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APPENDICES

APPENDIX I

AUTHENTICATION OF PLANT MATERIAL

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APPENDIX II

QUALITATIVE ANALYSIS OF PHYTOCHEMICALS

ALKALOIDS

Dragendroff’s Test:

8g of Bi (NO3)3.5H2O was dissolved in 20 ml of HN03 and 2.72g of potassium

iodide was dissolved in 50ml of water. These were mixed and allowed to stand. When

KNO3 crystals out, the supernatant was decanted off and made up to 100ml with distilled

water. The alkaloids were regenerated from the precipitate by treating with Na2CO3

followed by extraction of the liberated base with ether.

0.5ml of alcoholic solution of Caralluma adscendens (Roxb.) extract was added to

2.0 ml of HCl. To this acidic medium, 1.0 ml of reagent was added. An orange red

precipitate produced immediately indicates the presence of alkaloids.

Wagner’s Reagent (Iodine-potassium iodide solution)

Mixed 1.2g of iodide and 2.0g of sulphuric acid and the solution was diluted to

100ml.10ml of alcoholic extract of Caralluma adscendens (Roxb.) was acidified by adding

1.5% v/v of HCl and a few drops of Wagner’s reagent. Formation of yellow or brown

precipitate confirmed the presence of alkaloid.

Meyer’s Reagent (Potassium mercuric iodide)

1.36g of mercuric chloride was dissolved in 60ml of distilled water and 5.0g of

potassium iodide was dissolved in 10ml of water. The two solutions were mixed and

diluted to 100ml with distilled water. To 1 ml of aqueous solution of Caralluma adscendens

(Roxb.), a few drops of reagent were added. Formation of white or pale precipitate showed

the presence of alkaloids.

FLAVONOIDS

In a test tube containing 0.5ml of alcoholic extract of Caralluma adscendens

(Roxb.), 5-10 drops of dilute HCl and small piece of ZnCl or Magnesium, were added and

the solution was boiled for few minutes. In the presence of flavonoids, reddish pink or

dirty brown colour was produced.



SAPONINS

In a test tube containing about 5ml of ethanolic extract of Caralluma adscendens

(Roxb.), a drop of sodium bicarbonate was added. The mixture was shaken vigorously and

kept for 3 minutes. A honey comb like froth formed showed the presence of saponins.

CARBOHYDRATES

Fehling’s Test:

Solution A: 34.65g of copper sulphate was dissolved in water and made up to 500ml.

Solution B: 125g of potassium hydroxide and 173 of Roschelle salt (sodium potassium

tartarate) were dissolved in distilled water and made up to 500 ml.

To 2.0ml of aqueous solution of the Caralluma adscendens (Roxb.), mixture of

equal parts of Fehling’s solution A and B was added. The contents were boiled for a few

minutes. Formations of red or brick red precipitate indicate the presence of carbohydrates.

Benedict’s test:

173g of sodium citrate and 100g of sodium carbonate were dissolved in 850ml

water. To this solution, 17.3g of copper sulphate was added and made up to 1000ml with

water. To 0.5ml of aqueous solution of Caralluma adscendens (Roxb.), 5.0ml of Benedict’s

reagent was added and boiled for 5 minutes. Formation of a bluish green showed the

presence of carbohydrates.

Mollisch’s Test:

In a test tube containing 20ml of aqueous solution of Caralluma adscendens (Roxb.)

extract, 2 drops of freshly prepared 20% alcoholic solution of α-naphthol was added and

mixed. To this solution, 2.0ml of concentrated sulphuric acid was added so as to form a

layer below the mixture. Formation of the red violet ring at the junction of the solution

which disappeared on the addition of an excess solution indicated the presence of

carbohydrates.

PROTEINS

Million’s Test:

1 part of mercury was digested with 2 parts of HNO3 and the resulting solution was

diluted with 2 volumes of water.



To a small quantity of Caralluma adscendens (Roxb.) extract, 5 to 6 drops of

Million’s reagent was added. A white precipitate which turns red on heating was formed

and it indicates the presence of proteins.

PHENOLS

Ferric Chloride Test:

To 1.0ml of alcoholic solution of Caralluma adscendens (Roxb.) extract, 2.0ml of

distilled water followed by drops of 10% aqueous FeCl3 solution were added. Formation of

blue or green indicates the presence of phenols.

Lead Acetate Test:

1.0ml of alcoholic solution of Caralluma adscendens (Roxb.) extract was diluted to

5.0ml with distilled water and to this few drops of 1% aqueous solution of lead acetate was

added. A yellow precipitate formed which indicate the presence of phenols.

Liebermann’s Test:

A small quantity of alcoholic extract of the Caralluma adscendens (Roxb.) was

dissolved in 0.5ml of 20% sulphuric acid solution, followed by the addition of a few drops

of aqueous sodium nitrate solution. A red colour was obtained on dilution and it turned blue

when made alkaline with aqueous sodium hydroxide solution.

STEROID

Libermann-Burchard’s Test

To 1ml of ethanolic extract of Caralluma adscendens (Roxb.) 1ml of chloroform,

1ml of concentrated sulphuric acid was added followed by the addition of 2ml of acetic

anhydride solution. The green colour formed indicated the presence of steroids.

Salkowsi Reaction

To 2ml of ethanolic extract of Caralluma adscendens (Roxb.), 1ml of concentrated

sulphuric acid was added carefully along the sides of the test tubes. A red colour produced

in the chloroform layer confirmed the presence of steroids.



GLYCOSIDES

A small amount of ethanolic extract of Caralluma adscendens (Roxb.) was

dissolved in 1ml of water and the aqueous sodium hydroxide solution was added.

Formation of a yellow colour indicated the presence of glycosides.

RESINS

To 2ml of ethanolic extract of Caralluma adscendens (Roxb.), 5 to 10ml of acetic

anhydride was added, dissolved by gently heating, cooled and then 0.5ml of sulphuric acid

was added. The bright purple colour produced indicated the presence of resins.

TANNINS

Ferric Chloride Test:

To 1.0 to 2.0 ml of an aqueous solution of Caralluma adscendens (Roxb.), few

drops of 5% aqueous FeCl3 solution was added. A bluish black colour formed, disappears

on addition of a few ml of dilute H2SO4 and there was formation of a yellowish brown

precipitate indicated the presence of tannins.

Lead Acetate Test:

In a test tube containing 5.0ml of an aqueous of Caralluma adscendens (Roxb.)

extract, a few drops of 1% solution of lead acetate was added. A yellow or red precipitate

was formed indicated the presence of tannins.

THIOLS

To about 0.5ml of Caralluma adscendens (Roxb.) extract, enough Ammonium

sulphate was added to saturate the solution, 2.0-4.0 drops of 5% sodium nitro prusside was

then added followed by one or more drops of concentrated HNO3. Magenta colour was

developed. This showed the presence of thiols.

TERPENOIDS (Salkowski Test):

5.0 ml of Caralluma adscendens (Roxb.) extract was mixed with 2 ml of

chloroform, and concentrated sulphuric acid (3 ml) was carefully added to form a layer. A

reddish brown colouration of the inter face formed, confirmed the presence of terpenoids



CARDIAC GLYCOSIDES (Keller-Killani Test):

5 ml of Caralluma adscendens (Roxb.) extract was treated with 2 ml of glacial

acetic acid containing one drop of ferric chloride solution. This was over layered with 1 ml

of concentrated sulphuric acid. A brown ring of the interface indicated a deoxysugar

characteristic of cardenolides. A violet ring may appear below the brown ring, while in the

acetic acid layer, a greenish ring may form just gradually throughout thin layer.

APPENDIX III

QUANTITATIVE ESTIMATION OF PHYTOCHEMICALS

Estimation of Flavonoids (Bohm and Kocipai- Abyazan, 1994)

10 g of the plant sample was extracted repeatedly with 100 ml of 80% aqueous

methanol at room temperature. The whole solution was filtered through what man filter

paper No 42 (125 mm). The filtrate was later transferred into a crucible and evaporated into

dryness over a water bath and weighed to a constant weight and estimated colorimetrically.

Estimation of alkaloids (Harborne, 1973)

5 g of the sample was weighed into a 250 ml beaker and 200 ml of 10% acetic acid

in ethanol was added and covered and allowed to stand for 4 h. This was filtered and the

extract was concentrated on a water bath to one-quarter of the original volume.

Concentrated ammonium hydroxide was added drop wise to the extract until the

precipitation was complete. The whole solution was allowed to settle and the precipitate

was collected and washed with dilute ammonium hydroxide and then filtered. The

residuewas the alkaloid, which was dried and weighed.

Estimation of saponins (Obadoni and Ochuko, 2001)

The samples were ground and 20 g was taken in a conical flask and 100 ml of 20%

aqueous ethanol were added. The samples were heated over a hot water bath for 4 h with

continuous stirring at about 55°C. The mixture was filtered and the residue was re-extracted

with another 200 ml 20% ethanol. The combined extracts were reduced to 40 ml over water

bath at about 90°C. The concentrate was transferred into a 250 ml separating 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 a constant weight; the saponin content

was calculated as percentage.

Estimation of Total Phenols

(Malick and Singh, 1980)

Principle

Phenols reacted with phosphomolybdic acid in Folin-ciocalteau reagent in alkaline

medium and produced a blue-coloured complex (molybdenum blue) estimated

calorimetrically at 650 nm.

Reagents

80% ethanol

Folin-ciocalteau reagent

20% sodium carbonate

Procedure

Weighed exactly 0.5 to 1g of plant sample and ground it with a pestle and mortar in

10x volume of 80% ethanol. Centrifuged the homogenate at 10,000rpm for 20 minutes and

saved the supernatant. Extracted the residue with five times the volume of 80% ethanol,

centrifuged and pooled the supernatants to dryness. Dissolved the residue in known volume

of distilled water. Pipetted out different aliquots (0.2-2.0ml) into test tubes. Made up the

volume in each test tube to 3ml with water. Added 0.5ml of Folin-ciocalteau reagent. After

3 minutes added 2ml of 20% sodium carbonate solution to each test tube. Mixed thoroughly

and placed the tubes in a boiling water bath for exactly 1 minute, cooled and measured the

absorbance at 650nm, against a regent blank. The same procedure was repeated for all

standard gallic acid solutions and standard curve was obtained.



Estimation of Tannin (Schanderl, 1970)

Principle

Tannin like compounds reduced phosphotungstic molybdic acid in alkaline solution to

produce a highly coloured blue solution, the intensity of which was proportional to the

amount of tannins. The intensity was measured spectrophotometrically at 700 nm.

Materials

1. Folin-Denis reagent:

Dissolved 100mg of sodium tungstate and 20mg phosphomolybdic acid in 750ml distilled

water in a suitable flask and added 50ml phosphoric acid. Refluxed the mixture for 2 hours

and made up to 1 liter with water.

2. Sodium carbonate solution:

Dissolved 250g sodium carbonate in 100ml of water at 70-80°C. Filtered through glass

wool after allowing it to stand overnight.

3. Standard tannic acid solution

Dissolved 100mg tannic acid in 100ml of distilled water.

4. Working standard solution:

Diluted 5ml of the stock solution to 100ml with distilled water. 1ml contained 500 µg

tannic acid.

Procedure

A known amount of powdered sample was boiled with water and the supernatant

was collected and transferred 1ml of the sample extract to a 100ml flask and made up the

volume to 100ml.Added 5ml of Folin-Denis reagent, 10 ml sodium carbonate solution and

diluted to 100ml with water. Shaked well. Read the absorbance at 700nm after 30 minutes.

(If absorbance was greater than 0.7, make a 1:4 dilution of the sample).Prepared a blank

with water instead of the sample and treated in a similar manner.Prepared a standard graph

by using 0-100g tannic acid.



Calculation

Calculated the tannin content of the samples from the standard graph.

APPENDIX IV

GC MS ANALYSIS OF Caralluma adscendens (Roxb.)

Gas chromatography- mass spectrometry was carried out on a THERMO GC-

TRACE ULTRA VER: 5.0, THERMO MS DSQ11. The temperature program was from

100 to 250 0C. The chromatographic column was TR5-MS capillary standard non-polar

column (30 Mts × 0.25mm i.d.; film 0.25µm). The sample was injected into the non-polar

column. Helium was used as carrier gas at a flow rate of 1ml/minute. 1µl samples were

analyzed with the column held initially at 1000C for 1 minute. It was then increased to

2500C, with an 80C/minute and kept there for 10 minutes. The retention time of the sample

was 0.00-39.93. The low mass (m/z) was 50 and high mass was (m/z) 500. The run time of

the sample was 36.93 minutes.

Compound identification

Compound present in the extract of Caralluma adscendens (Roxb.) was identified

on the basis of their retention indices. Identification confirmation was by comparison of

their mass spectra with library on Wiley library data base.

Instrument Description

EQUIPMENT : THERMO GC - TRACE ULTRA VER: 5.0, THERMO MS DSQ II

COLUMN : TR 5 - MS CAPILLARY STANDARD NON - POLAR COLUMN

DIMENSION : 30 Mts, ID: 0.25 mm, FILM: 0.25 µm

CARRIER GAS: He, FLOW: 1 ML/Min

TEMP PROG : 80 - 250, RATE: 8/Min, HOLDING TIME: 10 Min @250



APPENDIX V

SUPER OXIDE RADICAL SCAVENGING ACTIVITY OF Caralluma adscendens

(Roxb.)

Liu et al., (1997)

Principle

Super oxide anions were generated in a non-enzymatic Phenazine methosulfate-

nicotinamide adenine dinucleotide (PMS-NADH) system through the reaction of PMS,

NADH, and oxygen. It was assayed by the reduction of nitro blue tetrazolium (NBT).

Reagents

Tris-HCl buffer (16 mM, pH 8.0)

Nitro Blue Tetrazolium NBT (50 µM) solution

Nicotinamide Adenine Dinucleotide NADH (78 µM) solution

Phenazine methosulfate PMS (10 µM)

Procedure

In these experiments, the super oxide anion was generated in 3 ml of Tris-HCl

buffer (16 mM, pH 8.0) containing 0.75 ml of NBT (50 µM) solution, 0.75 ml of NADH

(78 µM) solution and 0.3 ml of different concentrations of the extract. The reaction

was initiated by adding 0.75 ml of PMS (10 µM) to the mixture. After 5 min of incubation

at room temperature, the absorbance at 560 nm was measured in Spectrophotometer.

Results

The super oxide anion scavenging activity was calculated according to the

following equation: % Inhibition = [(A0-A1) / A0 x 100], Where A0 was the absorbance of

the control (blank, without extract) and A1 was the absorbance in the presence of the

extract.

APPENDIX VI

HYDROXYL RADICAL SCAVENGING ACTIVITY OF Caralluma adscendens

(Roxb.)

(Halliwell et al., 1987)

Principle

Hydroxyl radical scavenging assay was based on the measurement of the

competition between deoxy ribose and the extract for hydroxyl radicals generated from the

Fe 3 + / ascorbate/EDTA/ H2O2 system.



Reagents

Deoxyribose (3.75mM)

Hydrogen peroxide (1mM)

Potassium phosphate buffer (20mM pH 7.4)

Ferric chloride (0.1mM)

EDTA (0.1mM)

Ascorbic acid (0.1mM)

Procedure

The reaction mixture ,which contained plant extract, Deoxyribose (3.75mM),

Hydrogen peroxide (1mM), Potassium phosphate buffer(20mM pH 7.4), Ferric chloride

(0.1mM), EDTA (0.1mM) and Ascorbic acid (0.1mM) were incubated in a water bath at

37±0.5°C for 1hr. The extent of deoxyribose degradation was measured by a TBA method.

1 ml of TBA (1% W/v) and 1 ml of TCA (2.8% W/v) were added to the mixture and heated

in a boiling water bath at 100°C for 20 minutes. The absorbance of the resulting solution

was measured spectrophotometrically at 532 nm. All the analyses were done in triplicates

and average values were taken.

Result

Inhibition of deoxyribose degradation in percent was calculated according to the

equation I = (A0-A1/A0) x 100, Where A0was the absorbance of control reaction and

A1 was the absorbance of test compound.

APPENDIX VII

QUANTITATIVE DPPH RADICAL SCAVENGING ACTIVITY OF Caralluma adscendens (Roxb.)

(Shimada et al., 1992)

Principle

DPPH (1, 1- diphenyl-2-picrylhydrazyl radical) was a stable free radical having a

purple color. When free radical scavengers were added, DPPH was reduced and its color

was changed to yellow, based on the efficacy of antioxidants. The change in color was

monitored at 517nm.

Reagents

500µm DPPH in methanol

Phosphate buffer (pH=7.4)



Procedure

1ml of 500µm DPPH in methanol was mixed with equal volume of extract solution

in phosphate buffer (pH=7.4), Mixed well and kept in dark for 30 minutes. The absorbance

at 517nm was monitored in presence of different concentrations of extracts. Blank

experiment was also carried out to determine the absorbance of DPPH before interacting

with the extract.

Results

Scavenging of DPPH radical in percentage was calculated according to the equation

I = (A0-A1/A0) x 100 Where A0 was the absorbance of control reaction and A1 was the

absorbance of test compound.

APPENDIX VIII

FRAP ASSAY (FERRIC REDUCING ANTIOXIDANT POWER)

(Benzie and Strain, 1999)

Principle

FRAP assay measured the change in absorbance at 593 nm owing to the formation

of a blue colored Fe-II-tripyridyl triazine compound from colorless oxidized Fe-III formed

by the action of electron donating antioxidants.

Reagents

300 mM Acetate buffer (pH 3.6)

40 mM Dilute HCl

10mM 2, 4, 6-Tri Pyridyl -s-Triazine (TPTZ ) solution

20 mM Ferric chloride

Procedure

The FRAP reagent was prepared by adding 200ml of acetate buffer, 20ml

TPTZ;20ml Ferric chloride, and 24ml distilled water. Kept the reagent in desiccators and

discorded if the solution turns blue and prepared fresh solution after rinsing the bottle

thoroughly with demineralised water. Mixed and kept in water bath at 37ºC. Run a set of

blank and wash down cuvettes with distilled water. Added 7ml FRAP reagent vigorously to

each cuvette and mixed the contents thoroughly and set the spectrophotometer at 593nm.

The temperature was kept at 73ºC for 4minutes. After 4 minutes, zero the blank and pressed



run to record. All solutions were used on the day of preparation. Standard curve was

prepared using different concentrations (100–1000 µmol/L) of FeSO4 · 7H2O.

Result

The results were corrected for dilution and expressed in µmol Fe2 +/L.

APPENDIX IX

REDUCING POWER ASSAY

Oyaizu (1986)

Principle

The reducing ability of the reaction mixture was assayed by monitoring the

transformation of Fe3+ - Fe2+ in the presence of the extract at 700nm.

Reagents

0.2 M Phosphate buffer (pH 6.6)

1%, w/v Potassium hexa cyanoferrate [K3Fe (CN) 6]

10% Trichloro acetic acid (TCA) solution

0.1% w/v Ferric chloride (FeCl3) solution

Procedure

The extract (0.75 ml) at various concentrations was mixed with 0.75 ml of

phosphate buffer (0.2 M, pH 6.6) and 0.75 ml of potassium hexacyanoferrate (1%, w/v),

followed by incubating at 50° C in a water bath for 20 min. The reaction was stopped by

adding 0.75 ml of Trichloro acetic acid (TCA) solution (10%) and then centrifuged at

3000 rpm/min for 10 min. 1.5 ml of the supernatant was mixed with 1.5 ml of distilled

water and 0.1 ml of ferric chloride solution (0.1%, w/v) for 10 min. The absorbance at 700

nm was measured as the reducing power.

Result

Higher absorbance of the reaction mixture indicated greater reducing power.



APPENDIX X

NITRIC OXIDE RADICAL SCAVENGING ACTIVITY OF

Caralluma adscendens (Roxb.)

Marcocci et al., (1994)

Principle

Sodium nitroprusside in aqueous solution at physiological pH spontaneously

generates nitric-oxide which interacts with oxygen to produce nitrite ions that can be

estimated by the use of Griess Illosvoy reaction. Scavengers of nitric-oxide act against

oxygen, leading to reduced production of nitrite ions.

Reagents

25 mM sodium nitroprusside (SNP) solution

Griess reagent 1.5 ml 1% sulfanilamide in 5% H3PO4 and 0.1% Naphthyl Ethylene Diamine

dihydrochloride

Procedure

In this experiment, 2 ml of extract was added to 0.5 ml of 25 mM Sodium Nitro

Prusside (SNP) solution, and the mixture was incubated at room temperature (25 °C) for 90

min. The Griess reagent was then added to the mixture. The absorbance of the chromophore

formed during diazotization of the nitrite with sulfanilamide and subsequent coupling with

naphthyl ethylene diamine dihydrochloride was immediately recorded at 540 nm. All the

tests were carried out in triplicates.

Results

The nitric oxide radical scavenging activity was calculated according to the

following equation: % Inhibition = [(A0 – A1) / A0 ×100] Where A0 was the absorbance of

the control (blank, without extract) and A 1 was the absorbance in the presence of the

extract.



APPENDIX XI

HYDROGEN PEROXIDE SCAVENGING ACTIVITY OF


Ruch et al., 1989

Principle

The decrease in absorbance of H2O2 upon oxidation of H2O2 by hydrogen peroxide

scavenging species was monitored at 230nm.

Reagent

Hydrogen peroxide (40 mM) in phosphate buffer (pH 7.4).

Procedure

A solution of hydrogen peroxide (40 mM) was prepared in phosphate buffer (pH

7.4). Different concentration (20-100 µg/ml) of the extract was added to a hydrogen

peroxide solution (0.6ml, 40mM). Absorbance of hydrogen peroxide at 230nm was

determined after 10 minutes against a blank solution containing phosphate buffer without

hydrogen peroxide. The percentage of scavenging of hydrogen peroxide by the extract and

the standard compounds was calculated using following formula.

Percentage of Inhibition (%) = Absorbance of control-Absorbance of sample

Absorbance of control

APPENDIX XII

LIPID PEROXIDATION INHIBITION ACTIVITY OF Caralluma adscendens (Roxb.)

(Ohkawa et al., 1979)

Principle

Malondialdehyde (MDA), a secondary end product of the oxidation of

polyunsaturated fatty acids reacts with two molecules of Thiobarbituric acid (TBA)

yielding a pinkish red chromogen with an absorbance maximum at 532 nm.

Reagents

25 % (W/V) rat liver homogenate

40 mM Tris HCl buffer ( pH.7.0)

30 mM Pottasium chloride

0.06 mM Ascorbic acid



0.16 mM ferrous ions

8.1 % sodium dodecyl sulphate

20 % acetic acid (pH adjusted to 3.5 with NaOH)

0.8% Thio Barbituric acid (w/v) (prepared in 1.1% sodium dodecyl sulphate)

20% Trichloro acetic aid

Butanol-pyridine mixture (15: 1 V/V)

Procedure

0.1 ml of different concentration of the extract was incubated at 37°C with 25 % (

W/V) rat liver homogenate ( 0.1 ml) containing Tris HCl buffer ( 40 mM, pH.7.0), 0.1 ml

KCl ( 30mM),0.1 ml Ascorbic acid (0.06 mM) and 0.1 ml Ferrous ions ( 0.16 mM) in a

total volume of 0.5 ml for 1 hour. At the end of incubation period, 0.4 ml of the reaction

mixture was treated with 0.2 ml sodium dodecyl sulphate (8.1 %), 1.5 ml of Thiobarbituric

acid and 1.5 ml of acetic acid (20 %, pH 3.5). The total volume was then made up to 4 ml

by adding distilled water and kept in boiling water bath at 95° C for 1 hour. After the

mixture has been cooled, 1 ml of distilled water and 5 ml of butanol-pyridine mixture (15: 1

V/V) were added. Following vigorous shaking the tubes were centrifuged and the

absorbance of the upper layer containing the chromophore was read at 532nm.

Results

Inhibition of lipid peroxidation (%) by the extract was calculated according to 1 -

(E/ C) x 100, where C was the absorbance value of the fully oxidized control and E was

absorbance of the test sample.

APPENDIX XIII

TOTAL ANTIOXIDANT CAPACITY OF Caralluma adscendens (Roxb.)

(Prieto et al., 1999)

Principle

The antioxidant activity of the extract was evaluated by the phosphomolybdenum

method. The assay was based on the reduction of Mo (VI)-Mo (V) complex at acidc pH.

Reagents

Reagent solution 0.6 M sulphuric acid, 28mM sodium phosphate and 40mM ammonium

molybdate



Procedure

0.3 ml extract was combined with 3.0 ml of reagent solution (0.6 M sulphuric acid,

28mM sodium phosphate and 40mM ammonium molybdate). The tubes containing the

reaction solution was incubated at 95 ° C for min. Then the absorbance of the solution was

measured at 695nm using a spectrophotometer against blank after cooling to room

temperature. Methanol (0.3 ml) in the place of extract was used as the blank.

Result

The antioxidant activity was expressed as the number of gram equivalents of

ascorbic acid.

APPENDIX XIV

ABTS RADICAL CATION SCAVENGING ACTIVITY OF


Re et al., (1999)

Principle

ABTS radical cation decolorisation assay involves the generation of ABTS radical

cation chromophore by the oxidation of ABTS with potassium persulfate and the

decolorization of these radicals by active compounds was monitored at 734nm. It was

applicable for both hydrophilic and lipophilic compounds.

Reagents

4.9 mM potassium persulfate

14 mM ABTS (2, 20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) solution

Standard ascorbic acid

Procedure

ABTS radical cation was freshly prepared by adding 5 ml of a 4.9 mM potassium

persulfate solution to 5 ml of a 14 mM ABTS solution and kept for 16 h in dark. This

solution was diluted with distilled water to yield an absorbance of 0.70 at 734 nm and the

same was used for the antioxidant assay. The final reaction mixture of standard group was

made up to 1 ml with 950 µl of ABTS solution and 50 µl of Vit-C. Similarly, in the test

group 1 ml reaction mixture comprised 950 µl of ABTS radical cation solution and 50 µl of

the extract solutions. The reaction mixture was vortexed for 10 s and after 6 min

absorbance was recorded at 734 nm against distilled water by using an ELICO (SL150)



UV–Vis Spectrophotometer and compared with the control ABTS radical cation solution.

Ascorbic acid was used as reference antioxidant compound.

Result

The results were expressed as percentage of inhibition of ABTS radical cation.

APPENDIX XV

LIPID PEROXIDATION INHIBITION ACTIVITY OF


(Ohkawa et al., 1979)

Principle

Malondialdehyde (MDA), a secondary end product of the oxidation of

polyunsaturated fatty acids reacts with two molecules of thiobarbituric acid (TBA) yielding

a pinkish red chromogen with an absorbance maximum at 532 nm.

Reagents

25 % (W/V) rat liver homogenate

40 mM Tris HCl buffer (pH.7.0)

30 mM Pottasium chloride

0.06 mM Ascorbic acid

0.16 mM ferrous ions

8.1 % sodium dodecyl sulphate

20 % acetic acid (pH adjusted to 3.5 with NaOH)

0.8% Thio Barbituric acid (w/v) (prepared in 1.1% sodium dodecylsulphate

20% Trichloro acetic aid

Butanol-pyridine mixture (15: 1 V/V)

Procedure

0.1 ml of different concentration of the extract was incubated at 37°C with 25 % (

W/V) rat liver homogenate ( 0.1 ml) containing tris HCl buffer ( 40 mM, pH.7.0), 0.1 ml

KCl ( 30mM),0.1 ml ascorbic acid (0.06 mM) and 0.1 ml ferrous ions ( 0.16 mM) in a total

volume of 0.5 ml for 1 hour. At the end of incubation period 0.4 ml of the reaction mixture

was treated with 0.2 ml sodium dodecyl sulphate (8.1 %), 1.5 ml of thiobarbituric acid and

1.5 ml of acetic acid (20 %, pH 3.5). The total volume was then made up to 4 ml by adding



distilled water and kept in boiling water bath at 95° C for 1 hour. After the mixture has been

cooled, 1 ml of distilled water and 5 ml of butanol-pyridine mixture (15: 1 V/V) were

added. Following vigorous shaking the tubes were centrifuged and the absorbance of the

upper layer containing the chromophore was read at 532nm.

Results

Inhibition of lipid peroxidation (%) by the extract was calculated according to 1 -

(E/ C) x 100, where C was the absorbance value of the fully oxidized control and E was

absorbance of the test sample.

APPENDIX XVI

ESTIMATION OF REGUCED GLUTATHIONE GRUNERT AND PHILIPS, (1951)

Principle

Reduced glutathione was estimated by the modified sodium nitroprusside method.

Reagents

Saturated NaCl solution

2 % Metaphophoric acid

Procedure

The metaphosphoric acid extract of liver or fractions, was saturated with NaCl and

allowed to stand for 15-30 min and centrifuged at 3000 rpm for 10 min at 4°C. Taken 1 mL

of the aliquot of the supernatant and added to 3 mL saturated NaCl solution; allowed it to

stand for 10 min at 25°C. The nonspecific absorption in the sample was eliminated by

reading the sample against a blank containing 2% metaphosphoric acid and Sodium

nitroprusside. The colored complex developed was measured immediately at 520 nm on a

colorimeter using blank tube.

Result

The amount of reduced glutathione was expressed as µg/mg of protein

APPENDIX XVII

ESTIMATION OF CATALASE

JOHANSSON & BORG, (1988)

Principle

Catalase (CAT) activity was measured by monitoring decomposition of H2O2.



Reagents

250 mM Phosphate buffered saline (PBS)

12 M methanol

44 mM H2O2

Purpald (22.8 mM)

Potassium periodate (65.2 mM)

Procedure

The reaction was initiated by adding 50 µL of homogenized liver sample to the

reaction mixture containing 250 mM PBS with 12 M methanol and 44 mM H2O2 and

incubated at room temperature for 20 min. The reaction was terminated with addition of

Purpald (22.8 mM) and again incubated at room temperature for 20 min. After adding

potassium periodate (65.2 mM), the absorbance of the sample was measured at 550 nm

Result

Catalse concentration was estimated by a standard graph plotted using known

concentrations of formaldehyde and results expressed IU/mg protein.

APPENDIX XVIII

PREPARATION OF REAGENTS AND MEDIA

PREPARATION OF REAGENTS AND MEDIA:

SPIZIZEN’S SALT SOLUTION (10 X)

MgSO4.7H2O 0.02 g

Sodium Citrate 1 g

K2HPO4 14 g

KH2PO4 6 g

(NH4)2SO4 2 g

Warm Distilled Water 100 ml

This solution was sterilized by autoclaving at 121°C for 20 minutes.

HISTIDINE / BIOTIN SOLUTION (0.05 Mm)

D- Biotin 12.4 mg

L- Histidine 9.6 mg

Distilled Water 100 ml



Biotin was dissolved by heating the water and to this solution Histidine was added and

autoclaved for 20 minutes at 121°C and stored in glass bottle at 4°C.

HISTIDINE BIOTIN PLATES

Agar 1.5 g for 100 ml

Distilled Water 83.4 ml

10 X Spizizen’s Salt 10 ml

40% Glucose 5 ml

Sterile Histidine HCl.H2O 1 ml

Sterile 0.05 mM Biotin 0.6 ml

The agar and water were autoclaved for 20 minutes at 121°C and 10 ml of sterile

Spizizen’s salt solution, 5 ml Sterile 40% Glucose and Histidine were added to the hot agar

solution. After the solution was cooled slightly the sterile biotin was added and poured into

petridish.

NUTRIENT BROTH

Nutrient Broth 1.3 g


Nutrient Broth was dissolved in 100 ml distilled water and was autoclaved before using.

MINIMAL AGAR PLATES




40% Glucose 5 ml

The agar and water were autoclaved for 20 minutes at 121°C and 10 ml Spizizen’s

salt solution and 5 ml of sterile 40% Glucose were added and this solution was poured into

petridish and used for the experiment.

AMPICILLIN PLATE






40% Glucose 5 ml

Sterile Histidine HCl.H2O 1 ml

Sterile 0.05 mM Biotin 0.6 ml

Sterile Ampicillin solution 8 mg/ml

0.02N NaOH 0.4 ml

The agar and water were autoclaved for 20 minutes at 121°C and 10 ml of Sterile

Spizizen’s Salt Solution, 5 ml of Sterile 40% Glucose and 1 ml of Histidine were added to

hot agar solution. After the solution was cooled slightly, the Sterile Biotin and Ampicillin

solution were added and poured into Petri dish.

TOP AGAR

Agar 0.6 g

Sodium chloride 0.5 ml


The agar was dissolved in distilled water and autoclaved for 20 minutes at 121oC

and 2 ml of NaCl solution was added and distributed into sterile tubes.

APPENDIX XIX

CYTOTOXICITY ACTIVITY OF Caralluma adscendens (Roxb.) ON HT-29 CELL

LINES BY MTT ASSAY

(Philip et al., 1990)

Principle

The principle involved was the cleavage of tetrazolium salt 3-(4, 5 dimethyl

thiazole-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) into a blue coloured product

(formazan) by mitochondrial enzyme succinate dehydrogenase. The cells were then

solubilized with an organic solvent and the released, solubilized formazan reagent was

measured spectrophotometrically. Since reduction of MTT occur only in metabolically

active cells, the measurement of level of this mitochondrial activity gives the number of the

viable cells. The number of viable cells was found to be propotional to the extent of

formazan production.



Maintanence of Cell line culture

HT-29 Cell cultures were obtained from National Centre for Cell Sciences (NCCS),

Pune. Cells were grown in Minimal essential medium supplemented with 2mM L-

glutamine, 10% Fetal Bovine Serum, Penicillin (100µg/ml), Streptomycin (100µg/ml) and

Amphoterecin B (5µg/ml). The cells were maintained at 37°C in a humidified atmosphere

with 5% CO2 and sub cultured twice a week

Reagents

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

Trypsin phosphate versene glucose (TPVG)

EDTA

Glucose

Phosphate Buffered saline

Fetal Bovine Serum

Antibiotics

Propanol

Procedure

The monolayer cell culture was trypsinized using TPVG and the cell count was

adjusted to 1x 105 cells/ml using medium containing 10% new born calf serum. To each

well of the 96 well microtitre plate, 0.1ml of the diluted cell suspension was added. After

24hrs, partial monolayer was formed, the supernatant was flicked off, washed the

monolayer once and 100µl of different concentation of extracts were added to the cells in

microtitre plates. The plates were then incubated at 37°C for 3 days in 5% CO2 atmosphere

and microscopic examination was carried out and observations recorded every 24 hrs. After

72 hrs the drug solutions in the wells were discarded and 50µl of MTT was added to each

well. The plates were gently shaken and incubated for 3hrs at 37°C in 5% CO2 atmosphere.

The supernatant was removed and 50µl of propanol was added and the plates were gently



shaken to solubilize the formed formazan.The absorbance was measured using a microplate

reader at a wave length of 540nm.

RESULT

The percentage growth inhibition was calculated using the formula

% Growth inhibition = 100- (Mean OD of individual test group/ Mean OD of

control group)×100

CTC50 was determined by plotting the concentration Vs % growth inhibition.

APPENDIX XX

ANTIPROLIFERATIVE ACTIVITY OF Caralluma adscendens (Roxb.) ON HT-29 CELL LINES BY SRB ASSAY

(Philip et al., 1990).

Principle

SRB was a bright pink aminoxanthine dye with two sulfonic groups.Under mild

acidic conditions, SRB bound to protein basic amino acid residues in trichloro acetic acid

(TCA) fixed cells to provide a sensitive index of cellular protein content that was linear

over a cell density range of at least two orders of magnitude. The developed colour can be

measured at 540 nm spectrophotometrically.

Reagents

Sulforhodamine B (SRB), Fetal Bovine serum (FBS),Phosphate Buffered

Saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), Trypsin,EDTA,

Glucose, Trichloroacetic acid, Acetic acid, Tris base, Antibiotics, Dimethyl

Sulfoxide (DMSO)Propanol

Cell Lines and Culture Medium

HT-29 (, Colon cancer) cell culture was procured from National Centre for Cell

Sciences (NCCS), Pune, India. Stock cells of HT-29 were cultured in DMEM supplemented

with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin (100

µg/ml) and amphotericin B (5 µg/ml) in an humidified atmosphere of 5% CO2 at 37°C until



confluent.. The cells were dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA,

0.05% glucose in PBS). The stock cultures were grown in 25 cm2 culture flasks (Tarsons

India Pvt. Ltd., Kolkata, India).

Preparation of Test Solutions

For cytotoxicity studies, each weighed test drugs were separately dissolved in

distilled DMSO and volume was made up with DMEM supplemented with 2% inactivated

FBS to obtain a stock solution of 1 mg/ml concentration and sterilized by filtration. Serial

two fold dilutions were prepared from this for carrying out cytotoxic studies.

Procedure

The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0 x

105 cells/ml using medium containing 10% FBS. To each well of the 96 well microtitre

plate, 0.1 ml of the diluted cell suspension (approximately 10,000 cells) was added. After

24 h, when a partial monolayer was formed, the supernatant was flicked off, washed the

monolayer once with medium and 100 µl of different extract concentrations were added to

the cells in microtitre plates. The plates were then incubated at 37o C for 3 days in 5% CO2

atmosphere, microscopic examination was carried out and observations were recorded

every 24 h. After 72 h, 25 µl of 50% trichloro acetic acid was added to the wells gently in

such a way that it forms a thin layer over the extract to form an over all concentration of

10%. The plates were incubated at 4o C for 1 h. The plates were flicked and washed five

times with water to remove traces of medium, extract and serum, and air-dried. They were

stained with SRB for 30 min. The unbound dye was then removed by rapidly washing four

times with 1% acetic acid. The plates were then air-dried. Tris base (10 mM, 100 µl) was

then added to the wells to solubulise the dye. The plates were shaken vigorously for 5 min.

The absorbance was measured using microplate reader at a wavelength of 540 nm. The

percentage growth inhibition was calculated by formula.

The percentage growth inhibition was calculated using the formula

% Growth inhibition = 100- (Mean OD of individual test group/ Mean OD of control

group)×100

CTC50 was determined by plotting the concentration Vs % growth inhibition.



APPENDIX XX I

EFFECT OF Caralluma adscendens (Roxb.) ON DNA FRAGMENTATION IN HT-29

COLON CANCER CELL LINES

Agarose gel electrophoresis was carried out for the analysis of DNA fragmentation.

Reagents

HT-29 colon cancer cell lines

Roche apoptotic DNA ladder kit

TE buffer, pH 8.0 (10 mM Tris and 1 mM EDTA)

10X TBE buffer pH 8.3 (0.89 M tris, 0.89M boric acid and 20 mM EDTA)

Ethidium Bromide (10 mg/ml)

5X Gel loading buffer (50% glycerol, 1 mM EDTA, 0.25% bromophenol blue and 0.25 %

xylene cyanol.

Procedure

HT-29 cells (3 x 106 /ml) were seeded into 6 well plates and incubated at 37°C with

5% CO2 atmosphere for 24 h. The cells were washed with medium and were treated with

extract, standard drug and incubated at 37ºC, 5% CO2 for 24 hrs. After the incubation time

ended, the chromosomal DNA of cancer cells was prepared with Roche apoptotic DNA

ladder kit. Briefly, cells were harvested and lysed with lysis buffer for 10 min. Then the

samples were mixed with isopropanol before passing through the filter and washed. The

DNA was eluted from the filter and treated with RNAse at 37ºC for 30 min before loading

onto 2% agarose gel electrophoresis and run 50 V/cm for 3 hrs. The gel was visualized

under UV transilluminator and photographed.

APPENDIX XX II

EFFECT OF Caralluma adscendens (Roxb.) ON EXPRESSION OF CASPASE -3 IN HT29 COLON CANCER CELL LINES

(Jenny et al., 2010)

Principle

The caspase-3 colorimetric assay was based on the hydrolysis of the peptide

substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase-3, resulting in



the release of the p-nitroaniline (pNA) moiety. P-Nitroaniline has a high absorbance at 405 nm

(εmM = 10.5). The concentration of the pNA released from the substrate was calculated from

the absorbance values at 405 nm or from a calibration curve prepared with defined pNA

solutions.

Procedure

The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0

x 105 cells/ml using medium containing 10% FBS. To each well of the 6 well plate, 2ml of the

diluted cell suspension was added. After 24 h, when a partial monolayer was formed, the

supernatant was flicked off, washed the monolayer once with medium and different extract

concentrations prepared in DMEM were added to the cells. The plates were then incubated at

37o C for 3 days in 5% CO2 atmosphere, microscopic examination was carried out and

observations were recorded every 24 h. After 72 h, cells were scrapped and centrifuged at

2000rpm for 10 min to separate the pellet. The pellet was resuspended in 50 µl of chilled cell

lysis buffer and incubated cells on ice for 10min. Centrifuged at 15,000rpm for 1 min and

supernatant was transferred to a fresh tube. The vial was maintained in ice and protein

concentration of the samples was measured by Bradford assay. The samples were diluted to get

4 mg per ml. 50µl of samples was mixed with 50µl of 10mM Dithiothreitol (DTT), followed by

5µl of 4 mM DEVD-p-nitroanilide and incubated at 370C for 120 min. The absorbance was

measured using microplate reader at a wavelength of 405nm.

The activity of caspase-3 was expressed as OD/mg of protein.

APPENDIX XX III

EFFECT OF Caralluma adscendens (Roxb.) ON CELL MEMBRANE INTEGRITY AND VIABILITY OF HT-29 CELL LINES BY LDH LEAKAGE ASSAY

(Lacikova et al, 2009).

Principle

Cell death or cytotoxicity was classically evaluated by the quantification of plasma

membrane damage by the measurement of activity of lactate dehydrogenase (LDH) release.

LDH was a stable cytoplasmic enzyme present in all cells and rapidly released into the cell

culture supernatant upon damage of the plasma membrane. This assay was based on the

principle where, LDH oxidized lactate to pyruvate which then reacted with tetrazolium salt



INT ((2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) to form formazan.

The increase in the amount of formazan produced in culture supernatant directly correlates

to the increase in the number of lysed cells

Procedure

The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0 x

105 cells/ml using medium containing 10% FBS. To each well of the 96 well microtitre

plate, 0.1 ml of the diluted cell suspension (approximately 10,000 cells) was added. After

24 h, when a partial monolayer was formed, the supernatant was flicked off, washed the

monolayer once with medium and 100 µl of different extract concentrations were added to

the cells in microtitre plates. The plates were then incubated at 37o C for 3 days in 5% CO2

atmosphere, microscopic examination was carried out and observations were recorded

every 24 h. After 72 h, the supernatant was collected from individual wells, centrifuged at

2000rpm for 10 min. Collected the supernatant into clean vials. 100µl of each sample was

transferred to a fresh clean 96 well plate, added 100µl of reaction mixture of the kit and

incubated at room temperature for 30 min in dark. The absorbance was measured using

microplate reader at a wavelength of 490 nm. The percentage activity was calculated over

the untreated control samples.

APPENDIX XXIV

ESTIMATION OF SUPEROXIDE DISMUTASE

Maccord and Fridovich et al., (1969)

Principle

SOD was an endogenous enzymatic antioxidant which catalysed the dismutation of

super oxide free radical. This method was based on the inhibition of the spontaneous

oxidation of the adrenalin to adrenochrome by the enzyme superoxide dismutase.

Reagents

Adrenalin

Carbonate buffer pH 10.2

0.1mM EDTA



Procedure

To 0.5 ml of the homogenate, 1.5 ml of carbonate buffer and 0.5ml of 0.1mM

EDTA was added and mixed. To this 0.4ml of adrenalin was added at the time of

measurement of the optical density at 480nm.

Result

The antioxidant activity of SOD enzyme was expressed as Units/min/mg protein.

APPENDIX XXV

ESTIMATION OF CATALASE (CAT)

(SINHA, 1972)

Principle

Catalase causes rapid decomposition of hydrogen peroxide to water. The method

was based on the fact that dichromate in acetic acid reduced to chromic acetate when heated

in the presence of H2O2 with the formation of perchloric acid as an unstable intermediate.

The chromic acetate thus produced was measured colorimetrically at 610 nm.

Reagents

0.01M Phosphate buffer, pH 7.0

A: 0.1M Monobasic sodium phosphate

B: 0.1M Dibasic sodium phosphate

Mixed 39 ml of A and 61 ml of B was diluted to a total of 200 ml. 10 ml of this

solution was further diluted to 100 ml with distilled water.

0.2 M Hydrogen peroxide

Stock dichromate / acetic acid solution: Mixed 5 % potassium dichromate with

glacial acetic acid (1:3 by volume).

Working dichromate/acetic acid solution: The stock was diluted to 1:5 with water to

make the working dichromate / acetic acid solution.



Procedure

The assay mixture contained 0.5 ml of H2O2, 1.0ml of buffer and 0.4 ml of water.

0.2 ml of the enzyme was added to initiate the reaction. 2.0 ml of the dichromate / acetic

acid reagent was added after 0, 30, 60, 90 seconds of incubation. To the control tube the

enzyme was added after the addition of the acid reagent. The tubes were then heated for 10

min. and then color developed was read at 610 nm.

Result

The activity of catalase was expressed as µ moles of H2O2 utilized / min / mg

protein.

APPENDIX XXVI

ESTIMATION OF GLUTATHIONE PEROXIDASE (GPX)

Rotruct et al.,(1973)

Principle

The procedure was based on the reaction between left over glutathione in the

following reaction with the DTNB to form a compound which absorbs maximally at 412

nm

2 GSH+ H2O2 → GSSG+2 H2 O

Reagents

1. 0.4 M sodium phosphate buffer, pH 7.0

2. 10 mM Sodium azide

3. 2.5 mM Hydrogen peroxide

4. 4mM Reduced glutathione

5. 10% Trichloro acetic acid

6. 0.3 M Phosphate solution

7. 0.04% DTNB in 1% sodium citrate

8. Reduced glutathione standard

20 mg reduced glutathione was dissolved in 100ml of water.



Procedure

0.4ml of buffer, 0.1 ml of sodium azide, 0.2 ml of reduced glutathione, 0.1 ml of

hydrogen peroxide, 0.2 ml of enzyme and 1.0ml of water were added to a final incubation

volume of 2.0ml. The tubes were incubated for 0,30,60,90 seconds. The reaction was then

terminated by the addition of 0.5ml TCA. To determine the glutathione content, 2.0ml of

the supernatant was removed by centrifugation and added to 3.0ml disodium hydrogen

phosphate solution and 1.0ml of DTNB reagent. The colour developed was read at 412 nm.

The standards in range of 200-1000µg were taken and treated in similar manner.

The activity was expressed in terms of µmoles of GSH consumed/min/mg protein

APPENDIX XXVII

ESTIMATION OF GLUTATHIONE S-TRANSFERASE

(Habig et al., 1974)

Principle

Glutathione-S-transferase catalysed the reaction of 1-chloro2,4 dinitrobenzene

(CDNB) with the sulphydryl group of glutathione.

CDNB + GSH → CDNB-S- glutathione.

The conjugate, CDNB- glutathione absorbed light at 340 nm and the activity of the

enzyme estimated by measuring the increase in absorbance.

Reagents

0.5 M phosphate buffer, pH 6.5

30mM CDNB in 95% ethanol (30mg/5ml H2O)

30mM reduced glutathione (14mg/1.5ml H2O)

Procedurze

To 1.0ml of buffer, 0.1ml of sample, 1.7ml of water and 0.1ml of CDNB were

added and incubated at 37˚C for 5 min. After incubation, 0.1ml of reduced glutathione was

added. The increase in optical density of the enzyme was measured against that of the blank

at 340nm.



Result

The enzyme activity was calculated in terms of µ moles of CDNB conjugate

formed/min/mg protein.

APPENDIX XXVIII

ESTIMATION OF TOTAL REDUCED GLUTATHIONE

Moron et al., (1979)

Principle

The method was based on the reaction of reduced glutathione with DTNB to give a

compound that absorbs at 412 nm.

Reagents

1. Metaphosphoric acid

1.67g of glacial metaphosphoric acid, 0.2g EDTA and 30g sodium chloride in

100ml of water.

2. 0.4 M Na2HPO4

3. DTNB reagent

40mg of DTNB in 100ml of 1% trisodium citrate.

4. Standard glutathione

20mg of reduced was dissolved in 100 ml of water.

Procedure:

1.0 of the sample extract was treated with 4.0 ml of metaphosphoric acid

precipitating solution. After centrifugation, 2.0 ml of supernatant was taken. To this 2.0ml

0.4 M Na2HPO4 and 1.0 ml of DTNB reagent was added. A blank was set up with water

and treated in a similar manner. A series of standards were also carried out.

Result

The level of glutathione in the sample was expressed as m moles / mg protein.



APPENDIX XXIX

ESTIAMTION OF PROTEIN

Lowry et al., ( 1951)

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 color developed by the biuret reaction of the

protein with the alkaline cupric tartarate were measured at 660 nm.

Reagents

2 % sodium carbonate in 0.1 N NaOH (Reagent A)

0.5 % Copper sulphate in 1 % potassium sodium tartarate (Reagent B)

Alkaline copper reagent: Mixed 50 ml of A and 1.0 ml of B prior to use

Folin-Ciocalteau reagent: Mixed 1 part of reagent with 2 parts of water.

Stock standard: Weighed 50 mg of bovine serum albumin and made up to 50 ml in a

standard flask with saline.

Working standard: Diluted 10 ml of the stock of 50 ml with distilled water. 1.0 ml

of this solution contains 200 µg of protein.

Procedure

Pipetted out 0.2 to 1.0 ml working standard solution, 0.1 ml of the sample was

taken. The volumes in all the tubes were made up to 1.0ml with distilled water. Added 5.0

ml of alkaline copper reagent to each tube mixed well and allowed to stand for 10 mins.

Then added 0.5 ml of Folin-Ciocalteau reagent. Mixed well and incubated at room

temperature for 30 minutes. A reagent blank was also prepared. After 30 minutes, the blue

color developed was read at 660 nm.

The amount of protein was expressed as mg / g tissue.



APPENDIX -XXX

EXTRACTION OF LIPIDS

(Folch et al., 1975)

The tissues were washed with saline and dried between filter paper. A weighed

amount of tissue (500 mg) was homogenized with 7.0 ml of methanol in a Potter-Elvehjem

homogeniser and filtered through a Whatman No 1 filter paper into a conical flask. The

residue after filtration was scraped and homogenized in 14 ml choloroform. The residue

was once again scraped from the filter paper and ground with 10 ml of chloroform–

methanol mixture and the resulting filtrate was evaporated to dryness.

The dried lipid residue after evaporation was dissolved in 5.0 ml of chloroform-

methanol mixture. The redissolved lipid extract was mixed with 1.0 ml of 0.1 N KCl and

the contents were shaken well. The upper aqueous phase containing gangliosides and other

water soluble compounds were separated. The lower chloroform phase, containing neutral

and phospholipids was again washed 3 times with 2.0 ml of Folch’s reagent and the upper

aqueous phase was aspirated. The lower chloroform phase was made upto known volume

(2.0 ml) and aliquots were taken for the analysis of cholesterol, triglycerides, free fatty

acids and phospholipids.

APPENDIX XXXI

ESTIMATION OF CHOLESTEROL

(PAREKH AND JUNG, 1970)

Principle

Cholesterol reacted with ferric chloride in the presence of concentrated sulphuric

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

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

Reagents

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

in 100ml of glacial acetic acid.

Ferric chloride precipitating reagent: 10ml of stock ferric chloride reagent was taken

in 100ml of standard flask and made up to the mark with pure glacial acetic acid.



Ferric chloride diluting reagent: 8.5ml of stock ferric chloride was diluted to 100ml

with pure glacial acetic acid.

Standard cholesterol solution: 100mg of cholesterol was dissolved in 100ml glacial

acetic acid. The concentration of working standard was100µg /ml.

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

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

working standard was 100µg/ml.

Procedure

To 0.1ml of lipid extract added 4.9ml of ferric chloride precipitating reagent.

Centrifuged and to 2.5ml of supernatant added 2.5ml of ferric chloride diluting reagent.

Added 4.0ml of concentrated sulphuric acid and a blank was prepared simultaneously by

taking 5.0ml of diluting reagent and 4.0ml of concentrated sulphuric acid. A set of

standards (0.5 - 2.5 ml) were taken and made up to 5.0ml with FeCl2 diluting reagent. Then

added 4.0 ml of con.H2SO4. After 30min. the intensity of color developed was read at

450nm against reagent blank.

Result

The amount of cholesterol in tissue was expressed as mg / 100 g tissue.

APPENDIX -XXXII

ESTIMATION OF PHOSPHOLIPIDS

(ROUSER, 1970)

Principle

The organic phospholipid phosphorus was converted to inorganic phosphorus,

which reacts with ammonium molybdate to form phosphomolybdic acid, which on

reduction and reaction with ANSA forms a stable blue color and has absorption at 710nm

Reagent

70% Perchloric acid

3% Ammonium molybdate

3% Ascorbic acid



Standard: 35.1 mg of KH2PO4 was dissolved in 100ml of water. This contains 80µg of

phosphorous/ml.

Procedure

To 0.1ml of plasma or lipid extract, 1.0ml of perchloric acid was added and digested

on a sand bath until it become colorless. The volume was made up to 5.0ml with water.

Standards in the range 5-20µg were also taken and 0.8ml of perchloric acid was added and

made up to 5.0 ml with water. To all tubes, 0.5ml of ammonium molybdate was added

followed by 0.5ml of ascorbic acid solution and mixed well. The tubes were heated in a

boiling water bath for 6 min and the color developed was read immediately at 710

nm.Phosphorus content was multiplied by a factor 25, which gave the weight of

phospholipids. Phospholipids were expressed as mg/100ml in plasma and mg/g in tissues.

APPENDIX -XXXIII

ESTIMATION OF FREE FATTY ACID

(Horn and Mehanan, 1981)

Principle

Free fatty acids were extracted from lipids by Chloroform–heptane-methanol

(CHM) mixture. The free fatty acids formed a complex with cupric ions when mixed with

copper reagent. The coloured complex formed with copper was soluble in chloroform and

diethyl dithiocarbamate and was used as a color developer. The color developed was read at

430nm.

Reagent

Chloroform– heptane-methanol mixture (CHM mixture), the mixture was prepared

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

Activated silicic acid

Copper nitrate-triethanolamine solution: 9 volumes of aqueous 1M triethanolamine,

1 volume of 1 N acetic acid and 10 volumes of 6.45% Cu (NO3) 2H2O were mixed

with 33g of sodium chloride. The pH was adjusted to 8.1.

0.1% diethyl dithiocarbamate in n-butanol



Standard: A solution containing 200mg/100ml of palmitic acid was prepared in

CHM mixture. The solution was diluted 10 times for use (200µg/ml).

Procedure

To 0.2ml lipid extract, 6.0ml of CHM mixture and 200mg of activated silicic acid

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

Standard were also made upto 6.0ml with CHM mixture. Blank contained 6.0ml of CHM

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

a mechanical shaker for 20min. They were then centrifuged to give two separate phases.

2.0ml of the upper phase was transferred to another tube; 1.0ml of the color reagent was

then added and shaken well. The color developed was read at 430nm against a reagent

blank.

Free fatty acids were expressed as mg/100g in tissues.

APPENDIX -XXXIV

ESTIMATION OF LIPID PEROXIDATION PRODUCTS (LPO)

(NIEHIUS AND SAMUELSON, 1968)

Principle

Malondialdehyde had been identified as the product of lipid peroxidation that

reacted with thiobarbituric acid to give a red color absorbing at 535 nm.

Reagents

Stock TCA-TBA-HCl reagent: 15% w/v trichloroacetic acid, 0.375 w/v

thiobarbituric acid and 0.25 N HCl. The solution was heated mildly to assist the dissolution

of the TBA.

Procedure

To 1.0 ml of the sample, 2.0 ml of TCA- TBA-HCl reagent was added and mixed

thoroughly. The solution was heated for 15 min in a boiling water bath. After cooling, the

flocculent precipitate was removed by centrifugation at 1,000 g for 10 min. The absorbance

was determined at 535nm against a blank that contains all the reagents minus the sample.



Result

The results were expressed as nmoles of MDA formed/min/mg protein using an

extinction coefficient of the chromophore 1.56 x 105 Mcm and expressed as mmoles of

MDA formed/min/mg protein.

APPENDIX-XXXV

ASSAY OF TISSUE HYDROXY METHYL GLUTARYL COENZYME A REDUCTASE (HMG-COA REDUCTASE, EC.1.1.1.34)

RAO AND RAMAKRISHNAN (1975)

Principle

The ratio between HMG-CoA and mevalonate in tissues was taken as an index of

the activity of HMG-CoA reductase

Reagents

1. Saline-arsenate: 1g of sodium arsenate/ l in physiological saline.

2. Dilute perchloric acid: 50ml of perchloric acid was diluted up to 1 litre with water.

3. Hydroxylamine hydrochloride reagent (For Mevalonate): Equal volumes of

hydroxylamine hydrochloride reagent and water were mixed freshly before use.

4. Hydroxylamine hydrochloride reagent (For HMG-CoA): Equal volumes of

hydroxylamine hydrochloride reagent and 4.5M NaOH were mixed freshly before

use.

5. Ferric chloride reagent: 5.2g of trichloroaceticacid and 10g of ferric chloride were

dissolved in 50ml of 6.5N HCl and diluted to 100ml with distilled water

Procedure

Equal volumes of fresh 10% tissue homogenate and dilute perchloric acid were

mixed, kept for 5 minutes and centrifuged at 2000Xg for 10 minutes. To 1ml of filtrate,

0.5ml of freshly prepared hydroxylamine reagent (alkaline hydroxylamine reagent in the

case of HMG-CoA) was added, mixed and after 5min 1.5ml of ferric chloride was added

and shaken well. Readings were taken after 10min at 540nm against a similarly treated

saline-arsenate blank. The ratio of HMG-CoA to mevalonate was calculated. Lower ratio

indicates higher enzyme activity and vice versa.



APPENDIX XXXVI

ESTIMATION OF FUCOSE

Dische and Shettles (1948)

Principle

This method involved heating the sample with H2SO4 for 10 min followed by the

addition of cysteine hydrochloride. The absorbance of the colour developed was read at

430nm.

Reagents

1. 95% Ethanol

2. 0.1N NaOH

3. Sulphuric acid-water mixture: 6 volumes concentrated sulphuric acid and 1

volume water.

4. Cysteine reagent: 3g cysteine hydrochloride in 100ml distilled water.

5. Stock standard: 20mg fucose was dissolved in 100ml distilled water

6. Working standard: 10ml stock standard was diluted to 100ml with distilled water

to get a concentration of 20µg/ml

Procedure

In two tubes, each containing 0.1 ml sample (labelled as control and test), 5ml 95%

ethanol was added, mixed well and then centrifuged. The precipitate was dissolved in 1ml

NaOH. A series of standards with 1ml water was also set up along with the test. All the

tubes were kept in ice-cold condition and 4.5ml H2SO4-H2O mixture was added. The tubes

were kept in boiling water bath for 3min,cooled, 0.1ml cysteine reagent was added to all the

tubes except control and incubated for 60min at room temperature. The colour developed

was read at 396nm and 430nm against the blank. The fucose content was calculated from

the differences in the readings obtained at 396nm and 430nm and then the values were

substracted to obtain values excluding cysteine. This was read against standard curve

prepared using D (+/-) – fucose.

The fucose content was expressed as mg/g protein.



APPENDIX XXXVII

ESTIMATION OF ALKALINE PHOSPHATASE (ALP)

(KING AND ARMSTRONG, 1934)

Principle

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

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

liberated phenol was measured at 700nm with Folin -ciocalteau reagent.

Reagents

1. Sodium Carbonate - Sodium bicarbonate buffer, 100mmol/ L : Dissolved 6.36g

anhydrous sodium carbonate and 3.36g sodium bicarbonate in water and made to

a litre.

2. Disodium phenyl phosphate, 100 mmol/ L : Dissolved 2.18g in water, heated to

boil, cooled and made to a litre. Added 1.0ml of chloroform and stored in the

refrigerator.

3. Buffer - Substrate: Prepared by mixing equal volume of the above two solution.

This has a pH of 10.

4. Folin - ciocalteau reagent: Mixed 1.0ml of reagent with 2.0ml of water.

5. Sodium carbonate solution, 15 %: Dissolved 15 g of anhydrous sodium carbonate

in 100ml of water.

6. Standard phenol solution, 1g/L: Dissolved 1 g pure crystalline phenol in

100mmol/L HCL and made to litre with the acid.

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

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

Procedure

Pipetted 4.0ml of the buffer substrate into a test tube and incubated at 37°C for 5

mins. Added 0.2ml of sample and incubated further for exact 15 mins. Removed and

immediately added 1.8ml of diluted phenol reagent. At the same time a control was set up

containing 4.0ml buffer substrate and 0.2ml sample, to which 1.8ml phenol reagent was



added immediately. Mixed well and centrifuged. To 4.0ml of the supernatant added 2.0ml

of sodium carbonate. 4.0ml of working standard solution and for blank taken 3.2ml water

and 0.8ml of phenol reagent was taken. Then added 2.0ml of sodium carbonate. Incubated

all the tubes at 37°C for 15 min. Read the colour developed at 700 nm. The activity of

alkaline phosphatase in faecal sample was expressed as µmole of phenol liberated/ min/ mg

protein.

APPENDIX XXXVIII

ESTIMATION OF CARCINO EMBRYONIC ANTIGEN

Quantitative estimation of CEA was based on the solid phase enzyme linked

immunosorbent assay. CEA was assayed using UBI Magiwell enzyme immune assay kit.

Reagents

1. Microwell strips: Anti CEA antibodies coated wells.

2. Enzyme conjugate: Anti CEA antibodies conjugated to horse raddish Peroxidase.

3. Sample Diluent.

4. Reference standard set: Calibrated to 1.5,3,6,13 and 50 ng/ml in sample diluents.

5. Chromogen substrate: Buffer solution containing H2O2 and tetramethy benzidine

Procedure

25ml of standard, control and the test serum samples were added to the appropriate

wells. And then 100ml of enzyme conjugate were added in to each well and incubated for

60 min at room temperature. The wells were then rinsed five times in running tap water and

remove the water. Dispense 100ml of chromogen substrate solution in to each well and

incubate for 30 min at room temperature. Stop the reaction by adding 50ml of 1NH2SO4 to

each well and read OD at 480 nm with a microwell reader.

Result

The values were expressed as ng/ml



APPENDIX XXXIX

ESTIMATION OF ALPHA- FETOPROTEIN

Principle

Quantitative estimation of AFP was based on the solid phase enzyme linked

immunosorbent assay.

Reagents

Alpha feto protein standard solutions

Alpha feto protein polyclonal antibodies precoated Microwell strips

Biotinylated Alpha-Fetoprotein antibody

Avidin-Biotin-Peroxidase Complex (ABC)

Tetramethylbenzidine color developing reagent

1N H2SO4 stop solution

Procedure

100 µl of standard, control and test serum samples were added to the appropriate

wells and then after washing the wells with buffer,100 µl of biotinylated anti- Alpha-

Fetoprotein antibody was added to all the wells and incubated for 60 min at room

temperature.The plates were washed three times with Phosphate buffered saline and 100 µl

of Avidin-Biotin –Peroxidase solution was added to all the wells. After rinsing the wells

with appropriate buffer, dispensed 90µl of color developing solution into each well and

incubated for 30 min at room temperature.Stopped the reaction using 100 µl of 1NH2SO4

stop solution and read OD at 450 nm with a microwell reader.The standard curve can be

plotted as the relative O.D. 450 of each standard solution (Y) vs. the respective

concentration of the standard solution (X). The Alpha-Fetoprotein concentration of the

samples can be interpolated from the standard curve and the values were expressed as

ng/ml.



APPENDIX XL

HISTOPATHOLOGICAL TECHNIQUE

(CULLING METHOD, 1979)

The following steps were followed in histopathological technique.

1. Autopsy bits were preserved in 10% formalin solution for minimum one hour.

2. Dehydration of biopsy bits done by three changes of acetone (each 500 ml)

3. Cleaning of bits from acetone done by three changes of xylene (each 500ml) and

incubation of processed tissue bit in paraffin wax-two changes for three to four

hours in an incubator at 50-60°C.

5. Embedding of the tissue in paraffin was after incubation in melted paraffin.

6. Cutting of sections from autopsy bit embedded in wax and sections were taken on

the glass slide.Sections on glass slide were cleaned from wax by immersing in

xylene and sections were histochemically reacted with haematoxylin and eosin

staining to evaluate the morphology and cellular composition


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