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APPENDICES APPENDIX – I

Determination of Thrombolytic Activity

(Prasad et al., 2006)

Streptokinase

To the commercially available lyophilized streptokinase vial (15,00,000 IU) 5ml of sterilized

distilled water was added and mixed properly. This suspension was used as a stock from which 100µl

(30,000 IU) was used as a standard.

Procedure

Venous blood drawn from healthy volunteers (n=25) was transferred in different pre weighed

sterile microcentrifuge tube (500µl/tube) and incubated at 37ºC for 45 minutes. After clot formation,

serum was completely removed (aspirated out without disturbing the clot formed). Each tube with the

clot was again weighed to determine the clot weight (clot weight = weight of clot containing tube –

weight of tube alone). To the microcentrifuge tube containing clot, 100µl of streptokinase (30,000 IU)

or various dilutions of the aqueous fruit extract was added. Water was also added to one of the tubes

containing clot and this served as a negative thrombolytic control. All the tubes were then incubated

for 90 minutes at 37o C and the tubes were again weighed to observe the difference in weight after

clot disruption. Differences obtained in weight taken before and after clot lysis was expressed as

percentage of clot lysis. This test was repeated twenty five times with different concentrations of the

fruit extracts in blood samples of twenty five different healthy volunteers.

APPENDIX – II

Qualitative Analysis of Phytochemicals

(Raaman, 2006)

Identification of Carbohydrates

Molisch's Test

To 2ml of aqueous extract, few drops of 20% α-naphthol in ethyl alcohol were added. Then

about 1ml of concentrated sulphuric acid was added along the sides of the test tube. Reddish violet

ring appeared at the junction of two layers indicating the presence of carbohydrates.

Identification of Amino acids and Proteins

Millon's Test

To 2ml of the filtrate, 5-6 drops of MiIlon's reagent was added. Formation of red precipitate

indicated the presence of proteins and free amino acids.

Ninhydrin Test

To the filtrate lead acetate solution was added to precipitate tannins. It was then filtered and

the filtrate was spotted on a paper chromatogram, sprayed with ninhydrin reagent and dried at 1100C

for 5 minutes. Appearance of violet spots indicated the presence of proteins and free amino acids.

Biuret Test

To the ammoniated alkaline filtrate 2-3 drops of 0.02% copper sulphate solution was added

and formation of red colour indicated the presence of proteins and free amino acids.

Identification of Phenols

Ferric chloride Test

To 2ml of the extract, 2ml of ferric chloride solution was added and formation of deep bluish

green solution indicated the presence of phenols.

Identification of Saponins/Saponin Glycosides

Sodium bicarbonate Test

To a few ml of the ethanolic extract, few drops of sodium bicarbonate was added and shaken

well. Formation of honey comb indicated the presence of saponins.

Identification of Quinones/ Anthraquinones

Chloroform-Ammonia Test

To 0.5g of the plant sample, 10 ml of 5% sulphuric acid was added and allowed to boil. It was

then filtered and while hot, to the filtrate 5ml of chloroform was added and heated on a boiling water

bath. Two ml of the chloroform extract was mixed with 1ml of diluted 10% ammonia and the mixture

was shaken. A pink-red color in the ammoniacal layer showed the presence of anthracene derivatives.

Borntrager's Test

About 50 ml of the extract was heated with 10% ferric chloride solution and 1ml of

concentrated HCl. The extract was cooled, filtered and the filtrate was shaken with diethyl ether. The

ether extract was further extracted with strong ammonia and pink or deep red coloration of aqueous

layer indicated the presence of anthroquinone.

Identification of Alkaloids

Mayer’s Test

A fraction of the extract was treated with Mayer‟s reagent (1.36g of mercuric chloride and 5g

of potassium iodide in 100ml of distilled water) and observed for the formation of cream coloured

precipitate that indicates the presence of alkaloids.

Dragendorff's Test

To 1ml of the extract added 1ml of Dragendorff‟s reagent. Appearance of orange – red

precipitate indicated the presence of alkaloids.

Wagner's Test

To a fraction of the filtrate a few drops of Wagner's reagent was added. Appearance of

reddish-brown precipitate indicated the presence of alkaloids.

Identification of Flavonoids

The aqueous extract of the sample was reduced to dryness in a water bath. The residue was

treated with dilute NaOH followed by addition of dilute HCl. A yellow solution with NaOH which turned

colorless with dilute HCl confirmed the presence of flavonoids.

Pew Test

A piece of metallic Magnesium/ Zinc was added to 1ml of the extract, followed by addition of 2

drops of concentrated HCl. Formation of brown color confirmed the presence of flavonoids.

Identification of Tannins

Preparation of Extract

The plant material was suspended in methanol and allowed to stand overnight. It was refluxed

for 4 hours. It was then filtered and the residue was washed with methanol. The filtrate was allowed to

cool down, observed for any modification. This aliquot was used to assay tannins.

Braemer's Test

To 0.5 g of the methanolic/ethanolic extract, 10 ml of water was added and boiled. It was then

filtered. To the filtrate, few drops of 10% FeCl3 were added. A dark green, blue or brown color

indicated the presence of tannins.

Identification of Volatile oils

Two ml of the extract solution was shaken with 0.1 ml diluted sodium hydroxide and a small

quantity of dilute HCl. Formation of white precipitate indicated the presence of volatile oils.

Detection of Terpenoids

To 5ml of the extract, 2ml of chloroform was mixed and concentrated H2SO4 (3ml) was

carefully added to form a layer. A reddish brown coloration at the interface indicated the presence of

terpenoids

Identification of Glycosides

For detection of glycosides, 0.5g of sample was hydrolysed with concentrated

hydrochloric acid for 2 hrs on water bath, filtered and the hydrolysate was subjected to the

following tests.

Borntrager's Test

To 2 ml of the filtered hydrolysate, 3 ml of chloroform was added and shaken,

chloroform layer was separated and 10% ammonia solution was added to it. A pink colour

indicated the presence of glycosides.

Legal's Test

A few ml of ethanolic extract was dissolved in few drops of pyridine. Sodium nitroprusside

solution (2% w/v) was added and made alkaline using 20% sodium hydroxide. Presence of glycoside

was indicated by pink colour.

Kellar-Killani Test

The ethanolic extract was dissolved in glacial acetic acid containing a trace of ferric chloride.

Same amount of ferric chloride dissolved in concentrated H2SO4 was added along the sides of the test

tubes to settle at the bottom. Appearance of a reddish green colour at the junction of two reagents

within 2-5 minutes spreading slowly into the acetic acid layer confirmed the presence of cardiac

glycosides.

APPENDIX – III

Brine shrimp lethality assay

(Oladimeji et al., 2006)

Brine shrimp (Artemia salina) were obtained by hatching brine shrimp eggs in artificial sea

water (3.8% non-ionized sodium chloride solution) for 48 hours. 200µl of the fruit extracts of different

concentration were added to 10 ml of brine shrimp solution with 20 nauplii for each extracts in vials.

These vials were maintained at room temperature for 24 hours under the light and surviving

larvae were counted using a magnifying lens. Experiments were conducted along with potassium

dichromate as positive control. The mortality rate was calculated by the formula,

No of dead nauplii

% Mortality = × 100 Total no of nauplii

APPENDIX – IV

MTT Assay

(Scudiero et al., 1988)

Principle

MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) measures the metabolic

activity of the viable cells. The assay is non-radioactive and can be performed entirely in a

microtiterplate (MTP). It is suitable for measuring cell proliferation, cell viability or cytotoxicity. The

reaction between MTT and “mitochondrial dehydrogenase” produce water-insoluble formazan salt.

This method involves culturing the cells in a 96-well microtiterplate and then incubating them with MTT

solution for approximately 2 hours. During incubation period, viable cells convert MTT to a water-

insoluble formazan dye. The formazan dye in the MTP is solubilized and quantified with an ELISA

plate reader.

Reagents

1) Trypsin - 0.25%

2) FBS (Fetal bovine serum)

3) MTT

4) Dimethyl sulphoxide

5) Lysis buffer

6) RPMI

Procedure

Increasing concentrations of fruit extracts were added to the cells and incubated at 37˚C for

14 hrs in CO2 incubator with 5% CO2. The media was replaced with a fresh growth medium along with

20:1 of MTT reagent. It was again incubated for 4 hrs at 37˚C. After incubation, purple precipitate was

clearly visible under the inverted microscope then the growth medium was removed and 200µl of

0.1% 0.1N acidic isopropyl alcohol was added to the cells to dissolve the formazan crystals. Then the

covered plates were kept in the dark at 18-24˚C for overnight. The samples were drawn every 2 hrs

and the readings were observed at 570nm. If the reading is low the plate was returned for incubation.

Each experiment was conducted in triplicate form.

The average was calculated and compared with the control test samples. The percentage growth

inhibition was calculated using the following formula.

Control OD – Treated OD % Growth Inhibition = ------------------------------------- x 100

Control OD

APPENDIX – V

Determination of DPPH radical scavenging activity

(Mensor et al., 2001)

Principle

Antioxidants react with DPPH and convert it to diphenyl-picryl hydrazine by donating its OH

group. The degree of discoloration from purple to yellow colour can be measured at 519nm, which is a

measure of the radical scavenging potential of the extracts.

Reagents

1. DPPH (0.3mM in methanol)

2. Methanol

Procedure

The different solvent extracts and crude aqueous extract (5μl) was added with 0.5ml of

methanolic solution of DPPH and 0.495ml of methanol. The mixture was then allowed to stand at

room temperature for 30 minutes. DPPH methanol solution was used as positive control and methanol

alone acted as blank. After incubation, the conversion of purple colour to yellow colour was read at

518nm in a spectrophotometer. The per cent inhibition was calculated using the following formula;

A(Control) - A(Sample)

Scavenging activity (%) = 100 - x 100 A(Control)

APPENDIX – VI

Determination of nitric oxide scavenging activity

(Green and Hill, 1984)

Principle

An aqueous solution of sodium nitroprusside spontaneously generates nitric oxide at

physiological pH, which interacts with oxygen to produce nitrite ions, which is measured at 546nm.

Reagents

1. Sodium nitroprusside (100mM)

2. Phosphate buffered saline (PBS) (pH 7.4)

3. Griess reagent: (1% sulphanilamide, 2% phosphoric acid and 0.1% naphthyl ethylene

diamine dihydrochloride)

Procedure

Sodium nitroprusside (2.0 ml), phosphate buffered saline (0.5 ml) and each of the different

fruit extracts (0.5ml) were mixed and incubated at 25ºC for 30 minutes. Griess reagent (0.5ml) was

added and allowed to stand for 30 minutes. The control tube was prepared without fruit extracts. The

absorbance of the pink coloured chromogen was read at 546nm against a reagent blank.

A (Sample) Nitric oxide scavenging activity (%) = x 100 A (Control)

APPENDIX – VII

Estimation of superoxide scavenging activity

(Winterbourn et al., 1975)

Principle

The extent of superoxide generation was studied on the basis of inhibition of the production of

nitroblue tetrazolium formazon of the superoxide ion by the fruit extracts and is measured

spectrophotometrically at 560 nm.

Reagents

1. EDTA (0.1M containing 1.5 mg of sodium cyanide / 10 ml)

2. Nitroblue tetrazolium (NBT) (1.5 mM)

3. Riboflavin (0.12 mM)

4. Phosphate buffer (0.067 M, pH 7.6)

5. Dimethyl sulfoxide (DMSO)

Procedure

The assay mixture containing 0.02 ml of fruit extracts with 0.2 ml of EDTA, 0.1 ml of NBT,

0.05 ml of riboflavin and 2.63 ml of phosphate buffer was prepared. DMSO, instead of fruit extract,

was considered as control. All tubes were vortexed and the initial absorbance was read at 560nm.

The tubes were illuminated uniformly using a fluorescent lamp for 30 minutes. The absorbance was

read again at 560nm. The difference in optical density before and after illumination is the measure of

superoxide generation and the percentage inhibition was calculated using the formula,

A (After illumination) – A (Before illumination)

% Superoxide Scavenging = x 100

A (Control)

APPENDIX – VIII

Estimation of hydroxyl radical scavenging activity

(Elizabeth and Rao, 1990)

Principle

Hydroxyl radicals are generated from a Fe2+/ascorbate/EDTA/H2O2 system, which attacks

deoxyribose and eventually produces thiobarbituric acid reactive substances (TBARS). The ability of

the fruit extracts to inhibit TBARS formation is measured spectrophotometrically at 532nm.

Reagents

1. Deoxyribose (28mM)

2. FeCl3 (1mM)

3. EDTA (1mM)

4. H2O2 (10mM)

5. Ascorbate (1mM)

6. KH2PO4-KOH buffer (20mM, pH 7.4)

7. Thiobarbituric acid (1%)

8. HCl (25%)

Procedure

The reaction mixture contained deoxyribose (0.1ml), FeCl3 (0.1ml), H2O2 (0.1ml), ascorbate

(0.1ml), buffer (0.1ml) and 20μl of fruit extracts which corresponded to 10mg concentration. The total

volume was made up to 1ml with water. The tubes were capped tightly and incubated in a water bath

at 37ºC for one hour. The reaction was terminated by the addition of TBA (0.5ml) and HCl (0.5ml).

The tubes were heated in a boiling water bath for 20 minutes for colour development. The intensity of

the pink colour formed, as the indication of TBARS formation, was measured at 532nm. The per cent

TBARS produced for positive control (H2O2) was fixed as 100% and the relative per cent TBARS was

calculated for the extract treated groups.

A (Control) – A (Sample) Hydroxyl radical scavenging activity (%) = X 100

A (Control)

APPENDIX – IX

Estimation of hydrogen peroxide scavenging activity

(Ruch et al., 1989)

Principle

H2O2 scavenging activity was measured in terms of a decrease in the absorbance at 230nm

spectrophotometrically.

Reagents

1. H2O2 (40mM in 0.1M phosphate buffer)

2. Phosphate buffer (0.1M, pH 7.4)

Procedure

The fruit extract was diluted to a concentration of 10mg in 10μl. This extract (10μl

corresponding to 10mg) was added to 0.6ml of H2O2 solution and the final volume was made up to

3ml with the same buffer. After 10 minutes, the absorbance values at 230nm of the reaction mixtures

were recorded against a blank containing phosphate buffer without H2O2 for each sample. The

percent inhibition was calculated using the formula,

A (Control) – A (Sample)

H2O2 scavenging activity (%) = X 100

A (Control)

APPENDIX – X

Estimation of catalase

(Luck, 1974)

Principle

The UV light absorption of hydrogen peroxide solution can be easily measured between 230

and 250 nm. On decomposition of hydrogen peroxide by catalase, the absorption decreases with time.

The enzyme activity could be arrived at from this decrease.

Reagents

1. Phosphate buffer (0.067 M pH 7.0)

Dissolved 3.522 g of KH2PO4 and 7.268 g of K2HPO4.2H2O in distilled water and the volume

was made up to 1 litre.

2. Hydrogen peroxide – Phosphate buffer

Dissolved 0.16 ml of H2O2 (10% W/V) to 100 ml phosphate buffer, prepared fresh. The

absorbance of the solution should be about 0.5 at 240 nm with 1 cm light path.

Procedure

Enzyme Extract

The sample was homogenized in a prechilled mortar and pestle with M/150 phosphate buffer

(assay buffer diluted 10 times) at 1 - 4°C and centrifuged. The sediment was stirred with cold

phosphate buffer, allowed to stand in the cold with occasional shaking and then the extraction was

repeated once or twice. The extraction should not take more than 24 hr. The combined supernatants

were used for the assay.

Assay

Three ml of H2O2 Phosphate buffer was pipette out into the experimental cuvette. It was

mixed well with 0.01 – 0.04 ml sample using the flattened end of a glass rod. The time required (∆t) for

a decrease in absorbance from 0.45 to 0.40 (0.05 units) at 240 nm was noted. This value was used

for calculation. If ∆t was greater than 60 seconds, the experiment was repeated with increased

concentration of the sample.

The activity was calculated and expressed as units / g. One enzyme unit was calculated as

the amount of enzyme required to decrease the absorbance at 240 nm by 0.05 units.

Calculation

The concentration of H2O2 and thereby the catalase activity was calculated using the extinction

coefficient 0.036µ mole/ml.

APPENDIX – XI

Estimation of peroxidase

(Reddy et al., 1995)

Principle

In the presence of the hydrogen donor pyrogallol, peroxidase converts H2O2 to water and

oxygen. The oxidation of pyrogallol to the coloured product purpurogalli can be quantified

spectrophotometrically at 430nm. The formation of the product is proportional to the activity of the

enzyme and can be used as a measure of the same.

Pyrogallol + H2O2 Oxidized pyrogallol + H2O

Reagents

1. Pyrogallol (0.05M); Phoshphate buffer (pH 6.5) – 630 mg of pyrogallol in 100 ml of 0.1M

Phosphate buffer.

2. Hydrogen peroxide (1%)

Procedure

One gram of the sample was mascerated with 5 ml (w/v) 0.1 M phosphate buffer (pH 6.5) in a

homogeniser. The homogenate was then centrifuged at 300 g for 15 minutes. The supernatant was

used as the enzyme source. All the procedures were carried out at 0-5° C.

Three ml of 0.05 M pyrogallol solution and 0.5 to 1 ml enzyme extract was pipette out into a

test tube. The spectrophotometer was adjusted to read „0‟ at 400 nm. To this, 0.5 ml of 1% H2O2 was

added to the test cuvette. The change in absorbance for every 30 seconds upto 3 minutes was

recorded.

Calculation Change in absorbance / min = X

Weight of the fruit material taken = 300 mg

Volume of the extract taken for the assay = 0.02 ml

Change in absorbance for 1.5 ml extract = (X / 0.02) x 1.5 – Y

(i.e) Peroxidase activity in 300 mg fruit = Y

Peroxidase activity / g fruit = Y x (1000/300) Units

APPENDIX – XII

Assay of polyphenol oxidase

Esterbauer et al., (1977)

Principle

Phenol oxidases are copper proteins, which catalyze the aerobic oxidation of certain phenolic

substrates to quinines, which are auto-oxidized to dark brown pigments generally known as melanins,

which can be estimated spectrophotometrically at 495 nm.

Reagents

1. Reaction medium - Tris-HCl (50mM, pH 7.2), sorbitol (0.4M), NaCl (10mM)

2. Catechol (0.01M)

3. Phosphate buffer (0.1M, pH 6.5)

Procedure

Preparation of enzyme extract

The enzyme extract was prepared by macerating 5 g of leaf tissue in 20 ml reaction medium

containing tris-HCl. The homogenate was centrifuged at 2000 g for 10 minutes at 4ºC, the supernatant

was used for the assay.

Assay

Both phosphate buffer (2.5 ml) and catechol solution (0.3 ml) was pipetted out into the

experimental cuvette and the spectrophotometer was set at 495 nm. The sample (0.2 ml) was added

to the same cuvette and the changes in absorbance were monitored for every 30 seconds up to 5

minutes. One unit of either catechol oxidase / laccase is defined as the amount of enzyme that

transforms one μmole of dihydrophenol to one μmole of quinine/minute. The activity of PPO can be

calculated using the formula,

Enzyme unit = K x (ΔA / minute)

where, K for catechol oxidase = 0.272, K for laccase = 0.242

APPENDIX – XIII

Estimation of glutathione peroxidase

(Rotruck et al., 1973)

Principle

A known amount of enzyme preparation was used to react with hydrogen peroxide in the

presence of GSH for a specified time period when the screening was measured by the method of

Ellman.

Se – GPx 2 GSH + H2 O2 GSSH + 2H2 O

Reagents

1) 0.4 M Tris buffer, pH 7.0

2) 10 mM Sodium azide

3) 10 % TCA

4) 0.4 mM EDTA

5) 10 mM Hydrogen peroxide

6) 2 mM Glutathione

Procedure

To 2 ml of Tris buffer, 0.2 ml of EDTA, 0.1 ml of sodium azide and 0.5 ml of fruit extract were

added followed by 0.1 ml hydrogen peroxide, mixed well and incubated at 37˚ C for 10 minutes along

with the tube containing reagents except sample. After 10 minutes, the reaction was arrested by the

addition of 0.5 ml of 10% TCA, centrifuged and supernatant was assayed for glutathione by the

method of Ellman.

The activities are expressed as µg GSH consumed / minute.

APPENDIX – XIV

Estimation of superoxide dismutase

(Misra and Fridovich, 1972)

Principle

The assay of SOD is based on the inhibition of formation of NADH phenazine methosulphate -

nitroblue tetrazolium formazon, the extent of which can be assayed spectrophotometrically at 560 nm.

Reagents

1. Sodium pyrophosphate buffer (0.025M, pH 8.3)

2. Phenazine methosulphate (PMS) (186μM)

3. Nitroblue tetrazolium (NBT) (300μM)

4. NADH (700μM)

5. Glacial acetic acid

6. n-butanol

Procedure

Preparation of enzyme extract - Punica granatum aril and rind (0.5g) were ground with 3.0 ml of

sodium pyrophosphate buffer, centrifuged at 2000g for 10 minutes and the supernatant was used for

the assay.

Assay

The assay mixture contained in a total volume of 3.0 ml, 1.2 ml of sodium pyrophosphate

buffer, 0.1 ml of PMS, 0.3 ml of NBT, 0.2ml of enzyme preparations and 1.0 ml of water. NADH (0.2

ml) was added to start the reaction.

The assay mixture was incubated at 30º C for 90 seconds and the reaction was stopped by the

addition of 1.0 ml of glacial acetic acid. To this mixture, n-butanol (4ml) was added and allowed to

stand for 10 minutes and then centrifuged at 2000g for 5 minutes. The intensity of the chromogen in

the butanol layer was measured at 560nm against butanol as blank. The system devoid of enzyme

served as control. One unit of enzyme activity is defined as the amount of enzyme causing a 50%

reduction in NBT oxidation/minute.

APPENDIX – XV

Estimation of ascorbic acid

(Roe and Kuether, 1953)

Principle

Ascorbate is converted to dehydroascorbate by treatment with activated charcoal and bromine.

Dehydroascorbic 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

spectrophotomerically at 540 nm.

Reagents

1. 4% TCA

2. 9N H2SO4

3. 2% 2, 4 - dinitrophenyl hydrazine: 2 g of DNPH was dissolved in 100 ml of 9N H2SO4

4. 10% thiourea

5. 80% sulphuric acid

6. Stock standard solution: 100 mg of ascorbic acid was dissolved in 100 ml of 4% TCA

7. Working standard: 10 ml of the stock solution was diluted to 100 ml with 4% TCA

Procedure

About 1 g of the sample was homogenized in 4% TCA up to 10 ml and centrifuged at 2000 rpm

for 10 minutes. To the supernatant obtained, a pinch of activated charcoal was added, shaken well

and kept for 10 minutes. It was centrifuged once again and the charcoal residue was removed. The

volume of the clear supernatants was noted. 0.5 and 1.0 ml aliquots of this supernatant were taken

for the assay.

The assay volume was made up 2.0 ml with 4% TCA. 0.2 to 1.0 ml of the working standard

solution containing 20-100 µg of ascorbate respectively were pipetted out into clean dry test tube, the

volume of which were also made up to 2.0 ml with 4% TCA. To this, 0.5ml of DNPH reagent was

added to all the test tubes, followed by 2 drops of 10% thiourea solution and incubated at 37°C for 3

hours.

The osazones formed were dissolved in 2.5 ml of 85% sulphuric acid, in cold, drop by drop, with

no appreciable rise in temperature. To the blank alone, DNPH reagent and thiourea were added after

the addition of H2SO4. The tubes were incubated for 30 minutes at room temperature and the

absorbance was read spectrophotometrically at 540 nm. The content of ascorbic acid in the sample

was calculated using the standard graph.

APPENDIX – XVI

Estimation of α-tocopherol (Emmerie -Engel method, 1938 as described by Rosenberg, 1992)

Principle

Tocopherol can be estimated using Emmerie – Engel reaction which is based on the reduction

of ferric to ferrous ions by tocopherols, which then forms a red colour with 2, 2‟-dipyridyl. Tocopherol

and carotenes are first extracted with xylene and the extractives are read at 460 nm to measure

carotenes. A correlation is made for these after adding ferric chloride and reading at 520 nm.

Reagents

1. Absolute alcohol

2. Xylene

3. 2, 2‟- dipyridyl

4. Standard solution:

10 mg of α-tocopherol was dissolved in 10 ml absolute alcohol (91 mg of α -

tocopherol is equivalent to 100 mg of tocopherol acetate).

Extraction of fruit sample

The sample was homogenized with water in a blender. 2.5 g of the homogenised sample was

accurately weighed into a conical flask and 50 ml of 0.1 N H2SO4 was slowly added without shaking.

The mixture was stoppered and allowed to stand overnight. On the following day, the contents of the

flask were shaken vigorously and filtered through Whatmann No. 1 filter paper discarding the initial

10 – 15 ml of filtrate. Aliquots of the filtrate were used for the estimation.

Procedure

Into 3 stoppered centrifuge tubes (test, standard and blank), 1.5 ml of extract, 1.5 ml of

standard and 1.5 ml of water were added respectively. To the test and blank 1.5 ml of ethanol and to

the standard, 1.5ml of water was added. Then, 1.5 ml xylene was added to all the test tubes,

stoppered, mixed well and centrifuged. 1.0 ml of xylene layer was transferred into another stoppered

tube, taking care not to include any other ethanol or protein. 1.0 ml of 2, 2‟- dipyridyl reagent was

added to each tube, stoppered and mixed. 1.5 ml of the mixture was pipette out into colorimeter

cuvettes and the extinction of the test and standard was read against the blank at 460nm. For the

blank, 0.33 ml of ferric chloride solution was added.

The amount of vitamin E can be calculated using the formula,

Reading at 520 nm – Reading at 460 nm Amount of tocopherols in µg = Reading of standard at 520 nm × 0.29 × 15

APPENDIX – XVII

Estimation of tannins

(Schanderl, 1970)

Tannins are important polyphenolic compounds that act as antioxidants. 0.5 g of the sample

was weighed in a 250 ml conical flask and 75 ml of water was added to it. The flask was gently heated

first and then boiled for 30 minutes. The mixture was centrifuged at 2000 rpm for 20 minutes. The

supernatant was collected and made upto 100 ml and 1 ml of the same was transferred to a 100 ml

volumetric flask containing 75 ml alcohol, 5 ml of commercially available Folin‟s reagent, 10 ml of 35%

sodium carbonate were added and diluted to 100 ml with water. It was mixed well and the absorbance

was read at 700 nm after 30 minutes. The entire procedure was followed with a blank (water) and

standard (20-100 μg tannic acid).

APPENDIX – XVIII

Estimation of total phenols

(Malick and Singh, 1980)

Principle

Phenols react with phosphomolybdic acid in Folin - Ciocalteau reagent in alkaline medium and

produce blue coloured complex (molybdenum blue), which is read in a spectrometer at 650 nm.

Reagents

1. 80% ethanol

2. Diluted Folin – Ciocalteau reagent

3. 20% Sodium carbonate

4. Stock solution – 100 mg of gallic acid was made up with 100 ml distilled water

5. Working standard – 10 ml of stock standard was diluted to 100 ml. 1.0 ml of this contains 100

µg of gallic acid.

Procedure

Preparation of fruit extract

Preweighed fruit sample (0.5 g) was ground in 5 ml of 80% ethanol. The homogenate was

centrifuged at 10,000 rpm for 20 minutes. The supernatant was collected and the residue was re-

extracted again. After repeated centrifugation, the supernatants were collected and pooled. The

ethanol was evaporated and the residue was dissolved in distilled water and used further.

Estimation

Aliquots (0.2 to 2.0 ml) of the standard gallic acid solution were made up to 3 ml with distilled

water. Folin-Ciocalteau (0.5 ml) reagent was added to each test tube. After 3 minutes, 2.0 ml of 20%

sodium carbonate was added and mixed thoroughly. The tubes were heated in a boiling water bath for

exactly one minute and allowed to cool at room temperature. The blue colour developed was recorded

at 650nm against a reagent blank. The concentration of phenols in the sample was calculated from

the standard curve and expressed as mg phenols / g fruit.

APPENDIX – XIX

Estimation of flavonoids

(Zhishen et al., 1999)

Reagents

1. 5% sodium nitrite

2. 10% Aluminium chloride

3. 1mM sodium hydroxide

4. Standard solution: 0.011g of catechin dissolved in 100ml of water (110Ug/ml).

Procedure

0.1ml of methanolic extracts of fruit sample was added to 0.3ml of distilled water. To this

0.03ml of 5% sodium nitrite was added to the tubes and incubated for 5 minutes. To this 1mM sodium

hydroxide (0.2ml) was added and made up to 1ml with distilled water. The absorbance reading at

510nm was noted. The final absorbance of each sample was compared with a standard curve made

from catechin. From the standard graph, the amount of flavonoids present on the sample was

calculated.

APPENDIX – XX

Determination of Membrane Stability

(Shinde et al., 1999)

The membrane stabilizing activity of the extracts was assessed by using hypotonic solution-

induced and heat-induced human RBC haemolysis. To prepare the erythrocyte suspension, whole

blood was obtained from human and was taken in syringes (containing anticoagulant EDTA)

through intra venous method. The blood was centrifuged and blood cells were washed three times

with solution (154 mM NaCl in 10 mM sodium phosphate buffer (pH 7.4)) through centrifugation for

10 min at 3000 rpm.

Hypotonic solution induced haemolysis

The test sample consisted of stock erythrocyte (RBC) suspension (0.5 ml) mixed with 5 ml

of hypotonic solution (50 mM NaCl) in 10 mM sodium phosphate buffered saline (pH 7.4)

containing either the extract (1.0 mg/ml) or acetyl salicylic acid (0.1mg/ml). The control sample

consisted of 0.5 ml of RBCs mixed with hypotonic-buffered saline alone. The mixture was incubated

for 10 min at room temperature, centrifuged for 10 min at 3000 rpm and the absorbance of the

supernatant was measured at 540 nm.

The percentage inhibition of either haemolysis or membrane stabilization was calculated

using the following equation -

% Inhibition of haemolysis = 100 x (OD1 – OD2 / OD1),

OD1 = optical density of hypotonic-buffered saline solution alone (control)

OD2 = optical density of test sample in hypotonic solution.

Heat induced haemolysis

Isotonic buffer containing aliquots (5 ml) of the different extracts were put into two

duplicate sets of centrifuge tubes. The vehicle, in the same amount, was added to another tube as

control. Erythrocyte suspension (30 µL) was added to each tube and mixed gently by inversion. One

pair of the tubes was incubated at 54oC for 20 min in a water bath, while the other pair was

maintained at 0-5oC in an ice bath. The reaction mixture was centrifuged for 3 min at 1300 rpm

and the absorbance of the supernatant was measured at 540nm. The percentage inhibition or

acceleration of hemolysis in tests was calculated according to the equation:

% Inhibition of hemolysis = 100 x [1- (OD2 – OD1 / OD3 – OD1)]

OD1 = optical density of unheated test sample

OD2 = optical density of heated test sample

OD3 = optical density of heated control sample

APPENDIX – XXI

Estimation of Total Cholesterol

CHOD – POD Method

(Allain et al., 1974)

Principle

Cholesterol esterase (CHE) hydrolyses cholesterol ester to free cholesterol which is oxidized by

the cholesterol oxidase (CHOD) to 4-cholestenone and hydrogen peroxide. Hydrogen peroxide

formed reacts with 4-amino antipyrine and phenol in the presence of peroxidase to produce pink

colored compound called quinonimine

Cholesterol esters + H2O Cholesterol + fatty acid

Cholesterol + O2 4 - cholestenone + H2O2

2H2O2 + phenol + 4- aminophenzone Quinonimine + 4H2O

The intensity of the color formed is proportional to the cholesterol concentration in the sample. Reagent

Cholesterol standard: 200 mg/dl.

Procedure

One ml of the cholesterol reagent was pipetted out into a clean dry test tube and 20 µl of serum

sample was added to it. Standards were prepared by adding 1ml of reagent to 20 µl of the cholesterol

standard. It was then mixed well and incubated at 37˚ C for 10 minutes. The absorbance of the

samples and calibrator were measured against the blank at 505 nm.

Absorbance of test Total Cholesterol (mg/dl) = x 200 Absorbance of standard

CHE

CHOD

POD

APPENDIX – XXII

Estimation of Protein

(Lowry et al., 1951)

Principle

The aminoacid tyrosine and tryptophan present in the protein will react with the Folin-

Ciocalteau reagent. By the reduction of phosphomolybdic acid and phosphotungstic components it will

produce blue colour. Also the colour developed by the biuret reaction of the protein with the alkaline

cupric tartarate is measured by Lowry‟s method.

Reagents

1. Solution A: 1 % Copper sulphate

2. Solution B: 2% Sodium potassium tartarate.

3. Solution C: 2% Sodium carbonate in 0.1 N NaOH.

4. Solution D: Mixed just before use, 1 ml of solution A, 1 ml of solution B and 100 ml of

solution C.

5. Solution E: Folin-Ciocalteau reagent (Dilute the commercial reagent with an equal volume of

water on the day of use. This is a solution of sodium tungstate and sodium molybdate in

phosphoric and hydrochloric acids).

Procedure

A sample of 500 mg was extracted with 5-10 ml of 0.1M potassium phosphate buffer (pH 7.4).

It was centrifuged and an aliquot was pipetted out and made upto 1.0 ml with 0.1N NaOH. Standard

bovine serum albumin solution (40-200 µg) was also pipetted out and made upto 1.0 ml with 0.1N

NaOH. To this, 5 ml of alkaline copper reagent was added to all the tubes and allowed to stand for 10

minutes. Folin-Ciocalteau reagent (0.5 ml) was added to each tube and mixed well. The tubes were

allowed to stand for 30 minutes at room temperature. The blue colour developed was measured at

660 nm.

APPENDIX – XXIII

Estimation of HDL cholesterol

(Burstein et al., 1970)

Principle

Chylomicrons, LDL and VLDL (low and very low density lipoproteins) are precipitated from serum

by phosphotungstate in the presence of divalent cations such as magnesium. The HDL cholesterol

remains unaffected in the supernatant and is estimated using cholesterol reagent.

Phosphotungstate Serum / Plasma (LDL + VLDL + Chylomicrons) + HDL (supernatant) Mg2+ (precipitate)

Reagents

1. Cholesterol Reagent

2. HDL Cholesterol Standard (25 mg/dl)

3. Precipitating reagent

Procedure

Step – I

Precipitation of VLDL, LDL and chylomicrons

0.25 ml of serum and 0.5 ml of precipitating reagent were pipetted out into a clean and dry test tube. It

was mixed well and allowed to stand at room temperature for 10 minutes. It was then centrifuged for

10 minutes at 4000 rpm and the clear supernatant was used for HDL-cholesterol estimation.

Step – II

Assay of HDL cholesterol

1 ml of cholesterol reagent was pipette out in clean dry test tubes labelled blank (B), standard (S)

and test for HDL-Cholesterol (T). 0.05 ml of the supernatant from step - I was added to test (T). It was

mixed well and incubated at 37˚C for 10 minutes or (30˚C) for 12 minutes. The absorbance of the

sample and calibrator were measured against the blank at 505nm.

Calculation

Absorbance of Test HDL Cholesterol concentration (mg/dL) = x 25 x dilution factor Absorbance of standard

APPENDIX – XXIV

Screening of flavonoids using HPTLC

(Wagner et al., 1996)

HPTLC is a valuable tool for the investigation of herbal products with respect to different

aspects of their quality. The advantage of HPTLC over other techniques is that large number of

samples can be simultaneously analyzed using small volume of mobile phase unlike HPLC, thus

lowering analysis time and cost per analysis.

Instrumentation and Extraction

The fruit samples were centrifuged at 3000rpm for 5 minutes. The supernatant was collected

and used as test solution for HPTLC analysis. 5µl of the test solution and 5µl of standard solution was

loaded as 5mm band length in the 4 x 10 Silica gel TLC plate using a Hamilton syringe and CAMAG

LINOMAT 5 instrument. The samples loaded plate was kept in TLC twin trough developing chamber

(after saturation with solvent vapor) with respective mobile phase and the plate was developed in the

respective mobile phase up to 90mm.

The developed plate was dried by hot air to evaporate solvents from the plate. The plate

was kept in Photo-documentation chamber (CAMAG REPROSTAR 3) and the images were captured

in visible light, UV 254nm and UV 366nm. After derivatization with the appropriate reagents, the plate

was photo-documented in visible light and UV 366nm mode using photo-documentation chamber.

Finally, the plate was fixed in the scanner stage and scanning was done at UV 254nm. The peak

table, peak display and peak densitogram were noted.

APPENDIX – XXV

Extraction of rutin

(Hamad, 2012)

Principle

This method of extraction depends on the differences in solubility between glycosides and its

aglycone. The solubility of the glycoside and aglycones decreases after concentrating the aqueous –

alcoholic extract. Therefore they precipitate out of the solution and this mixture of precipitates are

further fractionated by dissolving the aglycone in the chloroform: ethylacetate: ethanol mixture leaving

the glycoside fraction alone which is later dissolved in hot methanol.

Procedure

The aril and rind of Punica granatum was extracted by soxhlet apparatus with 250 ml of 80%

ethanol till exhaustion. The extract was filtered and concentrated by evaporation under vacuum to

about 10 ml then mixed with 25 ml distilled water and extracted with petroleum ether (50ml x 3), then

with chloroform (50ml x 3).

After extraction, the aqueous layer was collected and left to stand in a cold place for 72 hours;

a yellow precipitate separated out of the solution. The precipitate was filtered and washed with a

mixture of chloroform: ethylacetate : ethanol (50:25:25). The undisolved part of the precipitate was

evaporated to dryness to give yellow powder.

APPENDIX – XXVI

Determination of UV absorption peaks

The isolated rutin was dissolved in methanol and its UV absorption peaks were determined

and compared with that of the standard rutin.

APPENDIX – XXVII

High Performance Liquid Chromatography (HPLC)

(Ashok and Saini, 2013)

HPLC analysis

The described HPLC procedure could be useful for the qualitative and quantitative analysis of

flavonoids in plant materials.

HPLC was performed on a liquid chromatograph HP1090 (Hewlett-Packard) with photodiode-

array detector. C18 – Reverse phase column (25cm × 4.6 mm length) with a 5 μm particle size was

used at the flow rate of 1 ml/min. The mobile phase was methanol with water 1:1. The injected volume

was 5 μl of methanolic solution of extracts and standards. The spectra were acquired at 280 and 360

nm. The identification of compounds was performed by comparison with retention time of pure

standards.

APPENDIX – XXVIII

Surface Plasmon Resonance (SPR)

Surface plasmon resonance was performed on a Biacore 2000 instrument from GE

Healthcare. All experiments used a CM5 sensor chip, which contains a carboxymethyl dextran

surface, onto which proteins were immobilised. All HBS-EP, a HEPES buffer containing EDTA and

Surfactant P20, was used as a running buffer.

Protein Immobilisation by Amine Coupling

All proteins were immobilised to the chip surface by amine coupling, whereby succinimide

esters react spontaneously with primary amine groups to link covalently to the dextran matrix. Binding

may occur at NH2 of amino acid side chains as well as the N-terminus of the protein. Prior to

immobilisation of the desired protein, pH scouting was performed to establish the most effective pH,

which turned out to be pH 4.0 for all immobilised proteins. Preparing the dextran surface of the CM5

sensor chip for amine coupling first requires reactions with 0.2 M 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide (EDC) and 0.05 M N-hydroxysuccinimide (NHS). This activates the

carboxyl groups on the surface to give reactive succinimide esters. The succinimide esters react

spontaneously with amine groups, allowing direct immobilisation of molecules. The protein was

prepared to a concentration of 50 μg/ml in HBS-EP buffer. The standard protocol for amine coupling

was followed using the manufacturer‟s instructions. Once the protein immobilisation was complete, 1M

ethanolamine was used to deactivate excess reactive groups.

Binding Analysis

All protein samples used as analyte for binding analysis were prepared in HBSEP running

buffer to a volume of 370 μl for 2 replications. Prior to analysis, all flow cells were normalised using

40% glycerol to compensate for variations in the reflectance characteristics between individual sensor

chips. The procedure followed the manufacture‟s instructions. Analyte was injected at a flow rate of 50

μl/min, which was fast enough to prevent mass transport effects. Regeneration of the chip after each

analysis was performed using 400 μl of 50 mM NaOH.

Analysis of Biacore Data

BIAevaluation version 3.1 was used to process and analyse the raw sensorgram data.

Sensorgrams were baseline corrected using the Y-transform function, by zeroing the signal in a region

before the start of injection. Only the association and dissociation phases of each sensorgram were

used for binding analysis. All curve fitting used simultaneous ka/kd calculations. Fitting simultaneous

ka/kd models to experimental data involves both generation of the rate equations for the model used

and finding the values for parameters in the rate equations that best fit the experimental data.

APPENDIX – XXIX

Enumeration of Red Blood Corpuscles

(Sanderson and Phillips, 1981)

The total erythrocyte count was determined accurately by diluting a measured quantity of red

blood corpuscles with a fluid isotonic solution.

Reagents

Red blood cell diluted fluid (Hayem‟s fluid) – 5g of sodium sulphate, 1g of sodium chloride,

0.5g of mercuric chloride were dissolved in 200 ml of distilled water.

Procedure

Blood was sucked exactly up to the 20µl mark in the RBC pipette and the diluting fluid was

drawn immediately up to the mark and mixed thoroughly. It was left for 2-3 minutes for proper mixing.

The Neubauer counting chamber was placed along with the cover glass in position. The capillary stem

of the pipette was emptied which contains only the diluting fluid. This was done by discarding first 3-5

drops.

Charging of the Counting Chamber

One drop of diluted blood was released into the groove of the Neubauer counting chamber. It

was left for the cells to settle for 2 – 3 minutes and the counting chamber was put under the

microscope and the ruled area was located.

Erythrocytes were counted in the 5 squares of the counting area of 1 mm square. The number

of cells in the 4-corner square was counted.

Calculation

The total number of cells found in 5 groups of 16 squares is multiplied by 10,000 to give the

number of cells in millions/mm of blood.

APPENDIX – XXX

Estimation of haemoglobin

(Drabkin and Austin, 1932)

Blood is diluted with an alkaline solution of potassium cyanide and potassium ferricyanide,

where haemoglobin is oxidized to methaemoglobin which is measured colorimetrically at 540nm.

Reagents

1. Drabkin‟s reagent: This reagent contains 0.05g of potassium cyanide, 0.20g of potassium

ferricyanide and 1g of sodium bicarbonate in 1 litre of distilled water.

2. Cyanmethaemoglobin standard: This was obtained commercially and had a concentration of 16g/dl.

Procedure

The reaction mixture contained 5.0 ml of Drabkin‟s reagent and 0.02ml of blood. The reaction

mixture was kept at room temperature for 5 minutes to ensure the completion of the reaction and read

at 540nm against a reagent blank containing reagent alone.

The haemoglobin content was expressed as g/dl blood.

APPENDIX – XXXI

Determination of Platelet count

(Sanderson and Phillips, 1981)

Reagents

Dacies fluid: This was prepared by dissolving 5.0g of sodium citrate and 1 ml of 40%

formaldehyde and made upto 100 ml with distilled water. To 19 ml of this solution 1 ml of 0.2% brilliant

cresyl blue solution was added just before use. The solution was filtered and used.

Procedure

Venous blood collected with EDTA was used for platelet count. 0.05 ml of sample was diluted

with 0.95 ml of Dacies fluid and mixed well and using a narrow bore Pasteur pipette, the counting

chamber was filled with the diluted blood. The cells were allowed to settle to the bottom of the

chamber for 15 min to prevent from drying, the chamber was placed in a petridish, which contained a

pieces of wet filter paper.

Using the 40X objective with reduced condenser aperture the platelets were counted in 1/5

sq.mm-5

of the small squares of the large center square. From this the number of platelets in cu.mm of

blood was calculated as,

Cells x Blood dilution x Chamber depth Platelet Count = Area of chamber counted

Platelet count is expressed as number of cells/mm.

APPENDIX – XXXII

Enumeration of White Blood Corpuscles

(Sanderson and Phillips, 1981)

WBC diluting fluid or Turk‟s fluid was used as the diluents which can destroy RBC‟S.

Reagents

WBC diluting fluid was prepared by mixing

1. Glacial acetic acid

2. Gentian violet 1%

3. Water 95 ml

Procedure

The method of counting is similar to RBC counting except that the count is made in 4 large

(1 mm) cover squares of the Neubauer counting chamber.

Calculation

The total number of cells in 4 squares is multiplied by a factor of 2500 to give the count/mm of

blood.

APPENDIX – XXXIII

Estimation of alanine transaminase (ALT)

(Reitman and Frankel, 1957)

Principle

SGPT catalyses the reversible transfer of amino group from L-alanine to alpha ketoglutarate

with the formation of pyruvate and glutamate. The pyruvate so formed is allowed to react with

2-4 dinitrophenylhydrazine (DNPH) to produce 2, 4- dinitrophenyl hydrazone derivative, which is

measured photometrically.

SGPT (pH 7.4) α – Keto glutarate + L Alanine L - Glutamate + Pyruvate

Alkaline Medium

Pyruvate + 2,4 DNPH 2,4 dinitrophenyl hydrazine

(Brown Colored)

Reagents

1. Tris buffer, pH 7.5 - 100mmol/l

2. L-alanine - 500mmol/l

3. 2-oxoglutarate - 15mmol/l

4. 2, 4 dinitrophenyl hydrazine reagent

5. Working sodium hydroxide (4N)

Procedure

Five hundred microlitre of buffered substrate was incubated at 37°C for 3 minutes and 0.1ml

of serum was added, mixed well and incubated at 37°C for 60 minutes. Then 0.5ml of DNPH reagent

was added, mixed well and kept at room temperature for 20 minutes and 0.5ml of 4N working sodium

hydroxide was added and kept at room temperature for 10 minutes. Blank and standards were also

processed in a similar way and the absorbance was measured spectrophotometrically at 505 nm.

Activity of SGPT was expressed as U/L.

APPENDIX – XXXIV

Estimation of alkaline phosphatase (ALP)

(King, 1965)

Principle

Alkaline phosphatase is an enzyme which catalyses the splitting of phosphoric acid from

certain monophosphoric esters. In this method disodium phenyl phosphate was hydrolyzed with the

liberation of phenol and formation of sodium phosphate. The amount of phenol formed was estimated

in a spectrophotometer at 650nm.

Reagents

1. Disodium phenyl phosphate (0.01M) – 1.09g of disodium phenyl phosphate was dissolved in

water and made up to 500ml. It was then boiled, cooled and little chloroform was added and

kept in refrigerator (Solution A).

2. Sodium carbonate-sodium bicarbonate buffer (0.1M) - 3.18g of anhydrous sodium carbonate

and 1.68g of sodium bicarbonate was dissolved in water and made up to 500ml (Solution B).

3. Buffered substrate for use - Equal volume of solution A and solution B was mixed which has

pH of 10.

4. Tricholoro acetic acid (20%) – Acid molybdate reagent - 5g of ammonium molybdate

dissolved in 5N sulphuric acid.

5. 1, 2, 4 – ANSA : 0.25% of 1,2,4 – ANSA was prepared by adding 0.5g of dry powder ANSA to

190 ml of 15% sodium bisulphate and 5ml of 20% sodium sulphite stoppered the bottle and

shaken until it dissolved.

6. Stock Phosphate solution – 2.194g of pure potassium dihydrogen phosphate was dissolved in

water and made up to 500 ml. Few drops of chloroform was added to it (1mg/1ml of

phosphate).

7. Working standard: Two ml of stock standard was diluted to 500ml.

Procedure

Six ml of buffered substrate was pipette out in test tube and placed in water bath at 370C for

few minutes. Then, 0.3ml of serum was added, mixed well and incubated for 15 minutes. At the same

time control and blank were also kept. For blank 0.3ml of water was added to 6ml buffered substrate.

For control 0.3ml of serum was added to 6ml of distilled water. Later, 1.2ml of 20% TCA was added

and shaken well. 5ml of the filtrate was taken in separate test tubes. To the blank and control, 0.8ml of

acid molybdate was added followed by 0.2ml of ANSA. It was then mixed well and allowed to stand for

10 minutes at 370C and the colour developed was read at 650nm.

1.0 to 4.0 ml of standard solution was pipetted out and made up to 5ml with distilled water.

0.8ml of acid molybdate was added followed by 0.2ml of ANSA. Standards were also read at 650nm.

Alkaline phosphatase activity in serum was expressed as U/L. The activity in tissue homogenate was

expressed as: mole of phenol liberated/min/mg protein.

APPENDIX – XXXV

Estimation of aspartate transaminase (AST)

(Reitman and Frankel, 1957)

Principle

Serum glutamine oxaloacetic transaminase catalyses the reversible transfer of an amino

group from aspartate to α-keto glutarate forming glutamate and oxaloacetate. SGOT catalyses the

following reaction:

SGOT (pH 7.4)

L – Aspartate + α – Keto glutarate Oxaloacetate + L – Glutamate

Alkaline Medium

Oxaloacetate + 2,4 DNPH 2,4 dinitrophenyl hydrazine

(BrownColored)

Reagents

1. Tris buffer, pH 7.5 - 100mmol/l

2. L-aspartate - 500mmol/l

3. 2-oxoglutarate - 15mmol/l

4. 2, 4 dinitrophenyl hydrazine reagent

5. Working sodium hydroxide (4N)

Procedure

Five hundred microlitre of buffered substrate was incubated at 37°C for 3 minutes and 0.1 ml

of serum was added, mixed well and incubated at 37°C for 30 minutes. Then 0.5ml of 2, 4 -

dinitrophenyl hydrazine (DNPH) reagent was added, mixed well and kept at room temperature for

20 minutes and 0.5ml of 4N working sodium hydroxide was added and kept at room temperature for

10 minutes. Blank and standards were also processed in a similar way and the absorbance was

measured spectrophotometrically at 505 nm. Activity of SGOT was expressed as U/L.

APPENDIX – XXXVI

Estimation of creatinine

(Owen et al., 1954)

Principle

Creatinine in alkaline medium react with picrate ions to form yellow orange complex whose

color intensity is measured at 492 nm.

Reagents

1. Picric acid – 35 mmol/l

2. Sodium hydroxide – 0.32 mmol

3. Creatinine standard – 2 mg/dl

4. Sodium tungstate

5. Sulphuric acid

Procedure

To 0.2 ml of serum 3 ml of water, 1 ml of 10% sodium tungstate and 2 ml of 2/3 N sulphuric

acid were added. It was then kept for 10 min and centrifuged. To 3.0 ml of supernatant, 1 ml of 0.04 M

picric acid solution and 1 ml of 0.75 N sodium hydroxide was added and allowed to stand for 20 min.

Blank and standards (10-50 μg) were treated similarly. The colour developed was read at 492 nm.

APPENDIX – XXXVII

Estimation of urea

(Netelson, 1957)

Principle

Urea is hydrolysed to ammonia and carbon dioxide in the presence of urease. Ammonia

reacts with 2 – oxoglutarate in the presence of NADH, which is oxidised and measured at 340nm.

Reagents

1. 10% TCA

2. Reagent A - 50 mg of ferric chloride, 0.2 ml of water, 1 ml of O-phosphoric acid and 2.5 ml of

water

3. Reagent B – 50 ml of concentrated sulphuric acid and 450 ml of water

4. Reagent C – 1 g of diacetyl monoxime in 50 ml water

5. Reagent D – 250 mg of thiosemicarbazide in 50 ml of water

6. Reagent I – 0.25 ml of Reagent A was mixed with 500 ml of Reagent B

7. Reagent II – 33.5 ml of Reagent C was mixed with 33.5 ml of Reagent D & diluted to 500 ml.

Procedure

To 0.2 ml of blood, 1.8 ml of 10 per cent TCA was added, mixed well and centrifuged after 10

minutes. The supernatant (0.5 ml) was taken and the volume was made upto 3 ml with water. Then 2

ml of reagent I was mixed with 2 ml of fresh reagent II. It was then mixed well, stoppered with

marbles and heated vigorously in a boiling water bath for 20 min. Blank and standards (10-50 μg)

were treated similarly. The tubes were removed, cooled and read against the blank at 340 nm.

APPENDIX – XXXVIII

Estimation of D-dimer

(Marder and Francis, 1983)

Fibrin degradation products (FDP), which are highly heterogeneous soluble fragments, are a

result of 2 simultaneous phenomena:

Fibrinogen is coagulated by thrombin and factor XIIIa to form stabilized fibrin,

The fibrin clot is dissolved by plasmin into soluble fragments released into the blood. The

terminal product of fibrinolysis is D-dimer.

Principle

The assay principle combines a two-step enzyme immunoassay sandwich method with a final

fluorescent detection (ELFA). The Solid Phase Receptacle (SPhR) serves as the solid phase with an

anti-FDP monoclonal antibody adsorbed on its surface.

Procedure

The sample was taken and transferred into the well containing an alkaline phosphatase –

labeled anti-FDP monoclonal antibody. The sample mixture was cycled in and out of the SPhR

several times to increase the reaction speed. The antigen binds to antibodies coated on the SPhR and

to the conjugate forming a “sandwich”. The remaining free antigen sites were saturated by cycling the

conjugate in the fifth well of the strip in and out of the SPhR. Unbound components were eliminated

during the washing steps.

Two detection steps were then performed successively. During each step, the substrate (4-

Methyl-umbelliferyl phosphate) was cycled in and out of the SPhR. The conjugate enzyme catalyzes

the hydrolysis of this substrate into a fluorescent product (4-Methyl-umbelliferone), the fluorescence of

which is measured at 450 nm. The intensity of the fluorescence is proportional to the concentration of

antigen present in the sample.

APPENDIX – XXXIX

Estimation of Fibrinogen and Clotting time

(Shaw, 1977)

Principle

Fibrinogen was estimated by measuring the clotting time of dilute plasma following the

addition of thrombin.

Reagents

1. Thrombin Reagent

The reagent contains a lyophilized preparation of approximately 100 NIH units/ml of bovine

thrombin with added stabilizers.

2. Fibrinogen Calibrator

The calibrator consists of lyophilized citrated normal human plasma assayed for fibrinogen using a

functional clotting assay.

3. Owren‟s Veronal Buffer

The buffer contains 28.4 mM barbital, 0.125 M sodium chloride and 0.05% sodium azide.

Specimen collection and handling

Specimen: Plasma obtained from whole blood collected with sodium citrate as an

anticoagulant is the specimen of choice. The concentration of the sodium citrate should be 3.8%

(0.129 M) or 3.2% (0.109 M).

Procedure

I. Calibration

1. All the reagents were allowed to equilibrate to room temperature (15-30°C).

2. Dilutions of Fibrinogen Calibrator were prepared as 1:5, 1:10, 1:20, 1:30 and 1:40 using Owren‟s

Veronal Buffer as follows:

1:5 1:10 1:20 1:30 1:40

Buffer (ml) 0.8 0.9 1.9 2.9 3.9

Fibrinogen Calibrator (ml) 0.2 0.1 0.1 0.1 0.1

a. 0.2 ml of calibrator dilution was incubated for 2 minutes at 37°C not exceeding 5 minutes.

b. 0.1 ml of thrombin reagent (room temperature) was added to immediately initiate the timed reaction.

c. Clotting time was calculated

d. The average clotting time obtained was plotted against the respective fibrinogen II. Testing

a. Sample plasma and controls were diluted in the ratio of 1:10 with Owren‟s Veronal Buffer (1 part

specimen and 9 parts buffer).

b. 0.2 ml of diluted specimens was added into a specimen cup and incubated for 2 minutes at 37°C

not exceeding 5 minutes.

c. 0.1 ml of thrombin reagent (room temperature) was added to initiate the timed reaction.

d. The clotting time was calculated. The concentration of fibrinogen was read from the standard curve

and was expressed as mg/dl.

APPENDIX – XL

Estimation of tissue plasminogen activator

(Kit method - Roche)

Tissue-type plasminogen activator (tPA) is one of two major physiologic activators of

plasminogen in plasma. The activation of plasminogen by tPA is dependent on the presence of a fibrin

cofactor. The binding of both tPA and plasminogen to fibrin is mediated in part through lysine binding

sites within the kringle structures of both enzyme and substrate, but also through the finger domain of

tPA. Activation of plasminogen by tPA occurs by cleavage after residue Arg560 to produce the two-

chain active serine protease plasmin.

Principle

Affinity-purified antibody to tPA is coated onto the wells of a microtitre plate. Any remaining

binding sites on the plastic wells are blocked with an excess of bovine serum albumin. The plates are

washed and plasma or other fluids containing tPA are applied. The coated antibody will capture the

tPA in the sample. After washing the plate to remove unbound material, a peroxidase conjugated

second antibody to tPA is added to the plate to bind to the captured tPA. After washing the plate to

remove unbound conjugated antibody, the peroxidase activity is expressed by incubation with

O-Phenylenediamine (OPD). After a fixed development time the reaction is quenched with the addition

of H2SO4 and the colour produced is quantified using a microplate reader. The colour generated is

proportional to the concentration of tPA present in the sample.

Reagents

1. Capture antibody (TPA–EIA-C)

2. Detecting antibody (TPA-EIA-D)

3. Coating Buffer: 50 mM Carbonate: 1.59g of Na2CO3 and 2.93g of NaHCO3 up to 1 litre. Adjust pH

to 9.6.

4. PBS (base for wash buffer and blocking buffer): 8.0g NaCl, 1.15g Na2HPO4, 0.2g KH2PO4 and

0.2g KCl, up to 1 litre. Adjust pH to 7.4, if necessary.

5. Blocking Buffer: PBS-BSA (1%, w/v) Dissolve 2.5 g of Bovine Serum Albumin (Sigma-RIA grade)

in 200 ml of PBS. Adjust pH to 7.4, if required, then make up to 250 ml with PBS.

6. Sample Diluent (HBS-BSA-T20): 5.95g HEPES (free acid), 1.46 g NaCl, 2.5 g Bovine Serum

Albumin dissolved in 200 ml H2O. Add 0.25 ml of Tween- 20, check and adjust pH to 7.2 with

NaOH, then make up to a final volume of 250 ml with H2O.

7. Substrate Buffer (Citrate-Phosphate buffer - pH 5.0) 2.6g Citric acid and 6.9g Na2HPO4 up to a

final volume of 500 ml with purified H2O.

8. OPD Substrate (o-Phenylenediamine.2HCl) TOXIC!: Make up immediately before use. Dissolve

5mg OPD in 12 ml substrate buffer then add 12 µl 30% H2O2.

9. Stopping Solution: 2.5 M H2SO4 (Where stock sulphuric acid is 18 M, add 13.9 ml to 86 ml H2O).

Procedure

1. Coating of plates: Dilute the capture antibody 1/100 in coating buffer (in a polypropylene tube) and

immediately add 100 µl per well in the plate. Incubate for 2 hours at ambient temperature or

overnight at 2-80C.

2. Blocking: Empty contents of plate and add 150 µl of blocking buffer to every well and incubate for

60 minutes at 220C. Wash plate X 3 with wash buffer.

3. Preparation of tPA Reference Standards: Reconstitute vials of tPA standard and tPA/PAI-1

deficient plasma according to manufacturer‟s instructions. After reconstitution, dilute the tPA

standard into tPA/PAI-1 deficient plasma to achieve six reference standard plasmas with final tPA

concentrations of 50, 25, 12.5, 6.25, 3.13 and 1.56 ng/ml respectively.

4. Samples: Reference plasmas prepared in step 3 and test plasmas are diluted 1/4 and 1/8 in HBS-

BSA-T20 sample diluent. Samples should be run in duplicate. Apply 100 µl/well and incubate plate

at 220C for 90 minutes. Wash plate X 3 with wash buffer.

5. Detecting Antibody: Dilute the detecting antibody 1/100 in HBS-BSA-T20 sample diluents and

apply 100 µl to each well. Incubate plate at 220C for 90 minutes. Wash plate X 3 with wash buffer.

6. OPD Substrate: Apply 100 µl of freshly prepared OPD substrate to every well. Allow colour to

develop for 5-10 minutes then stop colour reaction with the addition of 50µl/well of 2.5 M H2SO4.

The plate can be read at a wavelength of 490 nm.

APPENDIX – XLI

Estimation of Creatine phosphokinase

(Lott and Stang, 1980)

Principle

The creative phosphokinase enzyme is a dimer composed of subunits derived from either

muscle (M) or brain (B).

Creatine phosphate + ADP Creatine + ATP

ATP + D- Glucose ADP + G6P

G6P + NADP

+ D-6-phosphogluconate + NADPH + H

+

The rate of the NADPH formation is directly proportional to the catalytic CK activity. It is

determined by measuring the increase in absorbance.

Reagents

R1 Imidazole - 58 mmol/l, pH 6.0; N-acetylcysteine - 40 mmol/l; EDTA - 3 mmol/l;

AMP - 10 mmol/l; diadenosine pentaphosphate - 24 µmol/l; NADP+ - 9.5 mmol/l; Mg

2+ - 20

mmol/l; D-glucose - 40 mmol/l; preservative; stabilizer.

R2 EDTA - 3 mmol/l; HK (yeast) - ≥600 µkat/l; G6PDH (microbial) - ≥600 µkat /L;

ADP -12 mmol/L; creatine phosphate - 180 mmol/L; N- methyl diethanol amine -

69 mmol/L; preservative; stabilizer; detergent.

Procedure

Wavelength (sub/main) 546 / 630 nm

Reaction direction Increase

Units U/L (µkat/L)

Reagent Pipetting Diluent (H2O)

R1 61 µL 38 µL

R2 20 µL -

Sample Sample volumes Sample dilution Diluent

Normal 3 µL -

Decreased 3 µL 15 µL135 µL

Increased 6 µL -

Calculation

Conversion factor: U/L x 0.0167 = µkat/L

HK

CK

G6PDH

APPENDIX – XLII

Estimation of C – Reactive Protein

(Wadsworth, 1977)

C-reactive protein (CRP) is an acute phase protein synthesized in the liver. Its rate of

synthesis increases within hours of acute injury or the onset of inflammation and may reach as high as

20 times the normal levels. Serum CRP concentration provides useful information in patients with

myocardial infarction there being an excellent correlation between peak levels of CRP and creatine

phosphokinase.

Principle

The turbidimetric immunoassay for the determination of C-reactive protein in human serum is

based on the principle of agglutination reaction.

Procedure

The test specimen is mixed with activation buffer and Quantia – CRP reagent and allowed to

react. Presence of CRP in the test specimen results in the formation of an insoluble complex

producing turbidity, which is measured at 340 nm. The increase in turbidity corresponds to the

concentration of CRP in the test specimen.

The calibrator dilution is as shown below:

Test tube No 1 2 3 4 5

Calibrator dilution No D1 D2 D3 D4 D5

Volume of saline (µl) - 100 375 880 940

Volume of calibrator (µl) 100 100 125 120 60

Concentration of CRP (mg/dl) 10 5 2.5 1.2 0.6

APPENDIX – XLIII

Determination of Bleeding Time

(Brown, 1993)

Apparatus

Blood lancet, spirit, pieces of filter paper, stop watch

Procedure

With usual aseptic precautions, a puncture wound was inflicted. It is to be more deep than

usual and should be done in a standard manner. The time of puncture and first appearance of the

blood was noted. The blood was gently blotted with a filter paper. Care should be taken not to press or

wipe the wound. It was repeated with a fresh piece of filter paper for every 10 seconds till no blood

appears on the paper. The number of filter paper that shows the blot of blood on it was counted.

Number of blot x 10 seconds will be bleeding time. Similar experiments were carried out on a healthy

animal and the result obtained was considered as a control value. Normal bleeding time is 1 – 5

minutes.

APPENDIX – XLIV

Determination of activated partial thromboplastin time

(Analyser – Liqucelin E)

The arrest of bleeding depends upon primary platelet plug formed along with the formation of

a stable fibrin clot. Formation of this clot involves the sequential interaction of a series of plasma

proteins in a highly ordered and complex manner and also the interaction of these complexes with

blood platelets and materials released from the tissues.

Principle

Cephaloplastin activates the coagulation factors of the intrinsic pathway of the coagulation mechanism in the presence of calcium ions. APTT is prolonged by a deficiency of one or more of these clotting factors of the intrinsic pathway and in the presence of coagulation inhibitors like heparin. Procedure

Blood was collected with trisodium citrate and was centrifuged immediately for 15 minutes at

3000 rpm (approximately 2000 g) and the plasma was transferred into a clean test tube. Obtained

Fresh Normal Plasma (FNP) must be tested within three hours of blood collection.

Additional reagents required

a. Fresh normal pooled plasma.

b. CaCl2 (0.025 mol/l)

c. Physiological saline

Calibration Curve Method (For determination of heparin concentration):

1. Dilute heparin (as used for treatment) with physiological saline to a concentration of 10 U/ml.

2. Mix 0.2 ml of 10 U/ml diluted heparin with 1.8 ml of FNP to give a heparin standard of 1U/ml

concentration.

3. Dilute the heparin standard as prepared above (1U/ml) with FNP as follows

Test tube No 1 2 3 4 5 6 7

Heparin Standard (1U/ml) in ml 0.5 0.4 0.3 0.2 0.1 0.1 -

FNP in ml - 0.1 0.2 0.3 0.4 0.9 0.5

Heparin concentration (U/ml) 1.0 0.8 0.6 0.4 0.2 0.1 -

APPENDIX – XLV

Determination of euglobulin clot lysis time

(Nordby et al., 1980)

The euglobulin clot lysis time is a test that reflects the overall fibrinolytic activity of plasma.

Principle and Method:

Venous blood is collected into chilled tubes containing trisodium citrate as an anticoagulant

and placed on ice. The sample is then centrifuged at 4°C and the plasma sample is collected, diluted

with acetic acid and incubated on ice for 15 minutes. A precipitate forms [the euglobulin fraction of

plasma] which contains plasminogen, plasminogen activators [primarily t-PA] and fibrinogen. The

supernatant is collected by centrifugation in refrigerated centrifuge at 4°C. The supernatant is

discarded and the precipitate is dissolved in buffer. This is then clotted with thrombin and the time to

clot lysis is determined by inspection every 15 minutes. A control plasma sample collected at the

same time must be run in parallel.

APPENDIX – XLVI

Assay of catalase in experimental rats

(Sinha, 1972)

To 0.9 ml of 0.01M phosphate buffer (pH 7.0), 0.1 ml of tissue homogenate and 0.4 ml of

0.2M H2O2 were added. Two ml of dichromate acetic acid mixture (1:3 ratio of potassium dichromate

was mixed with glacial acetic acid. From this 1 ml was diluted again with 4 ml acetic acid) was added.

The tubes were kept in boiling water bath for 10 min and the colour developed was read at 620 nm.

Standards of H2O2 in the range of 2-10 μM were taken and preceded as test with blank containing

reagent alone.

The activity is expressed as μM of H2O2 consumed / min / mg protein.

APPENDIX – XLVII

Assay of superoxide dismutase in experimental rats

(Kakkar, 1984)

Assay mixture contained 1.2 ml of sodium pyrophosphate buffer (0.025M, pH 8.3), 0.1 ml of

186 μM phenazine methosulphate, 0.3 ml of 300 μM NBT and 0.2 ml of 780 μM NADH, appropriately

diluted enzyme preparation and water in a total volume of 3 ml. Reaction was started by the addition

of NADH. After incubation at 30°C for 90 seconds, the reaction was stopped by the addition of 1 ml

glacial acetic acid. Reaction mixture was stirred vigorously and shaken with 4 ml of n-butanol. The

mixture was allowed to stand for 10 minutes, centrifuged and the butanol layer was collected. The

colour intensity of the chromogen in the butanol layer was measured at 560 nm against butanol. A

system devoid of the enzyme served as control.

One unit of enzyme activity is defined as the enzyme concentration, which gave 50 per cent

inhibition of NBT reduction in 1 min under the assay conditions and expressed as specific activity in

units/mg protein.

APPENDIX – XLVIII

Assay of glutathione peroxidase in experimental rats

(Rotruck et al., 1984)

To 2 ml of 0.4 M Tris buffer (pH 7.0), 0.2 ml of 0.4 mM EDTA, 0.1 ml of 10 mM sodium azide

and 0.5 ml of tissue homogenate were added to the mixture, 0.2 ml of 2 mM glutathione followed by

0.1 ml of 20 mM hydrogen peroxide were also added. The contents were mixed well and incubated at

37°C for 10 min along with a tube containing all the reagents except sample. After 10 min the reaction

was arrested by the addition of 0.5 ml of 10% TCA, centrifuged and the supernatant was assayed for

glutathione by the method of Moron et al. (1979).

The activity is expressed as μg of GSH consumed / min / mg protein.

APPENDIX – XLIX

Estimation of vitamin C in experimental rats

(Omaye et al., 1979)

Tissue homogenate (0.5 ml) was mixed thoroughly with 1.5 ml of 6% TCA and centrifuged for

20 min at 3500 g. The supernatant was shaken vigorously with a pinch of acid-washed Norit and

filtered. To 0.5 ml of the filtrate, 0.5 ml of DNPH reagent (2.0 g of DNPH was dissolved in 100 ml of

9N sulphuric acid. To this 4.0 g of thiourea was added and mixed) was added and mixed well. The

tubes were allowed to stand at room temperature for 3 hr. Removed, placed in ice-cold water and

added 2.5 ml of 85% sulphuric acid and allowed to stand for 30 min. A set of standards containing 10-

50 μg of ascorbic acid were taken and processed similarly with a blank. The absorbance was read at

530 nm.

APPENDIX – L

Estimation of reduced glutathione in experimental rats

(Moron et al., 1979)

Principle

Reduced glutathione (GSH) was measured by its reaction with 5,5'- dithiobis-2-nitrobenzoic

acid (DTNB) (Ellman‟s reaction) to give a yellow coloured compound that absorbs at 412nm.

Reagents

1. TCA (5%)

2. Sodium phosphate buffer (0.2M, pH 8.0)

3. DTNB (0.6M in 0.2M sodium phosphate buffer)

Procedure

One gram of the tissue was homogenized in 5% TCA to give a 20% homogenate. The

precipitated protein was centrifuged at 1000 rpm for 10 minutes. The homogenate was cooled on ice

and 0.1 ml of the supernatant was taken for the estimation. The volume of the aliquot was made upto

1 ml with 0.2M sodium phosphate buffer (pH 8.0).

Two ml of freshly prepared DTNB solution (0.6 mM in 0.2 M phosphate buffer, pH 8.0) was

added to the tubes and the intensity of the yellow colour formed was read at 412 nm in a

spectrophotometer after 10 minutes. The standards (2-10 nmoles GSH in 1.0 ml of 5 per cent TCA)

were also treated in a similar manner.

APPENDIX - LI

Histopathological Examination

(Culling, 1979)

The rats were sacrificed by cervical dislocation and an autopsy was carried out to obtain liver,

heart and tail of the rats. Tissue samples were taken and preserved in 10% formalin solution for a

minimum of one hour. Formalin was removed from the tissue samples with running water.

Dehydration of the fixed tissue was done by giving three changes of acetone (each 100 ml). Cleaning

of tissue from acetone was followed by three changes of xylene (each 500 ml) in a total duration of

three hours. Incubation of processed tissue in melted paraffin was done by two changes for 3-4 hours

in an incubator maintained at 58-60˚C. Embedding of the tissue in paraffin wax was then done by

immersing the tissue in molten paraffin and then cooling it to harden the paraffin. Sections of the

paraffin embedded tissue were done using a microtome adjusted to 1-3 μ thickness. The paraffin

sections were carefully taken on glass slides. The sections were then cleaned by immersing in xylene.

The sections were stained with hematoxylin and eosin stain and screened to evaluate the morphology

and cellular composition.