The method proposed by Beutler (1984) was adopted for assaying the activity of

glutathione reductase.

Principle

Glutathione reductase catalyses the reduction of oxidized glutathione (GSSG)

to reduced glutathione (GSH) and is assayed by measuring in absorbance at 340nm.

Reagents

1. 0.3M Phosphate buffer PH -6.8

2. 25 mM EDTA

3. 12.5 mM oxidized glutathione

4. 1 mM NADPH

Procedure

The activity of the enzyme was determined by observing the change in

absorbance at 340nm. The reaction mixture contained 1.5 ml of buffer , 0.5 ml of

EDTA, 0.2 ml of GSSG and 0.1ml of NADPH. The reaction initiated by the addition of

0.2ml of enzyme extract.

The enzyme activity is calculated intermediate of micromoles of NADPH

oxidized /min/mg protein.

APPENDIX-16

ESTIMATION OF REDUCED GLUTATHIONE

The method proposed by Moron et al. (1979) was used for the estimation of

reduced glutathione.

Principle

Reduced glutathione (GSH) is measured by its reaction with DTNB (5,5’-

dithiobis-2-nitrobenzoic acid) (Ellman’s reaction) to give a yellow coloured product

that absorbs at 412 nm.

Reagents

1. Phosphate buffer ( 0.2M, pH 8.0)

2. DTNB (0.6mM in 0.2M phosphate buffer)

3. TCA (5% and 25%)

4. Standard GSH (10 nmoles/ml in 5% TCA)

Procedure

A 20% homogenate was obtained by homogenizing 0.5g of sample in 2.5 ml of

5% TCA. To precipitate the protein 125 µl of 25% TCA was added to 0.5 ml of tissue

homogenate. The precipitated protein was centrifuged at 1000rpm for 10 mins. The

homogenate was cooled on ice and 0.1 ml of the supernatant was taken for the

estimation. The supernatant was made up to 1 ml with 0.2M sodium phosphate buffer

(pH 8.0). 2.0 ml of freshly prepared DTNB solution was added to the tubes and the

intensity of the yellow colour formed was read at 412 nm in a spectrophotometer after

10 mins.

A standard curve of GSH was prepared using concentrations ranging from 2-10

nmoles of GSH in an electronic calculator set to the linear regression mode and the

values of the samples were read off it. The values are expressed as nmoles of GSH /g

leaf.

APPENDIX-17

ESTIMATION OF CAROTENOIDS

Weigh 5 to 10 g of the sample. Saponify for about 30 minutes in a shaking

water bath at 37 degree C after extracting the alcoholic KOH. Transferred the saponified

extract into a separating funnel (Packed with glass wool and calcium carbonate)

containing 10 to 15ml of petroleum ether and mixed gently which take up the carotenoid

pigments into the petroleum ether layer. Transferred the lower aquous phase to another

separating funnel and petroleum ether extract containing the carotenoid pigments to an

amber colored bottle. Repeat the extraction of the aqueous phase similarly with

petroleum ether, until it is colorless. Discard the aqueous layer. To the petroleum ether

extract added a small quantity of sodium sulphate to remove turbidity. Note the final

volume of the petroleum ether extract and diluted if needed by a known dilution factor.

The absorbance at 450nm was noted in a spectrophotometer using petroleum ether as a

blank.

Carotenoids(microgram)= P x 4x V x 100

W

P= Optical density of the sample

V= Volume of the sample

W=Weight of the sample

APPENDIX-18

ESTIMATION OF LYCOPENE

Weigh 5 to 10 g of the sample. Saponify for about 30 minutes in a shaking

water bath at 37 degree C after extracting the alcoholic KOH. Transferred the saponified

extract into a separating funnel (Packed with glass wool and calcium carbonate)

containing 10 to 15ml of petroleum ether and mixed gently which take up the carotenoid

pigments into the petroleum ether layer. Transferred the lower aquous phase to another

separating funnel and petroleum ether extract containing the carotenoid pigments to an

amber colored bottle. Repeat the extraction of the aqueous phase similarly with

petroleum ether, until it is colorless. Discard the aqueous layer. To the petroleum ether

extract added a small quantity of sodium sulphate to remove turbidity. Note the final

volume of the petroleum ether extract and diluted if needed by a known dilution factor.

The absorbance at 503nm was noted in a spectrophotometer using petroleum ether as a

blank.

3.1206 x OD sample x vol.made upx dilution x 100

(Lycopene mg/1000g) =

1 x weight of the sample x 100

APPENDIX-19

ESTIMATION OF VITAMIN A

The vitamin A level was determined by the method of Nield and Pearson (1963)

Principle

The method is based on the measurement of the interaction of vitamin A with

trifluro acetic acid, the intensity of which is a function of the concentration of vitamin

A which is measured at 620 nm. A correction for the absorbance contribution by

carotene is necessary.

Reagents

1. 2N KOH

2. 90% alcohol

3. Petrroleum ether

4. Trifluro acetic acid

5. Chloroform

6. TFA reagent: Mixed 1.0 ml of TFA and 2.0 ml of chloroform. Prepared

fresh.

7. Vitamin A stock standard (160µg/ml): Transfered 16 mg of all trans retinyl

acetate to 100 ml standard flask and made up with anhydrous chloroform.

8. Vitamin A working solution: Pipetted out 0.2-1.0 ml of stock and made up

to 100 ml with anhydrous chloroform with corresponding concentration of

3-15 µg respectively.

9. β-carotene stock standard (200µg/ml): Transferred 20 mg of β-carotene to

100 ml standard flask. Dissolved in approximately 4 ml of chloroform and

this was diluted to 100 ml with petroleum ether.

10. Carotene working standard: Pipetted out 0.05-0.2 ml of β-carotene stock and

made up to 1.0 ml with petroleum ether. It will have a concentration

corresponding to 1-4 µg respectively.

Procedure

To 1.0 ml of 10% homogenate 1.0 ml of saponification mixture (2N/KOH in

90% alcohol) was added and heated under gentle reflux for 20 min at 60°C. 25 ml of

water was added to teh mixture after cooling to room temperature and the solution was

transferred to a separating funnel. It was then extracted thrice using 25, 15 and 10 ml of

petroleum ether (40-60°C). The ether extracts were pooled and washed with 50-100 ml

of distilled water repeatedly until the wash water was free of alkali. The petroleum

ether extract was then dried by adding anhydrous sodium sulphate. The volume of the

extract was noted 3.0 ml of petroleum ether phase was transferred to a cuvette and read

at 420 nm against petroleum ether blank without delay to prevent evaporation of the

solvent and destruction of carotenoids by light. Marked this reading as A1. The β-

carotene working standards are measured at 450 nm.

The aliquots were evaporated to dryness at 60°C in a water bath. The residue

was taken immediately and 2.0 ml TFA reagent were added to it. The mixture was

rapidly transferred to a cuvette and the absorbance was measured at 620 nm exactly

after the addition of TFA reagent. Marked this reading as A2. The vitamin A working

standard was read at 620 nm.

A3 = A2-A1

A1 = Absorbance of carotene at 450 nm

A2 = Absorbance at 620 nm due to both carotene and vitamin A

A3 = Absorbance at 620 nm of vit A.

A3 x µg retinol calibarator / cuvette x 3 x total volume

Sample =

A620 retinol calibarator x 2 x gram

3 = Vol of petroleum ether from 1.0 ml extract

2 = Aliquot of the petroleum ether used for the assay

1 = 10% extract from initial sample.

The results are expressed as µg/mg protein.

APPENDIX-20

ESTIMATION OF ASCORBIC ACID

Ascorbic acid content in the leaves was estimated by the method of Roe and

Keuther (1943).

Principle

Ascorbate is converted to dehydroascorbate by treatment with activated

charcoal or bromine. Dehydro ascorbic acid then reacts with 2,4- dinitrophenyl

hydrazine to form osazones, which dissolves in sulphuric acid to give an orange

coloured solution, whose absorbance can be measured spectrophotometrically at

540nm.

Reagents

1. Trichloroacetic acid (4%)

2. Sulphuric acid (9N)

3. 2,4-dinitrophenylhydrazine reagent (2% in 9N sulphuric acid)

4. Thiourea solution (10%)

5. Sulphuric acid (85%)

6. Standard Ascorbate solution: 10 mg ascorbate in 100ml of 4% TCA.

Procedure

Ascorbate was extracted into 4% TCA by homogenizing 1g of sample in it and

the volume was made up to 10 ml with 4% TCA. The supernatant obtained after

centrifugation at 2000 rpm for 10 mins was treated with a pinch of activated charcoal,

shaken well and kept for 10 mins. Centrifugation was repeated once again to remove

the charcoal residue. The volumes of the clear supernatants obtained were noted.

Two different aliquots of the supernatant were taken for the assay (0.5 ml and

1.0 ml). The assay volumes were made up to 2.0 ml with 4% TCA. 0.2 to 1.0 ml of the

working standard solution containing 20-100 g of ascorbate respectively were pipetted

into clean dry test tubes, the volumes of which were also made up to 2.0 ml with 4%

TCA.

DNPH reagent (0.5ml) was added to all the tubes, followed by two drops of

10% thiourea solution. The osazones formed after incubation at 37°C for 3 hours, were

dissolved in 2.5 ml of 85% H2SO4, in cold, with no appreciable rise in temperature. To

the blank alone, DNPH reagent and thiourea were added after the addition of H2SO4.

After incubation for 30 minutes at room temperature, the samples were read at 540 nm

and the levels of ascorbic acid in the samples were determined using the standard graph

constructed on an electronic calculator set to the linear regression mode and expressed

as mg ascorbate /g leaf.

APPENDIX-21

DETERMINATION OF TOCOPHEROL

The levels of tocopherol in the leaves were estimated spectrophotometrically by

the method of Rosenberg (1992).

Principle

Tocopherols can be estimated using Emmerie-Engel reaction, which is based on

the reduction of ferric to ferrous ions by tocopherols, which forms a red colour with 2,

2’-dipyridyl. Tocopherols and carotenes were first extracted with xylene and read at

460nm to measure carotenes. A correction is made for this after adding ferric chloride

and read at 520nm.

Reagents

1. Absolute alcohol

2. Xylene

3. 2,2’-dipyridyl (1.2g in 1 litre of n-propanol)

4. Ferric chloride (1.2g in one litre of ethanol stored in brown bottle)

5. Standard solution of D, L- tocopherol, 10mg/L in absolute alcohol. (91mg of -

tocopherol is equivalent to 100 mg of tocopherol acetate).

6. Sulphuric acid (0.1N)

Procedure

A small volume of 0.1N sulphuric acid was used for homogenizing, 2.5g of

sample and the volume was finally made up to 50 ml by adding 0.1N sulphuric acid

slowly, without shaking and allowed to stand overnight. The contents of the flask were

shaken vigorously on the next day and filtered through Whatman No.1 filter paper.

Aliquots of the filtrate were used for the estimation. Into 3 stoppered centrifuge tubes

(test, standard and blank) 1.5ml of plant extract, standard and water respectively were

pipetted out. To all the tubes, 1.5ml each of ethanol and xylene were added, stoppered,

mixed well and centrifuged.

After centrifugation, the xylene layer was transferred into another stoppered

tube, taking care not to include any ethanol or protein. To 1.0 ml of xylene layer, 1.0 ml

of 2, 2’-dipyridyl reagent was added to each tube, stoppered and mixed. This mixture

was taken in the colorimetric cuvettes and the extinctions of the test and the standard

were read against the blank at 460nm. Then, in turn, beginning with the blank, 0.33 ml

of ferric chloride solution was added, mixed well and after exactly 15 mins, the test and

the standard were read against the blank at 520nm.

The levels of tocopherol in the leaf sample was calculated using the formula

Reading at 520nm – Reading at 450nm

Tocopherol ( g) = X 0.29 X 15

Reading of standard at 520nm

APPENDIX-22

DPPH RADICAL SCAVENGING ACTIVITY (Shimada et al., 1992)

Principle

DPPH radical is scavenged by antioxidants through the donation of a proton

forming the reduced DPPH. The colour change from purple to yellow after reduction

can be quantified by its decrease in absorbance at wavelength 517 nm.

Reagents

1. 0.2 mM DPPH

2. 80% Methanol

3. Butylated Hydroxy Anisole

Procedure

Various concentrations of ethanol & aqueous extract of the sample (4.0 ml)

were mixed with 1.0 ml of methanolic solution containing DPPH radicals, resulting in

the final concentration of DPPH being 0.2 mM. The mixture were shaken vigorously

and left to stand for 30 min, and the absorbance was measured at 517 nm. BHA was

used as control. The percentage of DPPH decolorization of the sample was calculated

according to the equation:

% decolorization = [1-(ABS sample/ABS control)] x 100

IC 50 value (mg extract/ml) is the inhibitory concentration at which DPPH

radicals were scavenged by 50 %. Ascorbic acid and BHA were used for comparison.

APPENDIX-23

ABTS RADICAL SCAVENGING ACTIVITY (Re et al., 1999)

Principle

ABTS decolourisation assay involves the generation of the AABTS+,

chromophore by the oxidation of ABTS with ammonium persulphate. It is applicable

for both hydrophilic and lipophilic compounds. The scavenging activity of the plant

extracts on ABTS radical action were measured at 734 nm.

Reagents

1. 7mM ABTS

2. 2.45 mM Ammonium per sulphate

3. ABTS solution: 7mM of ABTS was mixed with 2.45 mM ammonium per

sulphate and the mixture were allowed to stand in dark at room temperature for

12-16 hours before use. ABTS+ solution were diluted to an absorbance of

0.7±0.05 with ethanol at 734 nm.

4. Ethanol

Procedure

Samples were diluted to produce 0.2 to 1.0 mg/ml. The reaction was initiated by the

addition of 1.0 ml of diluted ABTS- to 10 µl of different concentration of ethanolic and

aqueous extract of the sample or 10 µl of methanol as control. The absorbance was read

at 734 nm and the percentage inhibition was calculated. The inhibition was calculated

according to the equation I = A1/A0 x 100, where A0 is the absorbance of control

reaction, A1 is the absorbance of test compound.

APPENDIX-24

SUPEROXIDE RADICAL SCAVENGING ACTIVITY (Liu t al., 1997)

The superoxide radical scavenging activity was analysed by the method of Liu

et al., 1997.

Principle

Superoxide radical was generated from the photo reduction of riboflavin and

was detected by NBT reduction method.

Reagents

1. 6µm EDTA

2. 3 µg NACN

3. 2 µM riboflavin

4. 2 µM NBT

5. 67 µM KH2PO4-Na2HPO4 buffer, pH7.8.

Procedure

The reaction mixture contained 6µm EDTA, 3 µg NACN, 2 µM riboflavin, 2

µM NBT, 67 µM KH2PO4-Na2HPO4 buffer, pH7.8 and various concentration of the

extracts in a final volume of 3.0 ml. The tubes were illuminated under incandescent

lamp for 15 mins. The optical density at 560nm was measured before and after

illumination. The inhibition of superoxide radical was determined by comparing the

absorbance values of the control with those of the treatments. ascorbic acid was used as

standard.

APPENDIX-25

NITRIC OXIDE RADICAL SCAVENGING ASSAY (Madan et al (2005))

Principle

The interaction of ethanolic extract of the sample with nitric oxide was

assessed by the nitrite detection method. Nitric oxide was generated from sodium nitro

prusside and measured by Griess illosvory reaction. Sodium nitroprusside in aqueous

solution at physiological pH spontaneously generated nitric oxide, which interacts with

oxygen to produce nitrite, which can be estimated by the use of Griess illosvory

reagent. In the present experiment, nitrite ion was measured by using Griess illosvory

reagent, which is modified by using naphthyl ethylene diamine dihydro chloride instead

of 1-naphthyl amine.

Reagents

1. Sodium nitroprusside solution (10mM; 0.2998gm of sodium nitroprusside was

accurately weighed and dissolved in distilled water to make up the volume to

100ml in a volumetric flask.

2. Naphthyl ethylene diamine dihydro chloride (NEDD) (0.1%) weighed

accurately 0.1gm of NEDD and dissolved in 60ml of 50% glacial acetic acid by

heating and made up the volume to 100ml in a volumetric flask with distilled

water.

3. Sulphanilic acid(0.33% w/v) reagent: 0.33g of sulphanilic acid was dissolved in

100mlof 20% glacial acetic acid by heating.

4. Phosphate buffer saline (PBS) pH.7

5. Dimethyl sulfoxide (DMSO), distilled.

Procedure

Nitric oxide generated from sodium nitroprusside in aqueous solution at

physiological pH interacts with oxygen to produce nitrite ions which are measured at

540nm. The reaction mixture (6.0 ml) containing sodium nitroprusside (4.0 ml)

phosphate buffer saline(PBS,1.0ml) and extract (1.0 ml at various concentrations) in

DMSO was incubated at 250c for 15 mins. After incubation 0.5 ml of the reaction

mixture was removed, 1.0 ml of sulphanilic acid reagent was added, mixed well and

allowed to stand for 5 mins for the completion of diazotization. Then add 1.0ml NEDD

and stand for 30 mins in diffused light. A pink coloured chromophore formed was

measured at 540nm against corresponding blank solution. Ascorbic acid was used as

standard.

APPENDIX-26

HYDROGEN PEROXIDE SCAVENGING ACTIVITY

Principle

Hydrogen peroxide H2O2 generated a singlet oxygen (O2) and a hydroxyl radical (OH-)

, which then become powerful oxidising agents, they can cross membranes and may

oxidize a number of compounds , while H2O2 itself cannot react , it can generate the

highly reactive hydroxyl radical (OH), through the fenton reaction . thus the scavenger

of H2O2 is an important anti –oxidant defense mechanism.

Fe2+ + H2O2 → Fe3+ + OH + OH-

the decomposition of H2O2 to water involves the transfer of electrons.

H2O2 + 2H+ + 2e

+ → 2 H2O2.

The Scavenging Of H2O2. Was measured at 230 nm in UV/Visible spectrophotometrically.

Reagents required:-

1.Standard solution:-

50mg of Ascorbic acid is dissolved in 50ml standard flask using distilled water.

(conc., 1mg/ml)

2.Extract solution:-

50mg of methanolic dried extract is dissolved in 50ml standard flask using distilled

water. (conc., 1mg/ml)

3. 43mM H2O2. Is prepared with PBS ( pH -7.4)

PROCEDURE:-

1.Prepare (50 - 250µg ) concentration of standard and extract solution. From

that take 3.4 ml of aliquot respectively.

2. Add 0.6 ml of H2O2 .

3. Incubate At Room Temparature For 10 Mins .

4. Read at 230 nm in UV /Visible spectophotometry.

5. 3.4 ml of buffer and 0.6 ml of H2O2 alone serves as blank solution.

APPENDIX-27

HYDROXYL RADICAL SCAVENGING ASSAY (Smirnoff and Cumbes, 1989)

Principle

OH radicals were generated from FeSO4 and hydrogen peroxide and detected by

their ability to hydroxylate salicylate and the hydroxylated salicylate complex was

measured at 562 nm.

Reagents

1. 1.5 mM Ferrous sulphate

2. 6 mM Hydrogen peroxide

3. 20 mM sodium salicylate

Procedure

The reaction mixture 3.0 m contained 1.0 ml of 1.5 mM FeSo4, 0.7 ml of 6 mM

hydrogen peroxide, 0.3 ml of 20 mM sodium salicylate and varying concentrations of

the extract. After incubation for 1 hour at 37°C, the absence of the hydroxylated

salicylate complex was measured at 562 nm. The percentage scavenging effect was

calculated as

Scavenging activity = [1-(A1-A2)/A0] x 100%

Where A0 was absorbance of the control (without extract) and A1 was the

absorbance in the presence of the extract, A2 was the absorbance without sodium

salicylate.

APPENDIX-28

DETERMINATIOM OF THE REDUCING POWER

principle

The reducing activity of a compound generally depends on the presence of

redutants, which exhibit antioxidant activity by breaking the free radical chain

through donating of a hydrogen atom. Fe3+/Fe

2+ transformation was investigated in the

presence of sample for the measurements of the reduced activity. The reducing capacity

estimated by the chelation of Fe2+ ions by the Decker and Welch method in which

ferrozine quantitatively forms complexes with Fe2+. In the presence of chelating

agents, the formation of this complex is disrupted there by impeding the formation of

red color imparted by the complex. The absorbance was measured at 700 nm on a UV /

Visible spectrophotometry.

Reagents required

1) Standard solution:-

50 mg of ascorbic acid is dissolved in 50 ml standadrd flask using distilled water (conc.

1mg/ml).

2) Extract solution:-

50 mg of methanolic dried crude extract is dissolved in 50 ml standard flask using

distilled water (conc. 1mg /ml).

3) 0.2M phosphate buffer (6.6).

4) 1% Potassium Ferricyaide.

5) 10% TCA.

6) 10%FeCl3.

7) Distilled water.

procedure

1) The standard solution and extract solution was prepared in the concentration range of

(100 -800 µg) in different test tubes from that 1.0 ml of sample is taken to different

tubes.

2) Add 2.5 ml of phosphate buffer (0.2M ,pH – 6.6).

3) To that tubes add 2.5 ml of 1% potassium ferricyanide.

4) Incubate all the tubes at 50oc for 20 mins.

5) After incubation add 2.5 ml of 10% TCA and centrifuge the tubes at 3000 rpm for 10 mins.

6) Collect 2.5ml of upper layer and add 2.5 ml distilled water and add 0.5 ml of 0.1%

FeCl3 to all the tubes.

7) The intensity of red color formation was read at 700 nm in a UV/Visible

spectrophotometry.

8) Maintained the control instead of sample solution, make with distilled water.

APPENDIX-29

DETERMINATION OF TOTAL ANTIOXIDANT ACTIVITY

Principle

The total antioxidant activity was determined by phosphomolybdenum method,

it it based on the reduction of MO (VI) to MO(V) by the sample and subsequence

formation of a green Phosphate/ MO(V) complex at acidic pH. The absorbence is

measured at 695nm using an UV/Vis spectrophotometrically. The antioxidant capacity

was expressed as Ascorbic acid equivalent(AAE) by using the standard Ascorbic acid.

reagents required

1.Standard solution:-

50mg of Ascorbic acid is dissolved in 50ml standard flask using distilled

water.(conc., 1mg/ml)

2.Extract solution:-

50mg of methanolic dried extract is dissolved in 50ml standard flask using

distilled water.(conc., 1mg/ml)

3.phosphomolybdenum Reagent:-

0.6M H2S04.

28mM sodium phosphate.

4mM ammonium molybdate.

procedure

1. Prepare (50-250µg) concentration of standard & extract solution, from that take 0.3ml

of each sample respectively.

2. To all the tubes add 3.0ml of Phosphomolybdenum reagent.

3. 0.3ml of water and 3.0 ml of reagent alone serves as blank.

4. All the tubes incubate at 97oC for 90minutes.

5. Cooled and the absorbance was measured at 695nm using an UV/Vis

spectrophotometrically against the blank . The antioxidant capacity was expressed as

Ascorbic acid equivalent(AAE) by using the standard Ascorbic acid.

APPENDIX-30

ESTIMATION OF HAEMATOLOGICAL PARAMETERS

ESTIMATION OF WBC

Principle

The glacial acetic acid lyses the red cells while the gentian violet slightly stains the

nuclus of the leucocytes .the blood specimen is diluted 1:20 in a wbc pipette with the

diluting fluid. The cells are counted under low power microscope by using a counting

chamber.

Reagents

1. Microscope

2. Improved neubauer chamber

3. WBC pipette

4. WBC diluting fluid:it is prepared as follows:

i. Glacial acetic acid: 2.0ml

ii.1%Gentian violet:1.0ml

iii. Distilled water: 97ml

Procedure

1. Draw blood up to 0.5 mark of a WBC pipette.

2. Wipe excess blood outside the pipette using cotton.

3. Mix the content in the pipette and after five minutes by discarding few drops,fill the

counting chamber and allow the cells to settle for 2-3minutes.

4. Focus on one of the ‘w’ marked areas by turning objective to low power(10x).

5. Count the cells in all four ‘w’ marked corners square

Calculation

No of white cells/µl of whole blood =No of cells counted 20

4 0.1

Clotting time

Principle

Blood is colleted in a capillary tube after afinger prick and the stop watch is started.the

formation of fibrin string is noted by breaking the capillary tube at regular intervals.the

is noted at thre first appearance of the fibrin string.

Requirements:

1.sterile lancet

2.capillary tubes

3.cotton

4.spirit/70%alcohol

5.stop watch.

Procedure

1. By using a piece of cotton ,apply the spirir to the patient’s finger tip.

2. Make a deep incision with the sterile lancet and start the stopwatch.

3. wipe off the first blood drop and collect the blood in the capillary tube upto 2/3 of its

length.

4. After every half minute, break off about 1cm of the capillary to find out whether

fibrin string has formed.

5. when the fibrin string appears ,stop the watch and note down the time.

Estimation of haemoglobin

Principle

When the blood is mixed with the drabkin’s reagent containing potassium cyanide and

potassium ferricyanide hb reacts with the ferricyanide to form methHb which is

converted into stable cyanomethHb(HicN)by the cyanide.the intensity of the colour is

propotional to Hb concentration and is compared with the a known cyanometHb

standard at 540nm(reen filter)

Requirements

1.Drabkin’s reagent

It contains:

1.Distilled water:1000ml

2.Pottasium ferricyanide:400mg

3.Potassium dihydrogen phosphate:280mg

4.Pottsium cyanide:100mg

5.Nonidet:1ml

2. Cyanomethemoglobin(HicN) standard .its OD is measured at 540nm.the reading is

obtained corresponds to 15g/dl,Hb.

3. Hb pipette (20 ml calibrated)

4. Test tubes.

5. Photophometer or spectrophometer.

Procedure

Mix the contents in the tube labelled as ‘Test’ thoroughly and wait for 5 minutes.

Read the absorbance of test by setting blank to 100% T at 540nm.

Read the absorbance of standard by pipetting it directly in a cuvette.

Calculation Hb (g/L) = OD of Test ×15

OD of Std.

Estimation of RBC

Principle:

The blood specimen is diluted 1:200 with the RBC diluting fluid and cells are counted

under high power (40X) by using a counting chamber

Requirements

1. Microscope.

2. Improved Neubauer chamber

3. RBC pipette

4. RBC diluting fluid: sodium citrate (3.0g) + Formalin(1.0ml) + Distilled water

(100ml).

Procedure

1. Mix the anticoagulated blood carefully by swirllling the bulb.

2. Draw blood in the RBC pipette upto 0.5 mark.

3. Wipe the excess blood using cotton.

4. Draw the diluting fluid upto 101 mark.

5. The pipette is rotated rapidly keeping it in horizontal position

6. After 5 minutes, by discarding few drops from the pipette and holding it slightly

inclined, small volume of the fluid is introduced under the coverslip which is

placed on the counting chamber.

7. Allow the cells to settle for 2-3 mins.

8. Place the counting chamber on the stage of the microscope

9. Switch to low power (10X) objective. Adjust light and locate the large square in

the centre with 25 small squares.

10. Now switch to high power objective (40X) objective.

11. The RBCs in 4 corner squares and in the centre square are counted.

Calculation

Total RBC/µl= NO of Red cells counted × Dilution

Area counted × Depth of fluid

APPENDIX-31

ESTIMATION OF GLUCOSE BY KIT METHOD (GOD/POD method)

Summary

Glucose is the major carbohydrate present in blood. Its oxidation in the cells is

the source of energy for the body. Increased levels of glucose are found in diabetes

mellitus, hyperparathyryoidism, pancreatitis, renal failure. Decreased levels are found

in insulinoma, hypothyroidism, hypopituitarism and extensive liver disease.

Principle

Glucose is oxidised to gluconic acid and hydrogen peroxide in the presence of

glucose oxidase. Hydrogen peroxide further reacts with phenol and 4- aminoantipyrine

by the catalytic action of peroxidase to form a red coloured quinoneimine dye complex.

Intensity of the colour formed is directly proportional to the amount of glucose present

in the sample

Glucose oxidase

Glucose +O2 +H2O gluconate + H2O2

Peroxidase

H2O2 + 4 Aminoantipyrine + Phenol red Quinoneimine dye + H2O

Normal reference values

Serum/plasma:(fasting): 70-110 mg/dl

2 hrs. p.p: upto 150mg/dl

CSF: 50-80 mg/dl

Reagents

Contents 2 X 150 ml 1000 ml

L1: Glucose Reagent 2 X 150 ml 1000 ml

S: Glucose Standard (100 mg/dl) 5 ml 5 ml

Procedure

Glucose is reported to be stable in the serum sample for 7 days when stored at

2-8°C.

Wavelength / filter : 505 nm / Green

Temperature : 37°C / R.T.

Light path : 1 cm

Pipette into clean test tubes labeled as blank (B), Standard (S), and Test (T):

Addition sequence B (ml) S (ml) T (ml)

Glucose Reagent

(L1)

1.0 1.0 1.0

Distilled water 0.01 - -

Glucose Standard (S) - 0.01 -

Sample - - 0.01

Mix well and incubate at 37°C for 10 min. or at R.T. (25°C) for 30 min. Measure the

absorbance of the standard and test sample against the blank, with in 60 min.

Calculations

Total glucose in mg/dl = Abs.T / Abs.S X 100

Note: To avoid glycolysis the serum should be separated from the clot as soon as

possible, and plasma should be collected in an EDTA + fluoride bulb (0.5 mg + 1mg

per ml of blood).

APPENDIX-32

ESTIMATION OF ALBUMIN BY KIT METHOD (BCG METHOD)

Summary

Albumin consists of approximately 60% of the total proteins in the body, the other

major part being globulin. It is synthesized in the liver and maintains the osmotic

pressure in blood. Albumin also helps in the transportation of drugs, hormones and

enzymes. Elevated levels are rarely seen and are usually associated with dehydration.

Decreased levels are seen in liver diseases (Hepatitis, Cirrhosis). Malnutrition, kidney

disorders, increaseed fluid loss during extensive burns and decreased absorption in

gastro-intestinal diseases.

Principle

Albumin binds with the dye Bromocresol Green in a buffered medium to form a

green coloured complex. The intensity of the colour formed is directly proportional to

the amount of albumin present in the sample.

Albumin + Bromocreasol Green → Green Albumin BCG complex


Serum, plasma (albumin) : 3.7-5.3 g/dl

Globulin : 2.3-3.6 g/dl

A/G Ratio : 1.0-2.3

Contents 150 ml 2 X 150 ml

L1: BCG Reagent 150 ml 2 X 150 ml

S: Albumin Standard (8 g/dl) 5 ml 5 ml

Procedure

Wavelength / filter : 630 nm / Red


Light path : 1 cm

Pipette into clean dry test tubes labeled as Blank (B) & Test (T):


BCG reagent 1.0 1.0 1.0


Albumin Standard

(S)

- 0.01 -

Sample - - 0.01

Mix well and incubate at 37°C for 5 min. measure the absorbance of the standard and

test sample against the blank.

Calculations

Albumin in g/dl = Abs.T / Abs.S X 4

Globulin in g/dl = Total Proteins (g/dl) – Albumin (g/dl)

A/G Ratio = Albumin / Globulin

APPENDIX-33

ESTIMATION OF UREA (Natelson et al., 1951)

Principle:

Urea reacts directly with Diacetyl Monoxime in the presence of thiosemi

carbazide to form a pink coloured product which is measured colorimetrically at 540 nm.

Reagents:

1. Diacety monoxime : 1.56g of Diacetyl monoxime was dissolved in 250ml of

distilled water.

2. Thiosemicarbazide: 41 mg of Thiosemicarbazide was dissolved in 250ml of

distilled water and stored in a brown bottle.

3. Ferric Chloride reagent: 324 mg of Ferric chloride was dissolved in 10ml of

56% of orthophosphoric acid and stored in a brown bottle. To 1 litre of 20%

sulphuric acid added 1 ml of Ferric Chloride reagent.

4. Stock standard: 100mg of Urea / 100ml

5. Working standard: 2.0ml of Stock standard was diluted to 100ml. 1ml of this

solution contains 20 g/ml.

Procedure:

To 0.5 ml of supernatant, 1.0 ml of Diacetyl Monoxime and 1.0ml of

thiosemicarbazide and 3.0 ml of acid reagent was added. Kept in a boiling water bath for 30

minutes. A blank was also set up with water. A series of standard were put up simultaneously

and treated as test. Cooled and read at 540 nm. The values are expressed in mg/dl.

APPENDIX-34

ESTIMATION OF CREATININE (Jaffe Owen et al., 1954)

Principle:

This method make use of the Jaffe’s reaction. The production of mahogony red colour with

an alkaline picrate solution. The intensity of the colour developed was read at 420nm.

Reagents:

1. Picric acid : 0.05 M

2. Sodium hydroxide : 0.75 N

3. Stock standard : Dissolved 100 mg of creatinine in N/10

Hydrochloric acid and made upto 100 ml

with the same.

4. Working standard : Diluted 2.0 ml of stock solution to 100 ml

with water. This contains 20 g of creatinine / ml.

Procedure:

To 4.0 ml of the supernatant, 1.0 ml of 0.15 N sodium hydroxide and 1.0ml of

picric acid was added standard graded volumes and a reagent blank was treated in a

similar manner. The colour development was read at 470nm.

The values are expressed as mg of creatinine / dl.

APPENDIX-35

ESTIMATION OF BILIRUBIN BY KIT METHOD

(Mod. Jendrassik & Grof’s Method)

Summary

Bilirubin is mainly formed the heme portion of aged or damaged RBC’s. it then

combines with albumin to form a complex which is not water soluble. This is

referred to as indirect or unconjugated bilirubin. In the liver this bilirubin complex is

combined with glucuronic acid into a water soluble conjugate. This is referred to as

conjugated or direct bilirubin. Elevated levels of bilirubin are found in liver diseases

excessive hemolysis / destruction of RBC (hemolytic jaundice) obstruction of the

biliary tract (obstructive jaundice) and in drug induced reactions. The differentiation

between the direct and indirect bilirubin is important in diagnosing the cause of

hyperbilirubinemia.

Principle

Bilirubin reacts with diazotized sulphanilic acid to form a coloured azobilirubin

compound. The unconjugated bilirubin couples with the sulphanilic acid in the presence

of a caffeine-benzoate accelerator. The intensity of the colour formed is directly

proportional to the amount of bilirubin present in the sample.

Bilirubin + Diazotized Sulphanilic acid → Azobilirubin Compound


Serum (Direct) : upto 0.2 mg/dl

(Total) : upto 1.0 mg/dl

Procedure

Wavelength / filter : 546 nm / Yellow - Green


Light path : 1 cm


Addition Sequence B (ml) T (ml)

Direct Reagent (L1) 1.0 1.0

Direct Nitrite Reagent (L2) - 1 drop

Sample 0.1 0.1



Total Bilirubin Assay



Total Bilirubin Reagent (L1) 1.0 1.0

Total Nitrite Reagent (L2) - 1 drop

Sample 0.1 0.1



Calculations

Total or Direct Bilirubin in mg/dl = Abs.T / Abs.S x 10

APPENDIX-36

ASSAY OF SGOT (AST) BY KIT METHOD

(Reitman & Frankel’s method)

Summary

kidneys. Injury to those tissues results in the release of the enzyme in blood

stream. Elevated levels are found in myocardial infarction, cardiac operations, hepatitis,

cirrhosis, acute pancreatitis, acute renal diseases, primary muscle diseases. Decreased

levels may be found in Pregnancy, Beri Beri and Diabetic Ketoacidosis.

Principle

SGOT converts L-Aspartate and α Ketoglutarate to Oxaloacetate and

Glutamate. The Oxaloacetate formed reacts with 2,4, Dinitrophenyl ydrazine to

produce a hydrazone derivative, which in an alkaline medium produces a brown

coloured complex whose intensity is measured. The reaction does not obey Beer’s law

and hence a calibration curve is plotted using a pyruvate standard. The activity of

SGOT is read off this calibration curve.

L-Asparate SGOT Oxaloacetate

+ → +

Α Ketoglutarate pH7.4 L-Glutamate

Oxaloacetate Alkaline 2,4,Dinitrophenyl Hydrazone

+ →

2,4 DNPH Medium (Brown coloured complex)


Serum : 8-40 Units/ml

Contents 40 assays

L1:Substrate Reagent 25 ml

L2:DNPH Reagent 2 X 12.5 ml

L3:NaOH Reagent (4N) 25 ml

S:Pyurate Standard (2mM) 5 ml

Reagent preparation

All reagents are ready to use except NaOH Reagent (4N) which has to be

diluted 1:10 with distilled / deionised water.

Working NaOH Reagent: Dilute the Sodium Hydroxide to 250 ml or for every 1.0 ml

of NaOH Reagent (4N) and add 9.0 ml of distilled water. The working sodium

Hydroxide reagent is stable at R.T. till the expiry mentioned, in a plastic bottle.

Procedure



Light path : 1 cm

Plotting of the calibration curve:

Pipette into five clean dry test tubes labeled as 1-5:

Addition sequence 1 2 3 4 5

Enzyme activity 0 (ml) 24 (ml) 61 (ml) 114 (ml) 190 (ml)

Substrate Reagent (L1) 0.50 0.45 0.40 0.35 0.30

Pyruvate Standard (S) - 0.05 0.10 0.15 0.20

Distilled Water 0.10 0.10 0.10 0.10 0.10

DNPH Reagent (L2) 0.50 0.50 0.50 0.50 0.50

Mix well and allow to stand at R.T. for 20 mins

Working NaOH Reagent (L3) 5.00 5.00 5.00 5.00 5.00

Mix well and allow to stand at R.T. for 10 min. Measure the absorbance of the tubes 2-

5 against tube1 (Blank). Plot a graph of the absorbance of tubes 2-5 on the ‘Y’ axis

versus the corresponding enzyme activity on the ‘X’.

Assay:



Substrate Reagent (L1) 0.50 0.50

Incubate at 37°C for 3 min

Sample - 0.10

Mix well and incubate at 37°C for 60 min

DNPH Reagent (L2) 0.50 0.50

Mix well and allow to stand at R.T. for 20 min

Distilled water 0.10 -

Working NaOH reagent (L3) 5.00 5.00

Mix well and allow to stand at R.T. for 10 min. measure the absorbance of the Test

(T) against blank and read the activity of the test from the calibration curve plotted earlier.

High concentrations of aldehydes and ketones in the sample or icteric or

lipemic, samples may cause slightly elevated results. It is recommended to run a sample

blank for these samples using serum instead of distilled water in the blank. High levels

of serum pyruvate may interfere with the results.

APPENDIX-37

ASSAY OF SGPT (ALT) BY KIT METHOD

(Reitman & Frankel’s method)

Summary

SGPT is found in a variety of tissues but is mainly found in the liver. Increased

levels are found in hepatitis, cirrhosis, obstructive jaundice and other hepatic diseases.

Slight elevation of the enzymes is also seen in myocardial infarction.

Principle

SGPT converts L-Alanine and α-Ketoglutarate to Pyruvate and Glutamate. The

Pyruvate formed reacts with 2,4,Dinitrophenyl hydrazine to produce a hydrazone

derivative, which in an alkaline medium produces a brown coloured complex whose

intensity is measured. The reaction does not obey Beer’s law and hence a calibration

curve is plotted using a pyruvate standard. The activity of SGPT is read off this

calibration curve.

L-Alanine SGPT Pyruvate

+ → +

α Ketoglutarate pH7.4 L-Glutamate

Pyruvate Alkaline 2,4,Dinitrophenyl Hydrazone

+ →

2,4 DNPH Medium (Brown coloured complex)


Serum : 5-35 Units/ml

Contents 40 assays

L1:Substrate Reagent 25 ml

L2:DNPH Reagent 2 X 12.5 ml

L3:NaOH Reagent (4N) 25 ml

S:Pyurate Standard (2mM) 5 ml

Reagent Preparation

All reagents are ready to use except NaOH Reagent (4N) which has to be

diluted 1:10 with distilled / deionised water.

Working NaOH Reagent: Dilute the Sodium Hydroxide to 250 ml or for every 1.0 ml

of NaOH Reagent (4N) and add 9.0 ml of distilled water. The working sodium

Hydroxide reagent is stable at R.T. till the expiry mentioned, in a plastic bottle.

Procedure



Light path : 1 cm

Plotting of the calibration curve:

Pipette into five clean dry test tube labeled as 1-5:

Addition sequence 1 2 3 4 5

Enzyme activity (U/ml) 0 (ml) 24 (ml) 61 (ml) 114 (ml) 190 (ml)

Substrate Reagent (L1) 0.50 0.45 0.40 0.35 0.30

Pyruvate Standard (S) - 0.05 0.10 0.15 0.20

Distilled Water 0.10 0.10 0.10 0.10 0.10

DNPH Reagent (L2) 0.50 0.50 0.50 0.50 0.50

Mix well and allow to stand at R.T. for 20 mins

Working NaOH Reagent (L3) 5.00 5.00 5.00 5.00 5.00

Mix well and allow to stand at R.T. for 10 min. measure the absorbances of the tubes 2-

5 against tube1 (Blank). Plot a graph of the absorbance of tubes 2-5 on the ‘Y’ axis

versus the corresponding enzyme activity on the ‘X’.

Assay:



Substrate Reagent (L1) 0.50 0.50

Incubate at 37°C for 3 min

Sample - 0.10

Mix well and incubate at 37°C for 60 min

DNPH Reagent (L2) 0.50 0.50

Mix well and allow to stand at R.T. for 20 min

Distilled water 0.10 -

Working NaOH reagent (L3) 5.00 5.00

Mix well and allow to stand at R.T. for 10 min. measure the absorbance of the

Test (T) against blank and read the activity of the test from the calibration curve plotted

earlier.

High concentrations of aldehydes and ketones in the sample or icteric or

lipemic, samples may cause slightly elevated results. It is recommended to run a sample

blank for these samples using serum instead ofdistilled water in the blank. High levels

of serum pyruvate may interfere with the results.

APPENDIX-38

ASSAY OF ALKALINE PHOSPHATASE

King, J. (1965a)

Principle

The method used was that of King and Armstrong in which disodium phenyl

phosphate is hydrolysed with the liberation of phenol and inorganic phosphate. The

liberated phenol is measured at 700nm with Folin-Ciocalteau reagent.

Reagents

1. Sodium carbonate – sodium bicarbonate buffer, 100 mM/l

2. Disodium phenyl phosphate, 100 mM/l: 2.18 g/l.

3. Buffered substrate: Mixed =equal volume of above to solutions. pH 10

4. Folin-Ciocalteau reagent: mixed 1.0 ml reagent with 2.0 ml of water.

5. Sodium carbonate solution, 15%: 15g/100ml water

6. Standard phenol solution: 1.0 g crystalline phenol/litre of 100 mM HCl

7. Working standard: Added 100 ml dilute phenol reagent to 5.0 ml of stock

standard and diluted to 500 ml with water. This contained 10 µg phenol/ml.

Procedure

Pipetted 4.0 ml of the buffer substrate into a test tube and incubated at 37°C for

5 min. Removed and immediately added 1.8 ml of diluted phenol reagent. At the same

time a control was set up containing 4.0 ml buffer substrate and 0.2 ml sample to which

1.8 ml phenol reagent was added immediately mixed well and centrifuged. To 4.0 ml of

the supernatant added 2.0 ml of sodium carbonate. Took 4.0 ml of working standard

solution and for the blank, take 3.2 ml water and 0.8 ml of phenol reagent, then added

2.0 ml of sodium carbonate. Incubated all the tubes at 37°C for 15 min. Read the colour

developed at 700 nm. The enzyme activity was expressed as units/l in serum, units/mg

protein in tissues.

APPENDIX-39

ASSAY OF GAMMA GLUTAMYL TRANSFERASE

GGT was estimated by the fixed time method of Orolowski and Meister (1963)

Reagents

1. Tris-HCL 120µM, MgCl2 12 mM, glycylglycine 90mM, pH 7.8, 14.54g Tris

hydroxyl methyl amino methane, 2.44g magnesium chloride and 11.89g glycine

were dissolved in 80 ml of water and the pH was adjusted to 7.8 at 37°C with

1M HCl and made upto 1L with water.

2. Substrat: (L-γ glutamyl p-nitroaniline 48 mM/L in 150 mM HCl)- 1.28g of L-

γglutamyl p-nitroaniline was dissolved with constant stirring in 100 ml of 150

mM HCl.

Procedure

To 2.0 ml of buffer, 0.2 ml of substrate was added and warmed to 37°C in a

water bath. Then 0.1ml of sample was added, mixed and incubated for exactly 10 mins

at 37°C. The reaction was then stopped rapidly by adding 2 ml of glacial acetic acid. A

control was simultaneously setup by incubating 2 ml of buffer and 0.2 ml of substrate

solution at 37°C for 10mins. After 10mins, 2 ml of acetic acid was added followed by

0.1 ml of sample and mixed. The absorbance of test and control were measured at 405

nm at the end of each minute for 5mins. The activity was calculated by using 9.9 the

absorption co-efficient of 4-nitroaniline at 405 nm.

The activity of GGT in serum was expressed as µmol p-nitroaniline formed/L

and in liver as nmol p-nitroaniline formed/min/mg protein.

APPENDIX-40

INHIBITION OF IN VITRO LIPID PEROXIDATION IN LIVER

HOMOGENATE (Okhawa et al. 1979)

Principle

The rat liver homogenate was used for the induction of lipid peroxidation,

mediated by FeSO4 as a pro-oxidant and the efficiency of the leaf extracts of sample in

inhibiting the in vitro lipid peroxidation was studied as per the method of Okhawa et al.

(1979) by the measurement of thiobarbituric acid reactive substances

spectrophotometrically at 535nm in the experimental mixture.

Reagents

1. Tris buffered saline (TBS) (10 mM Tris, 0.5 M NaCl, pH 7.4)

2. Ferrous sulphate (10 µM, prepared fresh in TBA)

3. Thiobarbituric acid (1% in TBS)

4. Alcohol (70%)

5. Acetone

6. Rat liver homogenate prepared in TBS (5%)

Procedure

A 5% rat liver homogenate was prepared in cold TBS and 50 µl of it was used

in the assay. Fresh plant tissue (0.5g) was weighed accurately and homogenized in 1

ml of cold TBS. Aliquots of 50µl of it were used in the assay. Ferrous sulphate at a

final concentration of 10 µmoles was added to the assay medium to induce oxidation.

The final volumes in the test tubes were made up to 500 µl with cold TBS.

Controls were prepared for each sample, containing the respective plant extract (50µl),

liver homogenate (50µl) and TBS to make up the final volume to 500µl. Pro oxidant

was not added to the control tubes.

A blank containing no plant extract, no liver homogenate but only FeSO4 and

TBS to make a final volume of 500 µl was also prepared. An assay medium

corresponding to 100% oxidant was prepared by adding all the other constituents except

the plant extract and the volume was made up to 500 µl with cold TBS. The

experimental medium corresponding to auto oxidation contained only the liver

homogenate and TBS to make up the final volume to 500 µl. All the tubes were

incubated at 37°C for one hour. Following the incubation period, 500 µl of 70% alcohol

was added to all the tubes to stop the reaction. 1.0 ml of 10% TBA was added to all the

tubes, followed by boiling in a hot water bath for 20 minutes. After cooling to room

temperature, the tubes were centrifuged. To the clear supernatants collected into tubes,

500 µl of acetone was added and the TBARS was measured at 535nm in a

spectrophotometer.

APPENDIX-41

ESTIMATION OF TRIGLYCERIDES BY KIT METHOD

(GPO / PAP Method)

Summary

Triglycerides are a form of fatty acid esters. They are produced in the liver by

binding glycerol and other fatty acids. They are transported by VLDL and LDL and act

as a storage source for energy. Increased levels are found in hyperlipidemias, diabetes,

nephrotic syndrome, hypothyroidism. Increased levels are risk factor for arteriosclerotic

coronary disease and peripheral vascular disease. Decreased levels are found in

malnutrition and hyperthyroidism.

Principle

Lipoprotein lipase hydrolases triglycerides to glycerol and free fatty acids. The

glycerol formed with ATP in the presence of glycerol kinase forms glycerol 3

phosphate which is oxidized by the enzyme glycerol phosphate oxidase to form

hydrogen peroxide. The hydrogen peroxide further reacts with phenolic compound and

4-aminoantipyrine by the catalytic action of peroxidase to form a red coloured

quinoneimine dye complex. Intensity of the colour formed is directly proportional to

the amount of triglycerides present in the sample.

Lipoprotein Lipase

Triglycerides → Glycerol + Free fatty acids

Glycerol Kinase

Glycerol + ATP → Glycerol 3 Phosphate + ADP

Glycerol 3 PO

Glycerol 3 Phosphate + O2 → Dihydroxyacetone phos. + H2O2

Peroxidase

H2O2 + 4 Aminoantipyrine + Phenol → Red Quinoneimine dye + H2O


Serum / plasma : 150-200 mg/dl

Contents 25 ml 2 X 75 ml

L1 : enzyme Reageent 1 20 ml 2 X 60 ml

L2 : Enzyme Reagent 2 5 ml 2 X 15 ml

S : Triglycerides Standard (200 mg/dl) 5 ml 5 ml

Procedure



Light path : 1 cm

Pipette into clean dry test tubes labelled as Blank (B) & Test (T):


Working reagent 1.0 1.0 1.0


Triglycerides Standard (S) - 0.01 -

Sample - - 0.01



Calculations

Triglycerides in mg /dl = Abs.T /Abs. S x 200

APPENDIX-42

ESTIMATION OF CHOLESTEROL

The serum cholesterol levels were determined using Zak’s method (Zak, 1977)

Principle

Cholesterol reacts with ferric chloride in the presence of concentrated sulphuric

acid to give a pink color. The intensity of color developed is directly proportional to the

amount of cholesterol present and was read at 540 nm in a colorimeter.

Reagents

1. Stock ferric chloride: 840 mg of pure dry ferric chloride was weighed and

dissolved in 100 ml of glacial acetic acid.

2. Ferric chloride precipitating reagent: 10 ml of stock ferric chloride reagent was

taken in 100 ml of standard flask and made upto the mark with pure glacial

acetic acid.

3. Ferric chloride diluting reagent: 8.5 ml of stock ferric chloride was diluted to

100 ml with pure glacial acetic acid.

4. Standard cholesterol solution: 100 mg of cholesterol was dissolved in 100 ml of

glacial acetic acid.

5. Working standard: 10 ml of stock was dissolved in 0.85 ml of stock ferric

chloride reagent and made up to 100 ml with glacial acetic cid. The

concentration of working standard is 100 µg/ml.

Procedure

To 0.1 ml of sample added 4.9 ml of ferric chloride precipitating reagent.

Centrifuged and to 2.5 ml of supernatant added 2.5 ml of supernatant added 2.5 ml of

ferric chloride diluting reagent. Added 4.0 ml of concentrated sulphuric acid. A blank

was prepared simultaneously by taking 5.0 ml of diluting reagent and 4.0 ml of

concentrated sulphuric acid. A set of standards (0.5 – 2.5 ml) were taken and made up

to 5.0 ml with FeCl2 diluting reagent. Then added 4.0 ml of con. H2SO4. After 30

mins, the intensity of colour developed was read at 540 nm against reagent blank.

The amount of cholesterol in the sample was expressed as mg/dl.

APPENDIX-43

ESTIMATION OF VLDL

Calculation of VLDL Cholesterol (mg/dl) (Freidewald’s formula):

VLDL cholesterol = triglycerides/5

Freidewald’s formula is reliable provided that:

No chylomicrons are present i.e. it is a fasting sample.

Triglyceride values are below 400 mg/dl

Type III hyperlipoproteinemia is absent

APPENDIX-44

ESTIMATION OF HDL CHOLESTEROL

(POLYETHYLENE Glycol method)

Summary

Lipoproteins are the proteins which mainly transport fats in the blood stream.

They can be grouped into chylomicrons, very low density lipoproteins, low density

lipproteins and high density lipoproteins. Chylomicrons and VLDL transport mainly

triglycerides, through VLDLs also transport some amount of cholesterol. LDL carries

cholesterol to the peripheral tissues where it can deposited and increase the risk of

arteriosclerotic heart and peripheral vascular disease. Hence high levels of LDL are

atherogenic. HDL transports cholesterol from the peripheral tissues to the liver for

excretion, hence HDL has a protective effect. The measurement of total and HDL

cholesterol and triglycerides provide valuable information for the risk assessment of

coronary heart diseases.

Principle

When the serum is reacted with the Polyethylene Glycol contained in the

precipitating reagent, all the VLDL and LDL are precipitated. The HDL remains in the

supernatant and is then assayed as a sample for cholesterol using the cholesterol

reagent.

Reagents

Contents 75 ml

L1: Enzyme Reagent 1 60 ml

L2: Enzyme Reagent 2 15 ml

L3: Precipitating Reagent 2.5 ml

S: HDL Cholesterol Standard (25mg/dl) 5 ml

Working reagent : Pour the contents of 1 bottle of L2 into bottle of L1. This working

reagent is stable for at least 8 weeks when stored at 2-8°C. upon storage the working

reagent may develop a slight pink colour however this does not affect the performance

of the reagent.

Procedure



Light path : 1 cm

Precipitation of VLDL & LDL:

Pipette into a clean dry test tube:

Precipitating reagent L3 0.1 ml

Sample 0.1 ml

Mix well and incubate at R.T. for 5 min. centrifuge at 2500-3000 rpm to obtain a clear

supernatant.

Cholesterol Assay:

Pipette into clean test tubes labeled as blank (B), Standard (S), and Test (T):


Working reagent 1.0 1.0 1.0


HDL Standard (S) - 0.05 -

Supernatant - - 0.05

Mix well and incubate at 37°C for 5 min. or at R.T. (25°C) for 15 min. measure the

absorbance of the standard and test sample against the blank, with in 60 min.

Calculations

HDL Cholesterol in mg/dl = Abs.T / Abs.S X 25 X 2

(where 2 is the dilution factor due to the deproteinization step)

APPENDIX-45

ESTIMATION OF LDL

Calculation of LDL Cholesterol (mg/dl) (Freidewald’s formula):

=Total cholesterol – (triglycerides/5) + HDL cholesterol

Freidewald’s formula is reliable provided that:

No chylomicrons are present i.e. it is a fasting sample.

Triglyceride values are below 400 mg/dl

Type III hyperlipoproteinemia is absent

APPENDIX-46

ESTIMATION OF FREE FATTY ACIDS

(Horn and Mehanan, 1981)

Principle

The free fatty acids were extracted from lipids by CHM mixture. The free fatty

acids form a complex with cupric ions when mixed with copper reagent, the coloured

complex formed with copper is soluble in chloroform and diethyl dithiocarbamate and

is used as a colour developer. The colour developed was read at 660 nm.

Reagents

1. Chloroform-Heptane-Methanol mixture (CHM mixture): the mixture was

prepared in the ratio of 200:150:7 (v/v)

2. Activated silicic acid

3. Copper nitrate-Triethanolamine Solution: 9 volumes of aqueous 1 M

triethanolamine, 1 volume of 1 N acetic acid and 10 volumes of 6.45%

Cu(NO3)2.3H2O were mixed with 33 g of sodium chloride. The pH was

adjusted to 8.1.

4. 0.1% Diethyl dithiocarbamate in n-Butanol

5. Standard: 200 mg palmitic acid / 100 ml CHM mixture. the solution was diluted

1 in 10 times for use (200 µg/ml).

Procedure

To 0.2 ml sample, 5.8 ml of CHM mixture and 200 mg of activated silicic acid

were added, mixed well and centrifuged. The supernatant was transferred to another

tube. Standard were also made up to 6.0 ml with CHM mixture, blank contained 6.0 ml

of CHM mixture. To all these tubes, 2.0 ml of copper nitrate-TEA solution was added

and mixed on a mechanical shaker for 20 min, they were then centrifuged to give two

separate phases, 2.0 ml of the upper phase was transferred to another tube, 1.0 ml of the

colour reagent was then added and shaken well, the colour developed was read at 430

nm against a reagent blank. Free fatty acids are expressed as mg/100 ml in serum and

mg/g in tissues.

APPENDIX-47

HISTOPATHOLOGICAL EXAMINATION

The livers were preserved in 20% commercial Formalin immediately on

removal from animal.

Tissue processing

Liver tissue is placed in 10% formal saline (10% formalin in 9% sodium chloride)

for 1 hr to rectify shrinkage due to higher concentration of Formalin. The tissue was

dehydrated by ascending grades of Isopropyl alcohol by immersing in 80%Isopropanol

overnight, 100% Isopropyl alcohol for 1 hr and second change of 100% Isopropyl alcohol

for 1hr. The dehydrated tissues were cleared in two changes of xylene, 1hr each. Then the

tissues were impregnated with histology grade paraffin wax (melting point 58-60°C) at

60°C for 2 changes of 1 hr each. The wax impregnated tissues were embedded in paraffin

blocks using the same grade wax. The paraffin blocks were mounted and cut with rotary

microtome at 3 micron thickness. The sections were floated on a tissue floatation bath at

40°C and taken on glass slides and smeared with equal parts of egg albumin and glycerol.

The sections were then melted in an incubator at 60°C and after 5 min the sections were

allowed to cool.

Tissue staining

The sections were deparaffinised by immersing in xylene for 10 min is

horizontal staining jar. The deparaffinised sections were washed in 100% Isopropyl

alcohol and stained in Ehrlich,s hematoxylin for 8 min in horizontal staining jar. After

staining in hematoxylin the sections were washed in tap water and dipped in acid

alcohol to remove excess stain (8.3 % HCl in 70% alcohol). The sections were then

placed in running tap water for 10 min for blueing (slow alkalization). The sections

were counter stained in 1% aqueous eosin (1 g in 100 ml tap water) for one min and the

excess stained was washed in tap water and the sections were allowed to dry. Complete

dehydration of stained sections was ensured by placing the sections in the incubator at

60°C for 5 min. When the sections are cooled, they were mounted in DPX mount

having the optical index of glass.

The architecture was observed at low power objective. The liver cell injury and

other aspects were observed under high power dry objective.

List of Publications

1. Pakutharivu T, Suriyavathana M (2009). In vitro antioxidant activity of Entada

pursaetha, Toddalia aculeata and Ziziphus mauritiana. Pharmacognosy

Journal 1: 246-250.

2. Suriyavathana M, Pakutharivu T (2011). Evaluation of acute and sub acute

Toxicity of Entada pursaetha, Toddalia aculeata and Ziziphus mauritiana.

World Journal of Life sciences and Medical Research 1:43-47.

Papers presented in Conference (Poster)

1. Suriyavathana M, Pakutharivu T. Phytochemical Analysis and Antioxidant

profile of Hippo-08 an Oral Ayurvedic Formulation. International Symposium

on Functional Foods and Health (2011), Periyar University. Salem-11.

Documents

APPENDIX-1 ESTIMATION OF TOTAL CARBOHYDRATE The total