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 (Na 2WoO4.2H2O), 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
Absorbance×100
Concentration (%) = g%
17

APPENDIX-9
ESTIMATION OF SAPONINS
20 g of crude taken from each plant were put into a conical flask and 100 cm 3 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 r e-
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 H 2 O2 -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.
P x 4x V x 100
Carotenoids(microgram)=
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% H2SO 4, in cold, with no appreciable rise in temperature. To
the blank alone, DNPH reagent and thiourea were added after the addition of H 2 SO4.
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 25 0c 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 H 2O2 itself cannot react , it can generate the
highly reactive hydroxyl radical (OH), through the fenton reaction . thus the scavenger
of H 2O2 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 H 2O 2 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 anti- oxidant activity by breaking the free radical chain
through donating of a hydrogen atom. Fe3+/Fe2+ 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 50 oc 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 97 oC 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 cells counted 20
No of white cells/µl of whole blood =
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.
OD of Test ×15
Calculation Hb (g/L) =
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
NO of Red cells counted × Dilution
Total RBC/µl=
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 1.0 1.0 1.0
(L1)
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
Normal reference values
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
Temperature : 37°C / R.T.
Light path : 1 cm
Pipette into clean dry test tubes labeled as Blank (B) & Test (T):
Addition sequence B (ml) S (ml) T (ml)
BCG reagent 1.0 1.0 1.0
Distilled water 0.01 - -
Albumin Standard - 0.01 -
(S)
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
Normal reference values
Serum (Direct) : upto 0.2 mg/dl
(Total) : upto 1.0 mg/dl
Procedure
Wavelength / filter : 546 nm / Yellow - Green
Temperature : 37°C / R.T.
Light path : 1 cm
Pipette into clean dry test tubes labeled as Blank (B) & Test (T):
Addition Sequence B (ml) T (ml)
Direct Reagent (L1) 1.0 1.0
Direct Nitrite Reagent (L2) - 1 drop
Sample 0.1 0.1
Mix well and incubate at 37°C for 5 min. measure the absorbance of the standard and
test sample against the blank.
Total Bilirubin Assay
Pipette into clean dry test tubes labeled as Blank (B) & Test (T):
Addition Sequence B (ml) T (ml)
Total Bilirubin Reagent (L1) 1.0 1.0
Total Nitrite Reagent (L2) - 1 drop
Sample 0.1 0.1

Mix well and incubate at 37°C for 10 min. measure the absorbance of the standard and
test sample against the blank.
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)

Normal reference values

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
Wavelength / filter : 505 nm / Green
Temperature : 37°C / R.T.
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:
Pipette into clean dry test tubes labeled as Blank (B) & Test (T):
Addition Sequence B (ml) T (ml)
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)
Normal reference values
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
Wavelength / filter : 505 nm / Green
Temperature : 37°C / R.T.
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:
Pipette into clean dry test tubes labeled as Blank (B) & Test (T):

Addition Sequence B (ml) T (ml)

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 FeSO 4 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
Normal reference values
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
Wavelength / filter : 505 nm / Green
Temperature : 37°C / R.T.
Light path : 1 cm
Pipette into clean dry test tubes labelled as Blank (B) & Test (T):

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

Working reagent 1.0 1.0 1.0
Distilled water 0.01 - -
Triglycerides 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
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
Wavelength / filter : 505 nm / Green
Temperature : 37°C / R.T.
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):
Addition sequence B (ml) S (ml) T (ml)
Working reagent 1.0 1.0 1.0
Distilled water 0.05 - -
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.