64

Subjected to bioassays for screening of extracts

The most active methanol extracts were fractionated on silica gel

column

Most potent fractions were chosen by bioassays and were subjected to sub column

Identification of active compound (s) & structural elucidation

The plant powders were soaked in Hexane, ethylacetate,

methanol & water and subjected to sequential extraction

MATERIALS AND METHODS

FIG.1. PHYTOCHEMISTRY

Tinospora cordifolia (Stem) Cassia fistula (Stem bark)

65

3.1. PHYTOCHEMISTRY

3.1.1. Collection of plant material

Tinospora cordifolia stem and Cassia fistula stem bark were

collected from Triunelveli District and Kodaikanal Hills, Tamil Nadu,

India respectively. The species were identified and authenticated at

the Department of Botany, Holy Cross College, Trichy, Tamil Nadu.

The stem and stem bark were shade-dried, cut into small pieces and

coarsely powdered. The coarse powder was used for extraction with

various solvents.

3.1.2. Preparation of plant extracts

2kg of dry powders were taken in individual aspirator bottle;

6 litres of hexane was used and the mixture was shaken occasionally

for 72 hours. Then the extract was filtered. This procedure was

repeated three times and all extracts were decanted and pooled. The

extracts were filtered before drying using Whattman filter paper no.2

on a Buchner funnel and the solvent was removed by vacuum

distillation in a rotary evaporator at 40˚C; the extracts were placed in

pre-weighed flasks before drying. The remaining plant residue was

successively extracted with ethyl acetate, methanol and water

sequentially as above (Saxena and Yadav, 1983).

3.1.3. Preliminary phytochemical screening

Preliminary phytochemical Screening of the plants was carried

out as per the methods and tests given by Dey and Raman, (1957).

66

Test for Terpenoids

Noller’s Test: The substance was warmed with tin and thionyl

chloride. Pink colouration indicated the presence of triterpenoids.

Test for flavonoids

Shinado’s Test: To the substance in alcohol, a few magnesium

turnings and few drops of concentrated hydrochloric acid were added

and boiled for five minutes. Red color showed the presence of

flavonoids.

Test for Glycosides

The substance was mixed with a little anthrone on a watch

glass. One drop of concentrated sulphuric acid was added made into

a paste and warmed gently over waterbath. Dark green color was the

indication of the presence of glycosides.

Test for Reducing Sugars

The substance was mixed with the Fehling’s solutions II and I.

Formation of red color was the indication of the presence of reducing

sugars.

Test for Polysaccharides

The extract (0.5g) was dissolved in distilled water and filtered.

The filtrate was mixed with iodine solution. Dark blue color formation

showed the presence of polysaccharides.

67

Test for Alkaloid

To the test substance a few drops of acetic acid were added,

followed by Dragendorff’s reagent and shaken well. Formation of

orange-red precipitate indicated the presence of alkaloid.

Test for Tannins

The substance is mixed with basic lead acetate solution.

Formation of white precipitate indicated the presence of tannins.

Test for Saponins

The substance was shaken with water. Copious lather formation

indicated the presence of saponins.

3.2. Thin Layer Chromatography

Thin Layer Chromatography (TLC) is the common method used

to detect the phytoconstituents (Marston et al., 1997). TLC is the

solid-liquid technique in which the two phases are, a stationary phase

(solid) and a mobile phase (liquid). Solids most commonly used in TLC

are silica gel (SiO2 x H2O) and alumina (Al2O3 x H2O).

5µl of a 100mg sample/ml solution was loaded on a pre-coated

silica gel 60 F254, 0.25 mm thick TLC plate (Merck) with suitable

solvent system. Separated compounds were visualized under visible,

ultraviolet light (254 and 360 nm, UV lamp) and iodine vapors. Plates

were also sprayed with 5% sulphuric acid in ethanol and 0.36%

sulphuric acid in methanol and heated for 3 min at 100˚C to allow for

the development of color changes (Carr and Rogers, 1986).

68

3.3. Column Chromatography

The active T.cordifolia stem methanol extract and C.fistula stem

bark methanol extracts (25gm) were chromatographed over separate

silica gel column (Acme’s silica gel, 100-200 mesh size, 750gm,

3.5 i.d. x 60 cm) and successively eluted with stepwise gradient of

hexane, ethyl acetate and methanol solvent system (5%, 10%, 20%,

30%, 50%, 70% and 100%). Finally the column was washed with

ethanol (100%).

A total of 128 fractions were collected from T.cordifolia extract

and 112 fractions were collected from C.fistula; each fraction was

spotted on a precoated Silica gel 60 F254, 0.25mm thick TLC plate

(Merck) and eluted in hexane: ethyl acetate (3:1). Fractions with

similar Rf values in TLC pattern were pooled together to get 5 fractions

each. The active fractions 3 of T.cordifolia and 4 of C.fistula were

rechromatographed on a silica gel column and eluted with a stepwise

gradient of ethyl acetate: metahnol (9:1) solvent system.

89 sub-fractions were obtained in T.cordifolia fractionation.

Sub-fractions with similar Rf values (11-69) were pooled and

confirmed as a pure compound. 76 sub-fractions were obtained in

C.fistula fractionation. Sub-fractions with similar Rf values (21-59)

were pooled and confirmed as a pure compound. The sub fractions

were assayed for plasma glucose lowering effect in STZ-diabetic rats.

We found that the compounds showed significant antihyperglycemic

effect compared to the other isolated fractions.

69

Tinospora cordifolia (2kg, dry and coarse powder)

Hexan

e

Ethyl acetate Methanol Water

(Active)

Column chromatography

Elution of compounds with increasing

polarity of solvents

128 Fractions were

obtained

Based on the TLC profiles, fractions were pooled together

and 5 major fractions were obtained

Fraction 3 significantly decreased the plasma glucose levels

All the fractions were utilized for

plasma glucose lowering effect

The active fraction-3 was utilized for compound elution with increasing polarity of solvents viz, ethyl acetate, methanol

Fig.2. Isolation of compounds

89 sub fractions were obtained and 11-69 were given the pure

compound.

All the fractions and compound were screened

for plasma glucose lowering effect

The compound (Sub Fr. 11-69) significantly

decreased the plasma glucose levels

COMPOUND I

70

Cassia fistula (2kg, dry and coarse powder)

Hexane Ethyl acetate Methanol Water

(Active)

Column chromatography

Elution of compounds with increasing

polarity of solvents

112 Fractions were

obtained

Based on the TLC profiles, fractions were pooled together

and 5 major fractions were obtained

Fraction 4 significantly decreased the plasma glucose levels

All the fractions were utilized for

plasma glucose lowering effect

The active fraction-4 was utilized for compound elution with

increasing polarity of solvents viz, ethyl acetate, methanol

Fig.3. Isolation of compounds

76 sub fractions were obtained and 21-59 were given the pure

compound.

All the fractions and compound were screened

for plasma glucose lowering effect

The compound (Sub Fr. 21-59) significantly

decreased the plasma glucose levels

COMPOUND II

71

3.4. Nuclear Magnetic Resonance (NMR) Spectroscopy

NMR is used as an analytical tool for predicting the structure of

the molecule based on the different environments of hydrogen atom by

measuring the magnetic moments of hydrogen atom. Purified sample

was subjected to NMR studies. Tetra Methyl Saline (TMS) was used as

standard, which shows chemical shift value at zero on the δ scale.

1H and 13C NMR spectra were recorded with a JEOL 300 MHz FT NMR

spectrometer (H1) 75 MHz (C13) and chemical shifts were recorded in

ppm.

3.5. Infrared Spectroscopy (IR)

IR spectra involve the energy transition between different

vibration energy levels. Mainly IR can be used to find the functional

groups present in a pure or mixture of compounds, because IR gives

details or gives a strong absorption pattern at a particular frequency

for a particular functional group. Solvents used for IR spectra are

CHCl3-CCl4. Liquid samples were taken in KBr crystals and solid

samples are ground with KBr and pellets were made and spectra were

taken. IR spectra were recorded in Shimadszu by KBr pellet method.

IR spectra were taken on a Perkin Elmer FT-IR (Spectrum One)

spectrophotometer.

3.6. Mass Spectrometry (MASS)

Using mass spectrophotometry the molecular mass of

a compound and its elemental composition can be easily determined.

High resolution Electron Impact Mass Spectroscopy (EI-MS) was

72

performed. Mass spectra were taken on a Jeol JMS-DX30

spectrometer.

3.7. ANTIDIABETIC ACTIVITY

3.7.1. Experimental animals

Healthy adult male albino Wistar rats, bred and reared at King’s

Institute of Preventive Medicine, Guindy, Chennai, were used for the

experiment. Weight-matched animals (140-160g) were selected and

housed in polypropylene cages layered with husk and kept in a

semi-natural light/dark condition (12 hours light/12 hours dark). The

animals were allowed free access to water and standard pellet diet (Sai

Durga Animal Feed, Pranav Agro Industries Ltd., Bangalore, India).

Animal handling and experimental procedures were approved by the

Institutional Animal Ethical Committee.

3.7.2. Experimental induction of diabetes

Diabetes was induced for the animals by a single intraperitonial

injection of STZ (60mg/kg. b.wt- Akbarzadeh et al., 2007) in freshly

prepared citrate buffer (0.1M, pH 4.5) after an overnight fast.

STZ- injected animals were given 20 % glucose solution for 24 hours

to prevent initial drug-induced hypoglycemic mortality. STZ injected

animals exhibited massive glycosuria (determined by Benedict’s

qualitative test) and hyperglycemia (by glucose oxidase method) within

a few days. Diabetes in STZ rats was confirmed by measuring the

fasting blood glucose concentration, 96 hours after injection with STZ.

73

The animals with blood glucose more than 350–450mg/dL were

considered diabetic and used for the experiment.

3.7.3. Preparation of plant extracts for oral administration

The plant extracts were poorly soluble in water. Hence, a

required quantity of the plant extracts was prepared in 0.1% carboxy

methyl cellulose (CMC) (1ml/kg. b.wt). Compounds were suspended in

vehicle solution dimethylsulfoxide (DMSO) (0.5%; 1ml/ kg. b.wt).

3.7.4. Administration of plant extracts

The different extracts of the parts of both the plants were

administered to the rats by force-feeding. This was executed by

inserting a baby oral feeding tube, which was connected to a syringe

containing the extract, into the gastric region of the rat. The animals

were fasted 30 min before and after the treatment to ensure maximum

bioavailability (Sridhar et al., 2005).

3.7.5. Experimental design

3.7.5.1. Preliminary study – determining the dosage for crude

One week after the induction of diabetes, rats with blood

glucose >350mg/dL were subjected to fasting for 16 hours. They were

divided into different groups, with 6 rats in each group. Hexane, ethyl

acetate, methanol and aqueous extracts ranging from 100 to

800mg/kg. b. wt, were administered to the animals and blood glucose

was determined at the end of 5 hours after the oral administration of

the extracts (Karunanayake et al., 1984). The lowest dose that brought

74

about the maximum antihyperglycemic effect for each plant extract

was given through oral intubations for the repeated administration.

The effective dose of all the extracts was found to be 250mg/kg. b.wt.

The selected doses of the plant extracts were given everyday till

completion of the experiment (i.e., 90 days).

3.7.5.2. Dose determination for compounds

A total of 90 rats were utilized and the animals were randomly

divided into 15 groups of six animals each as given below. Different

doses (5, 15, 20mg/kg. b.wt) of polysaccharide and catechin were

suspended in vehicle solution (Dimethylsulfoxide [DMSO] 0.5%;

1ml/kg.b.wt) and administered through oral route using an

intra-gastric tube for 15 days daily to the respective groups.

Group 1-Normal rats treated with vehicle alone

Group 2-Normal rats + Polysaccharide (5mg/kg.b.wt)

Group 3-Normal rats + Polysaccharide (15mg/kg.b.wt)

Group 4-Normal rats + Polysaccharide (20mg/kg.b.wt)

Group 5-Normal rats + Catechin (5mg/kg.b.wt)

Group 6-Normal rats + Catechin (15mg/kg.b.wt)

Group 7-Normal rats + Catechin (20mg/kg.b.wt)

Group 8-STZ induced diabetic rats treated with vehicle alone

Group 9-STZ induced diabetic rats + Polysaccharide (5mg/kg.b.wt)

Group 10-STZ induced diabetic rats + Polysaccharide (15mg/kg.b.wt)

Group 11-STZ induced diabetic rats + Polysaccharide (20mg/kg.b.wt)

Group 12-STZ induced diabetic rats + Catechin (5mg/kg.b.wt)

75

Group 13-STZ induced diabetic rats + Catechin (15mg/kg.b.wt)

Group 14-STZ induced diabetic rats + Catechin (20mg/kg.b.wt)

Group 15-STZ induced diabetic rats + Insulin (3-IU/kg.b.wt)

All doses were given 7 days after injection of STZ. No irritation

or restlessness was observed after each drug or vehicle

administration. No noticeable adverse effect (i.e., respiratory distress,

abnormal locomotion and catalepsy) was observed in any animal after

the drug administration. Fasting plasma glucose levels were estimated

every week to ascertain the status of diabetes in different groups of

rats. Oral administration of 20mg/kg b. wt dose of compound for

15 days showed significant plasma glucose lowering effect in

STZ-induced diabetic rats when compared to other doses (5 and

15mg/ kg.b.wt). This dose was fixed as effective dose and was selected

for further biochemical study.

3.7.5.3. Repeated administration of plant extracts, fractions and

compounds

One week after induction of diabetes in albino rats, the fasting

blood glucose levels of fasted rats were measured (Pushparaj

et al., 2000). Rats with plasma glucose more than 350–450mg/dL

were included in the study. Rats were divided into 7 groups of 6 each.

Groups 1 & 2, viz, normal and diabetic control rats received vehicle

alone (0.1% carboxy methyl cellulose (CMC) 1ml/kg body weight).

Groups 3 to 6 were treated with the effective dose of hexane,

ethylacetate, methanol and water extracts dissolved in vehicle solution

76

AFTER 90 DAYS OF CRUDE EXTRACTS TREATMENT

Male albino Wistar Rats (200-250gm-9 weeks)

STZ-induced (60mg/ kg b.wt)

through oral route for 90 days, respectively. Group 7 was treated with

insulin (3-IU/kg) for 90 days. Blood samples were collected

periodically.

Fig. 4: Methodology for screening of crude extracts

Normal

Biochemical Analysis

1. Plasma glucose

2. Kidney function

tests

3. Liver function tests

4. Lipid parameters

5. Tissue glycogen

RIA

1. Plasma insulin

2. C-peptide

Histology

1. Light microscopy of

pancreas

2. SEM of pancreas

3. TEM of pancreas

Diabetic

D+Hexane

D+ethyl acetate

D+Methanol

D+Aqueous

D+Insulin

77

Male Albino Wistar Rats (200-250gm-9 weeks)

Normal

Short term studies for

15 days

Blood Glucose

Dia +(Insulin

3 I.U /kg

b.wt)

Dia + Fra-2

Dia + Fra-1

Diabetic

Dia + Fra-5

Dia + Fra-4

Dia + Fra-3

STZ-induced (60mg/ kg b.wt)

A total of 78 rats were used for screening the fractions. The rats

were divided into 13 groups of 6 each. Groups 1 & 2, viz, normal and

diabetic control rats, received the vehicle alone (0.1% carboxy methyl

cellulose (CMC) (1ml/kg.b.wt). Groups 3 to 7 were treated with

fractions of T.cordifolia and groups 8-12 were treated with fractions of

C.fistula at the dose of 100mg/kg bw dissolved in 0.1% carboxy

methyl cellulose for 15 days. Group 15 was treated with insulin

(3- IU/ kg b.wt) for 15 days.

Fig. 5: Methodology for screening of fractions

78

A total of 42 rats were used to test the compounds activity. The

rats were divided into 7 groups of 6 each. Polysaccharide and catechin

were suspended in vehicle solution (Dimethylsulfoxide [DMSO] 0.5%;

1ml/kg.b.wt) and administered through oral route using an

intragastric tube for 60 days to the respective groups.

Group 1-Normal rats treated with vehicle alone

Group 2-Normal rats + Polysaccharide (20mg/kg.b.wt)

Group 3-Normal rats + Catechin (20mg/kg.b.wt)

Group 4-STZ induced diabetic rats treated with vehicle alone

Group 5-STZ induced diabetic rats + Polysaccharide (20mg/kg.b.wt)

Group 6-STZ induced diabetic rats + Catechin (20mg/kg.b.wt)

Group 7-STZ induced diabetic rats + Insulin (3-IU/kg.b.wt)

After 60 days of treatment, the 12 hours fasted animals were

anaesthetized between 7 am and 8 am, using ketamine

(24mg/kg.b.wt, intramuscular injection) and sacrificed by

decapitation. Blood was collected in two different tubes i.e., one with

whole blood for serum separation and another with anticoagulant-

potassium oxalate and sodium fluoride for plasma assays. Tissues

(pancreas, liver, kidney and gastrocnemius muscle) were surgically

removed, washed with cold physiological saline, cleared off the

adherent lipids and immediately transferred to ice-cold containers and

weighed.

79

Male albino Wistar Rats

(200-250gm-9 weeks)

STZ induced

(60mg/kg b.wt)

Fig. 6. Methodology for evaluation of activity of compounds

AFTER 60 DAYS OF COMPOUNDS TREATMENT

Antidiabetic Parameters

1. Plasma glucose

2 Kidney function tests

4. Liver function tests

5. Lipid profiles

6. Protein profile

7. Membrane bound enzymes

8. Carbohydrate enzymes

Glut-4 Expression

(gastrocnemius muscle)

1. m RNA expression

2. Protein expression

Normal Diabetic N+Polysaccharide

N+ Catechin

D+Polysaccharide

D+ Catechin

D+Insulin

RIA

1. Plasma insulin

2. C-peptide

3. 14C - Glucose

oxidation

80

Glucose oxidase

Peroxidase

3.8. BIOCHEMICAL ESTIMATIONS

3.8.1. Estimation of glucose (Diagnostic Kit–Reddy’s Laboratories,

Bachupally, Hyderabad, India)

Principle

Glucose was oxidized by the enzyme glucose oxidase to give

D-gluconic acid and hydrogen peroxide. Hydrogen peroxide, in the

presence of the enzyme peroxidase oxidized phenol, combine with

4-aminoantipyrine to produce a pink-coloured quinoneimine dye.

The intensity of the color produced was proportional to glucose

concentration in the sample.

D–glucose + H2O+O2 D-gluconic acid + H2O2

H2O2 + 4–Aminoantipyrine + Phenol

Quinoneimine dye + Phenol + H2O

Reagents

1. Glucose reagent

2. Standard (100mg/dL)

Procedure

For the estimation of glucose, 10µl each of serum and working

standard were incubated with 1ml of the reagent for 15 min at 37C

and the absorbance at 505nm was measured against a reagent blank.

For reagent blank, 10µl of distilled water was added to 1ml of the

reagent. The concentration of glucose in serum samples was

calculated as:

81

3.8.2. Estimation of haemoglobin

Haemoglobin in the blood was estimated by the method of

Drabkin and Austin (1932).

Reagents

1. Drabkin’s reagent: 200mg of potassium ferricyanide, 50mg of

potassium cyanide and 1.0g of Na2CO3 were dissolved in water

and made up to one litre. The reagent had a pale yellow color of

pH 9.6 and was stored in brown bottle.

2. Cyanmethaemoglobin standard solution: 16g/dL.

Procedure

To 0.02ml of blood, 5.0ml of Drabkin’s reagent was added,

mixed well and allowed to stand for 10 min. The solution was read at

540nm together with the standard solution, against a reagent blank.

Values were expressed as mg/dL for blood.

3.8.3. Estimation of glycosylated hemoglobin (Diagnostic Kit–Bio

Systems, Costa Brava, Spain)

Principle

The hemolysate was prepared first, in which the labile fraction

was eliminated, hemoglobin was retained by a cationic exchange

resin, HbA1a+b fraction was washed away and later, HbA1c was

Absorbance of sample

Absorbance of standard x 100 = mg/dL

82

specifically eluted and quantified by direct photometric reading at 415

nm.

Reagents

1. Reagent 1

2. Reagent 2

3. Reagent 3

4. Micro columns

Procedure

Whole blood was collected in a test tube that contained EDTA.

Columns and reagent were brought to room temperature for a few

min. Into a test tube, 50µl of blood and 200µl of reagent were

pipetted out. The contents in the test tube were mixed well and

allowed to stand at room temperature for 10-15 min, and this formed

the hemolysate. The column for the separation was prepared by

removing the upper cap from the column first and then the lower cap,

the rounded end of a pipette was used to push the upper disc down to

the resin surface; care was taken to avoid compressing it. The column

was allowed to drain completely.

50µl of the hemolysate was pipetted on the upper filter and the

column was allowed to drain to waste. The sample residue left above

the upper disc was removed by adding 200µl of reagent 2 and the

column was allowed to drain to waste. Again 2ml of reagent 2 was

added and the column was allowed to drain to waste. Next, the

83

column was placed over a test tube and 4ml of reagent 3 was added to

the column, the HbA1c was collected and the absorbance was read at

415nm against distilled water. In order to read the absorbance of the

total hemoglobin (AHbTOTAL), 12ml of reagent 3 was mixed with 50µl of

hemolysate and the absorbance was read at 415nm against distilled

water. HbA1c was calculated as:

3.8.4. Estimation of liver and skeletal muscle glycogen (Plummer,

1987)

Principle

Glycogen in liver and skeletal muscle was liberated when heated

with strong alkali. Released glycogen was precipitated by the addition

of ethanol and sodium sulphate (co-precipitant) to give a quantitative

yield of glycogen. The polysaccharide was then hydrolysed in acid and

the glucose released was estimated.

Reagents

1. Potassium hydroxide (30%).

2. Saturated Na2 SO4.

3. Ethanol (95% v/v).

4. HCl (1.2mol/L).

5. Phenol red indicator.

6. NaOH (0.5mol/L).

7. Reagents for the estimation of glucose.

AHbA1c

3 x AHbtotal

x 100 = % HbA1c

84

Procedure

1g each of liver and skeletal muscle was weighed into a

calibrated centrifuge tube that contained 2ml of KOH (30%) and

heated in a boiling water bath for 20 min with occasional shaking.

The tubes were cooled on ice and 0.2ml of saturated Na2SO4 was

added. Glycogen was precipitated by adding 5ml of ethanol (95% v/v).

The precipitate was separated by centrifugation and dissolved in 5ml

of water with gentle warming, and then diluted with distilled water to

10ml.

1ml samples of the glycogen solution were pipetted out into a

test tube, 1ml of HCl (1.2mol/L) was added to it, a marble was placed

inside the test tube and the test tube was heated on a boiling water

bath for 2hours. At the end of this period, 1 drop of phenol red

indicator was added and neutralized carefully with NaOH (0.5mol/L)

till the indicator changed from pink through orange to a yellow color.

Finally, it was diluted to 5ml with distilled water and preceded as for

the estimation of glucose. Duplicates were maintained.

3.8.5. Estimation of plasma insulin

(Radioimmunoassay kit – Diasorin, Italy).

Principle

The principle of the assay was based on the competition

between labelled insulin and insulin contained in standards or

specimens to be assayed for a fixed and limited number of antibody

binding sites. After the incubation, the amount of labelled insulin

85

bound to the antibody was inversely related to the amount of

unlabelled insulin present in the sample. The method adopted for

separation was based on the use of a precipitating reagent, in which a

second antibody is pre-precipitated and in excess.

Reagents

1. 125I-labelled insulin.

2. Insulin standards.

3. Insulin antiserum.

4. Precipitating reagent.

5. Control sample.

Procedure

Standards: 100µl of the standard from the respective standard

bottles (0-5) was pipetted out into different vials and 100µl of tracer

and 100µl of antiserum were added to it.

Sample: 100µl of plasma sample was pipetted out into a vial,

100µl of tracer and 100µl of antiserum were added to it.

Total activity: To measure the total activity, 100µl of tracer was

pipetted out into a vial.

The contents of the above vials were mixed with a vortex and

incubated for 1.5 hours at room temperature. The bottle of

precipitating reagent was allowed to reach the room temperature and

mixed well by repeated tilting. 1ml of precipitating reagent was

86

dispensed into all vials (except total activity vial). The contents of all

the vials were again vortexed and the vials were allowed to stand for

15 min at room temperature. The vials were centrifuged at 1500 rpm

for 15 min. The supernatant was discarded. The radioactivity of the

precipitate was measured. The mean net counts for each group of

tubes were computed.

The binding ability was evaluated as follows:

(B / T)0% = x 100

The mean counts for each standard and unknown sample was

expressed as a percentage of zero standard mean counts.

B / B0% = x 100

The percent values of each standard versus the insulin amount

expressed as µU/ml was plotted in linear-linear or semilog

co-ordinates to obtain a calibration curve. By interpolation of the

calibration curve, the insulin level of the samples was obtained.

3.8.6. Estimation of plasma C-peptide (Radioimmunoassay

kit – Missouri, USA).

Principle

The principle of the assay was based on the competition

between a fixed concentration of labeled tracer antigen and constant

Zero standard mean counts

Total activity mean counts

Standard or sample mean counts

Zero standard mean counts

87

dilution of antiserum such that the concentration of antigen binding

sites on the antibody is limited. Thus, the amount of tracer bound to

antibody will decrease as the concentration of unlabelled antigen

increases. This can be measured after separating antibody-bound

from free tracer and counting one or other or both fractions.

Reagents

1. Assay buffer.

2. Rat C-peptide antibody.

3. 125I – Rat C-peptide standards.

4. Labeled hydrating buffer.

5. Rat C-peptide standards.

6. Control.

7. Precipitating reagent.

Procedure

300µl of assay buffer was pipetteed out to the non specific

binding tubes. 200µl of assay buffer was pipetted out in the reference

tube. 100µl assay buffer pipetted out to all the other tubes (sample).

These tubes are vortexed and incubated for 20-24 hours at 4˚C. After

24 hours of incubation 100µl of 125I – Rat C-peptide tracer pipetted

out to all the tubes. These tubes are vortexed and incubated for 22-24

hrs at 4˚C. 1ml of cold precipitating reagent is added to all the tubes

except total count tubes. All the tubes are vortexed and incubated for

20 min at 4˚C. Tubes are centrifuged at 4˚C for 20 min at

2000-3000xg. Supernatant was decanted from all centrifuged tubes

88

except total count tubes, and all the tubes were counted in a gamma

counter for 1 min.

Calculation

The percentage of tracer bound

The percentage of total binding (%B/B0) for each standard and

sample.

%B/ B0 =

3.8.7. Estimation of plasma 14C-Glucose Oxidation

14C-Glucose Oxidation in skeletal muscle was estimated by the

method of Johnson and Turner, (1971) and Kraft and Johnson,

(1972).

Principle

The rate of glucose oxidation was measured in terms of release

of 14CO2, which is trapped by diethanolamine.

Reagents

1. Dulbecco’s Modified Eagle’s medium + Nutrient Mixture F-12

HAM (DMEM) (pH 7.4): 7.8g of DMEM/F-12 medium containing

HERPES (5MM), 600mg of sodium bicarbonate, 50mg of

benzathine penicillin, 50mg of streptomycin and 25mg of

fungicide (Amphotericin B), pH was adjusted to 7.4 and the

volume was finally made upto 500ml with distilled water.

Total binding counts

Total counts

x 100

Sample or standard

Total binding

x 100

89

2. 14C-glucose (Specific activity 310 mCi/mmol): 20µl of

14C–glucose was mixed with 180µl of DMEM/F-12 (pH 7.4).

3. Diethanoamine buffer: 10ml of 60% diethanolamine was mixed

with 4ml of 6N HCL and 3g of KHCO3 was dissolved in this

solution.

4. 1N H2SO4 : 1ml of (36n) concentrated sulphuric acid was made

upto 36ml with distilled water.

5. Scintillation Fluid: 4g of 2,5- Diphenyloxazole (PPO) and 400mg

of 1,4-bis (5-phenyloxazole-2-yl)-benzene (POPOP) were

dissolved in 1 liter of scintillation grade tolune, mixed well and

kept in dark.

Procedure

10mg of tissues were weighed and placed in a 2ml ampule

containing 170µl of DMEM (pH7.4), 10 IU penicillin in 10µl of DMEM

and 0.5µCi of 14C-glucose. Then the ampoules were aerated with a gas

mixture (5% CO2 and 95 % air) for 30 seconds and tightly closed with

cork containing CO2 trap. A piece of filter paper was inserted into the

rubber cork and 0.1ml of diehanolamine buffer (pH 9.5) was applied to

the filter paper before closing the ampoule. This closed system with

CO2 trap was placed in an incubator at 37°C. CO2 traps were replaced

every 2 hours. After removing second trap, 0.1ml of 1N H2SO4 was

added to the ampoule to halt further metabolism and release of any

residual CO2 from the sample. The system was again closed for 1h

before the third and final trap was removed. All the CO2 traps were

90

placed in the scintillation vials containing 10ml of scintillation fluid

and counted in a Beta counter. Results are expressed as CPM of 14C

released/10mg tissue.

3.8.8. KIDNEY FUNCTION TESTS

3.8.8.1. Estimation of protein (Lowry et al., 1951)

Principle

The aromatic amino acids such as tyrosine and tryptophan

present in protein reacted with Folin-Ciocalteu reagent to give a dark

blue color. The intensity of the color obtained was directly

proportional to the amount of protein present in the sample.

Reagents

1. 5% Trichloroacetic acid.

2. Reagent A–2g of sodium carbonate in 100ml of 0.1N sodium

hydroxide.

3. Reagent B–1g of copper sulphate in 100ml of distilled water.

4. Reagent C–2g of sodium potassium tartarate in 100ml of

distilled water.

5. Reagent D–Reagents B and C mixed in 1:1 ratio.

6. Reagent mixture–Reagents A and D in 50:1 ratio.

7. Folin–Ciocalteu’s reagent – Folin phenol reagent diluted 1:1 with

distilled water.

91

Procedure

0.5ml of the serum was mixed with 1ml of 5% trichloroacetic

acid, and centrifuged to precipitate the protein. The precipitate was

dissolved in 1N sodium hydroxide and made upto 10ml. To 1ml of the

sample, 5ml of reagent D was added, and after 10min incubation,

0.5ml of Folin–Ciocalteu’s reagent was added and mixed. After

30 min, the intensity of the blue color was read at 620nm against a

reagent blank. Protein content of serum sample was determined from

a standard curve.

Standard curve was prepared using bovine serum albumin

prepared at a stock concentration of 1mg/ml and diluted to obtain

serial dilutions at 50, 100, 150, 200 and 250µg/ml.

3.8.8.2. Estimation of urea (Fawcett and Scott, 1960)

Principle

The ammonia formed from urease action reacted with phenol in

the presence of hypochlorite to form an indophenol, which with alkali

gave a blue coloured compound. The nitroprusside acted as a

catalyst, increasing the rate of reaction. The intensity of the color

obtained was directly proportional to the amount of urea present in

the sample.

Reagents

1. Buffered urease solution.

2. Phenol–sodium nitroprusside solution.

92

3. Sodium hydroxide–sodium hypochlorite.

4. Urea solution – 1mg per ml.

Procedure

20µl each of serum and working standard were incubated with

200µl of urease–buffer solution at 37C for 15min, 5ml of the phenol

nitroprusside solution was added to it, followed by 5ml of the

hypochlorite reagent. It was placed in a water bath at 37C for

15 min and the absorbance was measured against reagent blank at

630nm. For reagent blank, 20µl of distilled water was added to 200µl

of urease buffer. Urea content in the sample was calculated as:

3.8.8.3. Estimation of uric acid

Uric acid in the serum urine was estimated by using the

diagnostic kit based on the enzymic method described by Caraway

(1955).

Uric acid in the sample is oxidized by uricase to allantoin. In

this reaction 1 mole of hydrogen peroxide is formed for every mole of

uric acid oxidized. Hydrogen peroxide reacts with 3, 5-dichloro-2-

hydroxybenzene sulfonic acid and 4-amino antipyrine to give

quinoneimine dye. Intensity of the color of this dye was proportional to

the concentration of uric acid in the sample.

Absorbance of sample

Absorbance of

standard

x 100 = mg/dL

93

Reagents

1. Enzyme reagent: 4-Aminoantipyrine (4mM), 3,5-dichloro-2-

hydroxybenzenesulfonate (2.0mM), microbial uriase (150

U/L), horseradish peroxidase (10,000 U/L).

2. Standard uric acid: 5.0mg/100ml

Procedure

To 1ml of the enzyme reagent, 25µl of plasma was added and

mixed by inversion. 25µl of standard and 25µl of distilled water (blank)

also processed simultaneously. The tubes were incubated at 37 C for

5 min and the color developed was read at 510nm.

The values were expressed as mg/dL of plasma and urine.

3.8.8.4. Estimation of creatinine (Diagnostic kit–Dr. Reddys’

Laboratories, Bachupally, Hyderabad, India)

Principle

Creatinine reacted with alkaline picrate to produce a

red-coloured complex; the rate of red colored complex formation was

directly proportional to the creatinine content in the sample.

Reagents

1. Working reagent

2. Standard (2mg/dL)

Procedure

For the estimation of creatinine, to 1ml of working reagent,

100µl of serum or working standard was added and a stopwatch was

94

Cholesterol ester

hydrolase

Peroxidase

Cholesterol oxidase

started. Initial absorbance, Ao, was read exactly after 30 seconds of

adding test and standard. Absorbance, A1, for test and standard was

read exactly after 60 seconds of adding test and standard to the

working reagent. Creatinine content in serum was calculated as:

Where, AT = A1T – AoT

AS = A1S – AoS

3.8.9. LIPID PARAMETERS

3.8.9.1. Estimation of total cholesterol (Diagnostic kit - Beacon

Diagnostics, Kabilpore, Navsari, India)

Principle

Cholesterol esters were hydrolysed to free cholesterol by

cholesterol ester hydrolase. The free cholesterol produced was

oxidized by cholesterol oxidase to cholest–4–en–3–one with the

simultaneous production of hydrogen peroxide, which oxidatively

coupled with 4-aminoantipyrine and phenol in the presence of

peroxidase to yield a chromogen with maximum absorption at

500nm.

Cholesterol ester + H2O Cholesterol + Fatty acids

Cholesterol + O2 Cholest-4-en-3-one + H2O2

2H2O2+ Phenol + 4–Aminoantipyrine Quinoneimine dye+ 4H2O Reagents

1 Working reagent

2 Standard (200mg/dL)

95

Glycerol Kinase

Lipase

L–α–Glycerol–Phosphate Oxidase

Procedure

In the assay of total cholesterol, 10µl each of serum and

working standard were incubated with 1ml of reagent for 5min at

37C and the absorbance at 500nm was measured against a reagent

blank. For reagent blank, 10µl of distilled water was added to 1ml of

working reagent. Concentration of cholesterol in serum samples was

calculated as:

3.8.9.2. Estimation of triglycerides (Diagnostic kit-Bio Systems,

Costa Brava, Spain)

Principle

Serum triglycerides were hydrolysed using lipase, and the

released glycerol was assayed in a reaction catalysed by glycerol

kinase and L–µ–glycerol–phosphate oxidase in a system that generated

hydrogen peroxide. The hydrogen peroxide generated was monitored

in the presence of horseradish peroxidase with 3, 5–dichloro–2–

hydroxylbenzenesulfonic acid/4-aminophenazone as the chromogenic

system. The high absorbance of the chromogen system at 500nm

gave the results.

Triglycerides + H2O Glycerol + Free fatty acids.

Glycerol + ATP L–α–Glycerol–phosphate + ADP

–α–Glycerol–Phosphate + O2

H2O2 + Dihydroxyacetonephosphate

Absorbance of sample

Absorbance of standard

X 200 = mg/dL

96

H2O2 + 3, 5–Dichlolo–2–hydroxybenzenesulfonic acid + 4–Aminophenazone

Peroxidase Quinonemonoimine dye + 2H2O.

Reagents

1. Working reagent

2. Standard (200mg/dL)

Procedure

For the estimation of triglycerides, 10µl each of serum and

working standard were mixed with 1ml of working reagent and kept at

room temperature for 15min and the absorbance was read at 500nm

against the reagent blank. For reagent blank, 10µl of distilled water

was added to 1ml of working reagent. Concentration of triglycerides

in serum samples was calculated as :

3.8.9.3. Estimation of serum HDL-cholesterol (Diagnostic

kit-Beacon Diagnostics, Kabilpore, Navsari, India)

Principle

In the presence of precipitating reagent, all lipoproteins of the

serum, except HDL fraction, were precipitated. After centrifugation,

the precipitate was discarded and the HDL-cholesterol content of the

supernatant was determined.

Reagents

1. Working reagent

2. Precipitating reagent

3. Standard (200mg/dL)

Absorbance of sample

Absorbance of standard

x 200 = mg/dL

97

Procedure

In the assay of HDL-cholesterol, 500µl of serum was added to

50µl of precipitating reagent and kept at room temperature for 10min.

Later, it was centrifuged at 3000 rpm for 10 min. The clear

supernatant was used for HDL-cholesterol estimation. 20µl of the

supernatant and 10µl of standard were incubated with 1ml of the

reagent for 5 min at 37C and the absorbance at 510nm was

measured against the reagent blank. For reagent blank, 10l of

distilled water was added to 1ml of the reagent. The amount of

HDL-cholesterol in the samples was calculated as:

3.8.9.4. Estimation of VLDL- and LDL-cholesterol (Friedwald

et al., 1972)

These were calculated using the formula

VLDL cholesterol = TG/5

LDL cholesterol = Total cholesterol –

(HDL cholesterol + VLDL cholesterol)

The values were expressed as mg/dL of plasma

3. 8.10. ESTIMATION OF MARKER ENZYMES

3.8.10.1. Assay of aspartate aminotransferase (AST, EC 2.6.1.1)

Plasma aspartate aminotransferase was assayed by using the

diagnostic kit based on the method of Reitman and Frankel (1957).

Absorbance of sample

Absorbance of

standard

x 100 x 1.1 = mg/dL

98

AST catalyses the transfer of amino group from L-aspartate to

α-ketoglutarate with the formation of oxaloacetate and glutamate. The

amount of oxaloacetate was measured by converting it into pyruvate

by treating with aniline citrate and then reacting the pyruvate with

2,4-dinitrophenylhydrazine to form 2,4-dinitrophenylhydrazone

derivative which is brown colored in alkaline medium. The absorbance

of this hydrazone derivative is correlated to AST activity.

Reagents

1. Buffered substrate: 2.66g of aspartate and 38mg of

α-ketoglutarate were dissolved in 20.5ml of 1N sodium

hydroxide, with gentle heating. This was made up to 100ml with

phosphate buffer (0.01 M, pH 7.4).

2. Aniline-citrate reagent: 50g of citric acid was dissolved in

50ml of distilled water and mixed with equal volume of

redistilled aniline.

3. Dinitrophenylhydrazine (DNPH) color reagent: 1.0mM DNPH in

2.0N hydrochloric acid.

4. Sodium hydroxide: 0.4N.

5. Pyruvate standard: 2.0mM.

Procedure

0.5ml of buffered substrate was added to 0.1ml of plasma and

placed in a water bath at 37C. To the blank tubes, 0.1ml distilled

water was added instead of serum. Exactly an hour later, 2 drops of

aniline citrate reagent and 0.5ml of DNPH reagent were added and

99

kept at room temperature for 20 min. Finally, 5.0ml 0.4N sodium

hydroxide was added. A set of standards was also treated in the same

manner and read at 520nm after 10 min.

The results were expressed as IU/L of plasma.

3.8.10.2. Assay of alanine aminotransferase (ALT, EC 2.6.1.2)

Plasma alanine aminotransferase was assayed by using the

diagnostic kit based on the method of Reitman and Frankel (1957).

ALT catalyses the transfer of amino group from L-alanine to

-ketoglutarate with the formation of pyruvate and glutamate. The

pyruvate so formed, is allowed to react with 2,4-dinitro

phenylhydrazine to produce 2,4-dinitrophenylhydrozone derivative

which is brown colored in alkaline medium. The absorbance of this

hydrazone derivative is correlated to ALT activity.

Reagents

1. Buffered substrate: 1.78g of DL-alanine and 38mg of

-ketoglutarate were dissolved in buffer. 0.5ml of sodium

hydroxide was added and the volume was made up to

100ml with phosphate buffer (0.01M, pH 7.4).

2. All other reagents were same as used for the assay of

aspartate transaminase.

100

Procedure

Procedure was same as that used for the assay of aspartate

transaminase except the incubation time which was reduced to

30 min (60 min for AST).

The results were expressed as IU/L of plasma.

3.8.10.3. Estimation of alkaline phosphatase (ALP, EC 3.1.2.3.1)

Plasma alkaline phosphatase was estimated by using the

diagnostic kit based on Kind and King’s method (1954).

ALP catalyses disodium phenyl phosphate into phenol and

disodium hydrogen phosphate at pH 10. Phenol so formed reacts with

4-aminoantipyrine in alkaline medium in the presence of oxidizing

agent potassium ferricyanide to form a red colored complex whose

absorbance is proportional to the enzyme activity.

Reagents

1. Buffered substrate: 0.01M Disodium phenyl phosphate

dissolved in carbonate-bicarbonate buffer (0.1M, pH 10).

2. Color reagent: 4-aminoantipyrine, sodium hydroxide and

potassium ferricyanide.

3. Phenol standard: 10mg%.

Procedure

The incubation mixture containing 1.0ml of buffered substrate,

3.1ml of deionised water and 0.1ml of plasma, was incubated at

101

37C. exactly after 15 min, 2.0ml of color reagent was added to all the

tubes. The control tubes received the enzyme after the addition of

color reagent. 0.1ml of standard and 0.1ml of distilled water (blank)

were also treated simultaneously and the color developed was read at

510nm.

The enzyme activity was expressed as IU/L of plasma.

3.8.10.4. Estimation of acid phosphatase (ACP, EC 3.1.3.2)

Plasma acid phosphatase level was measured based on Moss

(1984).

Breakdown of orthocarboxyphenyl phosphate liberates salisilic

acid which in turn increases the absorbance at 300nm thus

determining the reaction mixture. Under suitable conditions of

temperature (25˚C) and pH 5. One unit catalyzes 1µMoles of

orthocarboxyphenyl phosphate per minute.

Ortho phosphoric monoester + H2O Alcohol + Phosphoric acid.

Reagents

1. 0.1M sodium acetate buffer pH 5.

2. 3.65mM orthocarboxyphenyl phosphate

Procedure

2ml of 0.1M sodium acetate buffer and 0.5ml carboxyphenyl

phosphate were added to the cuvette. It was shaken well and blank

absorbance was recorded at 300nm. To this 10µl of dilute enzyme

102

3500 x enzyme con. in the sample

solution added and absorbance was recorded at 300nm for the sample

solution.

Calculation

Amount of ACP present in the sample = OD value per minute

The enzyme activity was expressed as µl/ml of plasma.

3.8.11. ESTIMATION OF ALBUMIN

Albumin in the serum was estimated by Biuret

method (Reinhold, 1953).

Proteins form a purple coloured complex with cupric ions in

alkaline solution. The reaction takes its name from the simple

compound biuret which reacts in the same way. The intensity of the

purple color is proportional to the amount of protein present in the

sample.

Reagents

1. Stock Biuret reagent: 45g of sodium potassium tartarate was

dissolved in 400ml of 0.2N sodium hydroxide and 15g of

copper sulphate was added and stirred. 5.0g potassium

iodide was then added, dissolved and made up to 1 litre with

0.2N sodium hydroxide.

2. Dilute Biuret reagent: 200ml of stock Biuret reagent was

diluted to 1 litre with 0.2N sodium hydroxide containing 5.0g

potassium iodide/l.

103

3. Standard egg albumin: 500mg/100ml distilled water (small

quantity of alkali was added to dissolve albumin).

4. Sodium sulphite solution: 28%.

Procedure

0.5ml of serum was taken in a test tube and 6.5ml of sodium

sulphite solution was added and mixed. To the mixture, 3.0ml of ether

was added, stoppered, shaken well for 20s and then centrifuged for

5 min. 3.0ml of the clear supernatant was taken for the estimation of

albumin and treated with 5ml of Biuret reagent simultaneously,

2.0ml of standard egg albumin were mixed with 1.0ml of water and

treated with 5.0ml of Biuret reagent. The purple color developed was

read at 540nm after 15 min using reagent blank.

Values were expressed as g/dL of serum.

3.8.12. ESTIMATION OF GLOBULIN

Serum globulin concentration was calculated using the

following formula after the estimation of total protein and albumin.

Globulin = Total protein – albumin

3. 8.13. ESTIMATION OF GLYCOPROTEIN COMPONENTS

3. 8.13.1. Estimation of total hexoses

Total hexoses in the plasma and tissues were estimated by the

method of Niebes (1972).

104

Reagents

1. Orcinol – sulphuric acid mixture: 1.6g of orcinol was

dissolved in 100ml of water. 1.0ml of this was mixed with

7.5ml H2SO4: H2O mixture (3:2 v/v). This was prepared fresh

before use.

2. 5mg of galactose and 5.0mg of mannose were dissolved in

100ml of water. This had a concentration of 100g/ml.

Procedure

0.2ml of the plasma or homogenate was mixed with 8.5ml of

orcinol–H2SO4. The tubes were then heated at 80C for 15 min, cooled

and read at 540nm after 20 min. Standard and blank containing

0.2ml of 0.2 N H2SO4 were also processed similarly.

Total hexose content was expressed as mg/dL of plasma or

mg/100g of tissue.

3. 8.13.2. Estimation of hexosamine

Hexosamine in the plasma and tissues was determined by the

method of Elson and Morgon (1933).

Reagents

1. Ethanol: 95%

2. Hydrochloric acid: 3N

3. Sodium hydroxide: 3N

4. Acetyl acetone reagent: 1ml of acetyl acetone in 50ml of 0.5N

sodium carbonate, freshly prepared.

105

5. Ehrlich reagent: 0.8g of p-dimethylaminobenzaldehyde

(recrystallized as the hydrochloride) dissolved in 30ml of

methanol and 30ml of con. HCl.

6. Glucosamine standard: 0.05mg/ml of free glucosamine in

water.

Procedure

To 0.1ml of plasma or homogenate in a test tube graduated at

10ml, 5ml of 95% ethanol was added and mixed well, centrifuged for

15 min, decanted, and the precipitate was suspended in 3ml of 95%

ethanol, centrifuged and decanted. To the precipitated protein, 2ml of

3N HCl was added and hydrolysed in a boiling water bath for 4 hours.

The hydrolysate was neutralised with 3N NaOH. 1ml of the

acetyl acetone was added to 1ml of the aliquot, 1ml of the water

(blank) and 1ml of standard. The tubes were capped with marbles to

prevent evaporation and placed in a boiling water bath for 15 min. The

tubes were cooled under tap water. 5ml of 95% ethanol was added

and mixed well. To these tubes 1ml of Ehrlich reagent was added and

mixed well. This was diluted to 10ml with 95% ethanol. Absorbance

was measured at 530nm after 30 min.

Hexosamine content was expressed as mg/dL of plasma or

mg/100g of tissue.

106

3.8.13.3. Estimation of sialic acid

Sialic acid in the plasma and tissues was estimated by the

method of Welmer et al. (1952).

Reagents

1. TCA: 5%

2. Acid mixture: 90ml of glacial acetic acid and 10ml of

concentrated sulphuric acid.

3. Diphenylamine reagent: 1g of diphenylamine recrystallized

from ethanol was dissolved in 100ml of mixture.

4. Sialic acid standard: 0.2mg/ml

Procedure

4.8ml of 5% TCA was added slowly to 0.2ml of plasma or

homogenate, and 0.2ml of orosomucoid standard in a separate tube.

The test tube was placed in a boiling water bath for exactly 15 min

with a glass marble to prevent evaporation; the tubes were cooled by

immersion in water and filtered. 2ml of clear filtrate in each of tubes

was pipette out and 4ml of DPA reagent was added into one of each

pair of tubes and 4ml of acid-mixture containing without DPA into

another.

The reagent blank was prepared by adding 2ml of 5% TCA and

4ml of DPA reagent. The tubes were mixed well, capped with a glass

marble and immersed in a boiling water bath for exactly 30 min. The

tubes were cooled in water and the absorbance was determined at

107

530 nm with a reagent blank set at zero. Sialic acid content was

expressed as mg/dL of plasma or mg/100g of tissue.

3.8.13.4. Estimation of fucose

Fucose in the plasma and tissues was estimated by the method

of Dische and Shettles (1948).

Reagents

1. Sulphuric acid reagent: Con. H2SO4 and distilled H2O were

mixed in the ratio of 6:1.

2. Cysteine hydrochloride reagent: 3% cysteine hydrochloride in

water 0.1N NaOH

Procedure

To 2.2ml of plasma or homogenate, 4.8ml of sulphuric acid

reagent was added and heated in a boiling water bath for 3 min. The

sample was cooled and 0.1ml of cysteine hydrochloride reagent was

added, 0.5ml of 0.1N NaOH was also treated in the same way for

blank, after 25 min the optical density was measured at 393 and

430nm.

Fucose content was expressed as mg/dL of plasma or mg/100g

of tissue.

108

3.8.14. MEMBRANE-BOUND ENZYMES

3.8.14.1. Determination of total ATPases (ATP-phosphohydrolase)

(EC: 3.6.1.3)

The activity of the enzyme in the erythrocytes and tissues was

estimated according to the method of Evans (1969).

Reagents

1. Tris-HCl buffer: 0.1M, pH 7.0

2. Sodium chloride: 0.1M

3. Potassium chloride: 0.1M

4. Adenosine triphosphate: 0.01M ATP

5. TCA: 10%

Procedure

A 2ml incubation volume of test consisted of 1.5ml of Tris-HCl

buffer, sodium chloride, potassium chloride, adenosine triphosphaste,

and suitable aliquot of the enzyme. A control was also run

simultaneously without the enzyme. The tubes were incubated at

37C for 15 min. The reaction was arrested by 10% trichloroacetic

acid. The enzyme was then added to the control. The tubes were

centrifuged at 3000g for 10 min. The inorganic phosphorus liberated

was estimated by Fiske and Subbarow (1925) method.

The total ATPases activity was expressed as mol of Pi

liberated/h/mg of protein for erythrocytes and tissues.

109

3.8.14.2. Determination of (Na+ K+ )- ATPase [E.C. 3.6.1.3]

Sodium and potassium dependent ATPase activity in the

erythrocytes and tissues was assayed by the method of Bonting

(1970).

Reagents

1. Tris-HCl buffer: 184mM, pH 7.5.

2. Magnesium sulphate: 50mM.

3. Sodium chloride: 160mM.

4. Potassium chloride: 50mM.

5. EDTA: 1mM.

6. ATP: 4mM.

7. TCA: 10%.

Procedure

The incubation volume of the test consisted of 1ml of Tris-HCl

buffer and 0.2ml each of magnesium sulphate, potassium chloride,

sodium chloride, EDTA, ATP and suitable aliquot of the enzyme.

Control without enzyme was also treated similarly. The tubes were

incubated at 37C for 15 min. The reaction was arrested by 10%

trichloroacetic acid followed by the addition of the enzyme to the

control. The tubes were centrifuged at 3000xg for 10 min. The

inorganic phosphorus was estimated by the method of Fiske and

Subbarow (1925).

The enzyme activity was expressed as mol of Pi liberated/h/mg

of protein for erythrocytes and tissues.

110

3.8.14.3. Determination of Mg2+- ATPase [E.C. 3.6.1.4]

The activity of the enzyme in the erythrocytes and tissues was

estimated by the method of Ohnishi et al. (1982).

Reagents

1. Tris-HCl buffer: 125mM, pH. 7.6.

2. Magnesium chloride: 25mM.

3. ATP: 0.01M.

4. TCA: 10%.

Procedure

A 0.5ml of incubation volume of the test consisted of 0.1ml each

of Tris-HCl buffer, magnesium chloride, ATP, distilled water and

enzyme preparation. Control without enzyme was run simultaneously.

The tubes were incubated at 37C for 15 min. The reaction was then

arrested by 10% trichloroacetaic acid followed by 0.1ml of the enzyme

of the control. The tubes were centrifuged at 3000g for 10 min. The

inorganic phosphorus was estimated by the method of Fiske and

Subbarow (1925).

The enzyme activity was expressed as mol of Pi liberated/h/mg

of protein for erythrocytes and tissues.

3.8.14.4. Determination of Ca2+ATPase [E.C.3.6.1.5]

The activity of Ca2+-ATPase activity in the erythrocytes and

tissues was measured according to the method of Hjerten and Pan,

(1983).

111

Reagents

1. Tris-HCl buffer: 125mM, pH 8.0.

2. Calcium chloride: 50mM.

3. ATP: 10mM.

4. TCA: 10%.

5. Fiske and Subbarow reagents.

Procedure

The incubation mixture contained 0.1ml of buffer, 0.1ml of

calcium chloride, 0.1ml of ATP, 0.1ml of water and 0.1 of enzyme

(Tissue homogenate or erythrocytes membrane). The contents were

incubated at 37C for 15 min. The reaction was then arrested by the

addition of 0.5ml of ice-cold 10% TCA. The amount of phosphorus

liberated was estimated by the method of Fiske and Subbarow.

The enzyme activity was expressed as mol of Pi liberated/h/mg

of protein for erythrocytes and tissues.

3.8.15. Enzymes of carbohydrate metabolism in serum & tissue

3.8.15.1 Assay of glucokinase (Brandstrup et al., 1957)

Principle

Glucokinase was assayed by the estimation of glucose-6-

phosphate formed from glucose.

Reagents

1. Tris HCl buffer – 0.1M, pH 7.4.

2. Glucose – 0.005M.

112

3. Adenosine triphosphate (ATP) – 0.072M.

4. Magnesium chloride – 0.05M.

5. Potassium dihydrogen phosphate – 0.0125M.

6. Potassium chloride – 0.1M.

7. Sodium fluoride – 0.5M.

8. TCA-10%.

9. Tris-HCI buffer -0.01M, pH 8.0.

10. Reagents for the estimation of glucose.

Procedure

1g of liver was homogenized in 0.1M Tris HCl buffer, pH 7.4, at

4C in a potter-Elvehjam homogenizer for 3min. The homogenate was

centrifuged at 2500 rpm for 10min.

The reaction mixture in a total volume of 5.3ml contained 1ml of

glucose solution, 0.5ml of ATP solution, 0.1ml of magnesium chloride

solution, 0.4ml of potassium dihydrogen phosphate solution, 0.4ml of

potassium chloride solution, 0.4ml of sodium fluoride solution and

2.5ml of Tris-HCl buffer. The mixture was pre-incubated at 37C for

5min. The reaction was initiated by the addition of 2ml of tissue

homogenate. 1ml of the reaction mixture was immediately removed

to the tubes containing 1ml of 10% TCA which was considered as zero

time. A second aliquot was removed after 30 min incubation at 37C.

The protein precipitate was removed by centrifugation and the

residual glucose in the supernatant was estimated. Protein content of

the samples was estimated by the method of Lowry et al. (1951). The

113

enzyme activity was expressed as µ Moles of glucose phosphorylated /

min / g protein.

3.8.15.2 Assay of glucose–6–phosphatase (Baginsky et al., 1992)

Principle

Glucose–6–phosphatase was assayed by estimation of inorganic

phosphate liberated from glucose–6–phosphate.

Reagents

1. Sucrose (250mM).

2. Sucrose/EDTA buffer (0.25M/mM, pH 7).

3. Glucose –6– phosphate (100mM).

4. Imidazol buffer (100mM, pH 6.5).

5. Na2HPO4 (1.5mM).

6. TCA (10%).

7. Ascorbate (2%).

8. Ammonium molybdate (1%).

9. Sodium arsenite (2%).

10. Sodium citrate (2%).

Procedure

On a precooled watch glass, 1g of liver was chopped into small

pieces and homogenized in a Potter-Elvehjem homogenizer for 2min

with ice-cold sucrose (15ml, 250mM). The homogenate was

centrifuged at 2C for 30 min at 9000 rpm.

114

Four tubes labeled as sample, control, blank and standard were

taken. To each tube was added 0.1ml of sucrose/EDTA buffer

(0.25M/mM, pH 7), 0.1ml of glucose–6–phosphate (100mM) and 0.1ml

of imidazol buffer (100mM, pH 6.5). This was followed by the addition

of 0.1ml of liver homogenate supernatant to the sample tube, 0.1ml of

distillated water to the blank and 0.1ml of Na2HPO4 (1.5mM) to the

standard tube. All the tubes were incubated at 37C for 1min and the

enzyme activity was terminated by adding 2ml TCA/ascorbate

(10%/2% : w/v). Liver homogenate supernatant (0.1 ml) was added

to the control tube only. It was followed by centrifugation of all tubes

at 3,000 rpm for 10 min. To 1ml of the clear supernatant were added

0.5ml ammonium molybdate (1%) and 1ml of sodium arsenite/sodium

citrate (2%/2%: w/v). The tubes were then allowed to stand for a

minute at room temperature and absorbance was read at 700nm. The

amount of inorganic phosphate liberated by the enzyme was

calculated by comparing with the absorbance values of the standard.

Enzyme activity was expressed as µmole of Pi liberated/min/mg

protein. Protein content of the samples was estimated by the method

of Lowry et al. (1951).

3.8.15.3 Assay of fructose 1, 6-bisphosphatase

(Fructose 1, 6-bisphosphate phosphohydrolase: EC 3.1.3.11)

Fructose 1, 6-bisphosphatase was assayed by the method of

Gancedo and Gancedo (1971).

115

Reagents

1. Tris-HCl buffer: 0.1M, pH 7.0

2. Substrate: Fructose 1,6- bisphosphate, 0.05M

3. Magnesium chloride: 0.1M

4. Potassium chloride: 0.1M

5. EDTA solution: 0.001M

6. TCA: 10%

7. Molybdic acid: 2.5% ammonium molybdate in 3N

sulphuric acid.

8. ANSA reagent: As above.

9. Phosphorus stock Standard: 35.1mg of potassium

dihydrogen phosphate was dissolved in 100ml of distilled

water (80g/ml).

Procedure

The assay medium in a final volume of 2.0ml contained 1.0ml

buffer, 0.4ml of substrate, 0.1ml each of magnesium chloride, 0.2ml

potassium chloride, 0.1ml of EDTA and 0.2ml of enzyme source.

The incubation was carried out at 37C for 15min. The reaction was

terminated by the addition of 1.0ml of 10% TCA. The suspension was

centrifuged and the phosphorus content of the supernatant was

estimated according to the method described by Fiske and Subbarow

(1925).

To 1ml of an aliquot of the supernatant, 0.3ml of distilled water

and 0.5ml of ammonium molybdate were added. After 10min, 0.2ml

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of ANSA was added. The tubes were shaken well, kept aside for 20

min and the blue color developed was read at 620nm.

The values were expressed as mol of Pi liberated/h/mg protein.

3.8.16 Assay of Hepatic Glycogen synthase and Glycogen

phosphorylase

Hepatic Glycogen synthase and Glycogen phosphorylase were

assayed by the method of Leloir and Goldmberg (1960) and Cornblath

et al. (1963), respectively.

Reagents

1. Tris HCL buffer: 0.05M, Ph 7.5.

2. NADP+ : 0.1M.

3. Magnesium chloride: 0.1M.

4. Potassium chloride:0.1M.

5. EDTA solution: 0.001M.

6. TCA: 10%.

7. Molybdic acid: 2.5% ammonium molybdate in 3N

sulphuric acid.

8. Phosphorous stock standard: 35.1mg of potassium

dihydrogen phosphate was dissolved in 100ml of distilled

water (80µg/ml).

Procedure

The activity of glycogen synthase was estimated by coupling it

with pyruvate kinase activity. It was measured by the amount of

117

uridine diphosphate (UDP) formed from UDP-glucose in the presence

of glycogen and glucose-6-phosphate. Pyruate kinasecatalyzes the

transfer of phosphate from phosphoenolpyruate to UDP and the

pyruate liberated was estimated colorimetrically (Leloir and

Goldmberg, 1960). The property of synthesizing glycogen from

glucose-1-phosphate liberating inorganic phosphate is made use in

the assay of glycogen phophorylase activity (Cornblath et al., 1963).

The activity of glycogen synthase enzyme was expressed as

µmoles of UDP formed/h/mg protein and the activity of glycogen

phosphorylase enzyme was expressed as µmoles of Pi liberated/h/mg

protein.

3.9. GLUT-4 GENE EXPRESSION ANALYSIS

3.9.1. Isolation of total RNA

Total RNA was isolated from control and experimental samples

using TRIR kit (total RNA isolation reagent) from ABgene, UK. Briefly,

100mg fresh tissue was homogenized with 1ml TRIR and the

homogenate were transferred immediately to a microfuge tube and kept

at -80°C for 60 min to permit the complete dissociation of

nucleoprotein complexes. Then, 0.2ml of chloroform was added,

vortexed vigorously for 1min and placed on ice at 4°C for 5 min. The

homogenate were centrifuged at 12,000×g for 15 min at 4°C. The

aqueous phase was carefully transferred to a fresh microfuge tube and

an equal volume of isopropanol was added, vortexed vigorously for 15

seconds and placed on ice at 4°C for 10 min. The samples were

118

centrifuged at 12,000×g for 10min at 4°C. The supernatant was

discarded and RNA pellet was washed with 1ml of 75% ethanol by

vortexing and subsequent centrifugation for 5 min at 7,500×g (4°C).

The supernatant was removed and RNA pellets were mixed with 50µl of

autoclaved Milli-Q water and dissolved by heating in a water bath for

10 min at 60°C. The concentration and purity of RNA were determined

spectrophotometrically at absorbance 260/280nm. The purity of RNA

obtained was >1.75. The yield of RNA is expressed in microgram (µg).

3.9.2 Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

analysis

Total RNA sample was used for the synthesis of cDNAs by

Qiagen One-step Reverse Transcriptase-Polymerase Chain Reaction

kit. This provides a convenient format for highly efficient and specific

RT-PCR using total RNA/mRNA. The kit contains optimized

components that allow both reverse transcription and PCR

amplification to take place in what is commonly referred to as a

“One-Step” reaction.

Principle

The reverse transcriptase-polymerase chain reaction is one of

the most powerful techniques in molecular biology. RT-PCR selectively

amplifies the first strand of cDNA that has been synthesized in vitro by

RNA polymerase from mRNA templates by reverse transcription. The

cDNA is first denatured by heating in the presence of a large molar

excess of two oligonucleotide primers and four dNTPs. The reaction

119

mixture is then cooled to a temperature that allows the oligonucleotide

primers to anneal to their target sequences, after which the annealed

primers are extended with DNA polymerase. The cycle of denaturation,

annealing and DNA synthesis is then repeated for the required

number of times.

Kit components

1 QIAGEN One Step RT-PCR Enzyme Mix–50μl (contains

Omniscript Reverse Transcriptase, Sensiscript Reverse

Transcriptase and Hot Star Taq DNA Polymerase).

2 QIAGEN One Step RT-PCR buffer (5x) – 250μl.

3 dNTP Mix 10mM each–50μl.

4 RNase- free water – 1.0ml.

5 Oligonucleotide primers.

The following gene specific oligonucleotide primers were used for

the generation of cDNAs.

Primers for Rat GLUT4 (Liu et al., 2006) Amplicon Size – 150 bp

Sense primer : 5'-GGG CTG TGA GTG AGT GCT TTC-3'

Anti-sense primer : 5'-CAG CGA GGC AAG GCT AGA-3'

Primers for Rat β-actin (Houghton et al., 2006) Amplicon Size –

374 bp

Sense primer : 5’- GCC ATG TAC GTA GCC ATC CA-3’

Anti-sense primer : 5’- GAA CCG CTC ATT GCC GAT AG-3’

120

For the first strand cDNA synthesis, 2µg of RNA template was

added with master mix containing 10µl of RT-PCR buffer, 2µl of dNTP

mix, 2µl of RT-PCR enzyme, appropriate volume of 6µM (genes of

interest) and 1.5µM (house keeping gene) of primers, respectively and

the master mix was made up to 50µl with RNase-free water. RT-PCR

was performed using the thermal cycler (eppendorf) programmed as

follows:

Reverse Transcription 50°C for 30 min

PCR Activation 95°C for 15 min

Denaturation 94°C for 1.5 min

Annealing 60°C for 1.5 min

Extension 72°C for 3.0 min

No. of cycles 35

Final Extension 72°C for 10 min

Finally, the reaction mixture containing the RT-PCR products

was separated/resolved in 2% agarose gel along with 100bp marker

DNA. Gels were visualized in Gel documentation apparatus (Bio Rad)

and the cDNAs of interest were normalized to that of the house

keeping gene or internal control (β-actin), which is simultaneously

co-amplified in the same reaction vial along with gene of interest.

121

3.9.3. Agarose Gel Electrophoresis

Agarose gel electrophoresis is simple and highly effective method

for separation, identification and purification of DNA (Sambrook et al.,

1989).

Principle

The separation is carried out under an electric field applied to

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

negatively charged phosphate along the backbone of DNA. The rate of

migration of a linear DNA is inversely proportional to the ratio of its

molecular weight. Thus, the larger molecules travel at a much slower

speed when compared to smaller one.

Reagents

1. Tank buffer (Tris Boric acid EDTA – TBE- 5x): Tris (54g) and

boric acid (27.5g ) were dissolved in 800ml of autoclaved distilled

water and 20ml of 0.5M EDTA was added and final volume was

made up to 1000ml with autoclaved double distilled water.

2. 1x TBE buffer (pH 8.0): 60ml of 5x buffer was mixed with 200ml

of double distilled water; pH adjusted to 8.0 and made up to

300ml with autoclaved double distilled water.

3. Ethidium bromide (10mg/ml).

4. Agarose (2%): 1g of standard agarose dissolved in 50ml of 1x

buffer.

122

5. Gel loading dye:

0.1 M EDTA - 2ml

0.01 M Tris- HCl - 0.1ml

0.25% Bromophenol blue - 25mg

50% Glycerol - 5ml

To this, 2.9ml of autoclaved double distilled water was added to

make the final volume of 10ml.

Procedure

The agarose mix (2%) was prepared in 1x–TBE buffer in a

microwave oven to get a clear homogeneous solution. The solution was

cooled to 55C and 2µl ethidium bromide was added. Then the

solution was poured in to the casting tray with the combs placed.

After allowing it to settle for 30 min, the comb was removed carefully.

The gel was kept completely submerged in the buffer. The electrodes

were connected and were allowed to run at 60mA as a pre run for

about 15min. Then 5μl of PCR products was taken from each reaction

tube mixed with 10µl gel loading dye and then loaded to each well. 2µl

of 100 base pair DNA molecular marker mixed with 10µl gel loading

dye was simultaneously loaded in the first well. The power supply was

turned on and the current adjusted to 60mA. The gel was run

(3 hours) till the dye reaches the end of the gel. Then the gel

containing cDNA was visualized in Gel documentation apparatus

(Bio Rad) and quantified. The band intensity of Glut-4 was normalized

to that of β–actin and the internal control.

123

3.10. GLUT-4 PROTEIN EXPRESSION ANALYSIS

3.10.1. Preparation of Sample

Plasma membrane fraction from skeletal muscles

(Gasrtocnemius) of control and experimental animals were prepared

as described by Dombrosokki et al., (1996).

Reagents

1. Protease inhibitor: Commercially available protease inhibitor

cocktail (Sigma Chemical Company, USA) was used.

2. Buffer – A (pH 7.0): 84mg of sodium bicarbonate (NaHCO3) was

dissolved in 75ml of distilled water and pH adjusted to 7. To

this, 85.575g sucrose, 1.742mg phenylmethylsulfonylfluoride

(PMSF) and 32.5mg sodium azide (NaN3), protease inhibitors

were added and made upto 100ml with distilled water.

3. Buffer – B (pH 7.0): 84mg of sodium bicarbonate (NaHCO3) was

dissolved in 75ml of distilled water and pH adjusted to 7. To

this, 1.742mg PMSF and 32.5mg sodium azide and protease

inhibitor were added and made upto 100ml with distilled water.

4. Sucrose gradient (w/w): A discontinuous sucrose density

gradient prepared by layering successive decreasing sucrose

density solution upon one another. The preparation and

centrifugation of a discontinuous gradient containing sucrose

solution from 25, 32 and 35%. This gradient gives good

separation of plasma membrane and total membrane (or)

cytosolic fractions.

124

25% sucrose (w/w) : For 20ml of 25% sucrose, 5g of

sucrose was dissolved in 15g of buffer B.

32% sucrose (w/w) : For 20ml of 32% sucrose, 6.2g of

sucrose was dissolved in 13.6g of buffer B.

35% sucrose (w/w) : For 20ml of 35% sucrose, 7g of

sucrose was dissolved in 13g of buffer B.

Preparation of sucrose gradient

The gradient was prepared by layering progressively less dense

sucrose solution upon one another. Briefly, sucrose solutions were

added into the polyallomer tube (Ultracentrifugation tube) slowly and

steadily, starting with the 35% solution. Once the 35 % solution had

drained into the tube, the 32% solution could be loaded on top of the

35% solution. This procedure was continued with the 25% solution.

There was enough space left at the top of the tube upon which to load

the 0.5ml of sample.

Sub-cellular fractionation

Skeletal muscles from control and experimental animals were

simultaneously processed for the preparation different fractions. All

steps were carried out on ice or at 4C. Tissue (~1 g) was first cleaned

of all visible fat, nerve, and blood vessels and minced in buffer – A.

The minced muscle was homogenized (1g/15ml of buffer-A) using a

polytron equipped homogenizer at a precise low setting. The resulting

homogenate was centrifuged at 1,300xg for 10 min. The resultant

supernatant was centrifuged at 1,90,000xg for 1 hour. The resultant

125

supernatant was saved, and sampled as a cytosolic fraction. The pellet

was resuspended in buffer – A and applied on discontinuous sucrose

gradients (25%, 32% and 35% wt/wt) and centrifuged at 1,50,000xg

for 16 hours. Plasma membrane at 25–32% interface was recovered,

diluted with buffer − B, and centrifuged at 1,90,000xg for 1hour.

Plasma membrane fraction (pellet) were resuspended in buffer − A and

kept at −80˚C until used. Protein concentration in the sample was

determined prior to the western blot analysis.

3.10.2 Estimation of Total Protein

Protein concentration was determined as per the method of

Lowry et al. (1951) with bovine serum albumin (BSA) as the standard.

3.10.3 Western Blot Analysis

Separation of proteins

Proteins were separated by SDS-Polyacrylamide gel

electrophoresis as described by Laemmli (1970).

Principle

SDS-PAGE involves the separation of protein based on their

size. By heating the sample under denaturing and reducing

conditions, proteins become unfolded and coated with SDS detergent

molecule, acquiring a high net negative charge that is proportional to

the length of the polypeptide chain. When loaded on to a gel matrix

and placed in an electric field, the negatively charged protein

molecules migrate towards the positively charged electrode and are

126

separated by a molecular sieving effect. A molecular weight protein

marker that produces bands of known size is used to help identify

proteins of interest.

Reagents

1. Acrylamide/Bis (30% T, 2.67% C)

29.2g acrylamide and 800mg N’N’–bis-methylene-acrylamide

were dissolved in 100ml of deionized water. Filtered and stored at

4˚C in the dark (30 days maximum).

2. 10% (w/v) SDS

10g SDS was dissolved in 90ml water with gentle stirring and

brought to 100ml with deionized water.

3. 1.5 M Tris – HCl (pH 8.8)

18.15g Tris base was dissolved in 80ml deionized water.

Adjusted to pH 8.8 with 6N HCl and brought total volume to

100ml with deionized water and stored at 4˚C.

4. 0.5 M Tris – HCl (pH 6.8)

6g Tris base was dissolved in 60ml deionized water. Adjusted to

pH 6.8 with 6N HCl and brought total volume to 100ml with

deionized water and stored at 4˚C.

5. 10% APS (Fresh daily)

100mg ammonium per sulfate was dissolved in 1ml of deionized

water.

6. N’ N’ - Tetramethyl ethylene diamine (TEMED)

Commercially available

127

7. Sample Buffer (SDS Reducing Buffer)

1.25ml 0.5M Tris-HCl (pH 6.8), 2.5ml glycerol, 2ml 10% (w/v)

SDS were added to 0.2ml of 0.5% (w/v) bromophenol blue and

brought total volume to 9.5ml with 3.55 ml deionized water. Stored

at room temperature. 50μl β - mercaptoethnaol was added to 950

μl of sample buffer prior to use.

8. 10X electrophoresis buffer (pH 8.3)

30.3g Tris base, 144.0g Glycine and 10g SDS were dissolved in

800ml deionized water and brought total volume to 1litre with

deionized water and stored at 4˚C.

Procedure

Gel formulation (10 ml)

10% running gel was prepared by mixing the reagents as shown

below.

Deionized H2O – 4.1ml

30% degased acrylamide/bis – 3.3ml

1.5 M Tris – HCl (pH 8.8) – 2.5ml

10 % (w/v) SDS – 0.1ml

Gently mixed and degassed the mixture for 15 min. 50µl of 10%

APS and 5µl TEMED were added prior to pouring the gel and swirled

gently to initiate polymerization. This mixture was poured into 1mm

thickness gel casting plate setup and allowed to 20–30 min for

polymerization.

128

5% stacking gel was prepared by mixing the reagents as given below.

Deionized H2O – 3.4ml

30% degased acrylamide/bis – 830µl

1.5 M Tris – HCl (pH 6.8) – 630µl

10 % (w/v) SDS – 50µl

Gently mixed and degassed the mixture for 15 min. 50µl of 10%

APS and 5µl TEMED were added prior to pouring the gel and swirled

gently to initiate polymerization. This mixture was added on top of the

10% running gel. Then, comb (1mm thickness) was inserted and

allowed to form the well.

Separation of proteins

Equal volume (25μg) of samples (plasma membrane and

cytosolic protein) were diluted 1:2 with sample buffer, heated at 95˚C

for 4 minutes and then cooled on ice for 5 minutes. Samples were

loaded to 10% SDS–PAGE in a Bio – Rad miniature slab gel apparatus.

The prestained protein molecular weight markers used were

β–galactosidase (117kDa), bovine serum albumin (85kDa), ovalbumin

(49kDa), β–lacto globulin (25kDa) and lysozyme (19kDa) carbonic

anhydrase (34kDa), β (Fermentas). Electrophoresis of protein was

performed at 100V (constant) until the dye front reaches the bottom of

the running gel.

129

Transfer of proteins to the membrane and immunoblotting

Reagents

1. Transfer buffer

3g Tris, and 25g glycine were dissolved in 800ml of double

distilled water and made upto 1000ml with 200ml of methanol.

2. Polyvinylidene difluoride (PVDF) membrane (Amersham

Biosciences Ltd., UK)

3. TBS

4g of NaCl and 10ml of 1M Tris HCl (pH 7.6) were dissolved in

450ml of distilled water, adjusted the pH to 7.6 and made upto

500ml with distilled water.

4. TBS-T

500μl of Tween-20 was dissolved in 500ml of TBS and checked

the pH 7.6

5. 5% Blocking solution

500mg of blocking reagent was dissolved in 10ml of TBS-T

solution.

Procedure

After the separation of protein by SDS-PAGE, the stacking gel

was cut and discarded; the separating gel was briefly rinsed in

distilled water 2-3 min and then equilibrated in cold transfer buffer

under gentle agitation during 5-10 min. In the mean time, fibre pad

and Whattman paper and PVDF membrane were soaked in cold

transfer buffer. Transfer sandwich was assembled in the following

order from anode(+) to cathode(-).

130

a. + ve end

b. Fibre pad

c. Filter paper soaked in transfer buffer

d. PVDF Membrane

e. Gel

f. Filter paper soaked in transfer buffer

g. Fibre pad

h. - ve end

The setup was placed in the transfer apparatus filled with cold

transfer buffer and subjected to an electric current at 100V for 1hour

under cold condition. After the transfer, the PVDF blot was removed

from the transfer system blocked the unreacted sites on the

membrane to reduce the amount of non-specific binding during

subsequent steps in the assay using 5% blocking solution for 1 hour.

After the blocking is over, decanted the blocking solution and rinsed

the membrane in TBS-T and incubated for 1h in GLUT4 antibody at

room temperature, which was diluted 1:1000 with TBS-T. Following

incubation, the blot was washed for three times (5 minutes each) with

TBS-T and TBS. After washing, the blot was incubated for 1 hour with

horseradish peroxidase conjugated rabbit secondary antibody, which

was diluted 1:5000 with TBS-T. Following incubation, the blot was

washed for three times (5 minutes each) with TBS-T and TBS. Drained

the excess wash buffer from the washed blot and placed them, protein

side up on a sheet of Saran Wrap™. The detection reagent mixture [an

equal volume of detection solution 1 with detection solution 2

131

(Enhanced Chemiluminescence, Amersham Biosciences, UK)] was

pipetted on to the blot and incubated for 30-60 seconds and drained

off excess reagent. The blotted protein was quantified using Quantity

one software system (Bio-Rad).

Stripping and reprobing membrane

Membrane was stored wet wrapped in Saran Wrap™ in a

refrigerator (2-8˚C) after immune detection. The membrane was

submerged in stripping buffer [62.5mM Tris-HCl (pH 6.7); 2% SDS;

100mM 2- mercaptoethanol] and incubated at 50˚C for 30 min with

occasional agitation. Then the membrane was washed for 2 times

(each 10 min) in TBS-T at room temperature using large volume of

wash buffer. The membrane was blocked by immersing in 5% blocking

solution for 1 hour at room temperature. After the blocking is over,

decanted the blocking solution and rinsed the membrane in TBS-T

and incubated for 1hour in β-actin antibody at room temperature,

which was diluted 1:5000 with TBS-T. Repeated the immunodetection

protocol as detailed previously.

3. 11. HISTOLOGICAL STUDIES

3. 11. 1. Light microscopic studies–Paraffin method (Humason,

1979)

Following solutions were used:

1. Physiological saline (0.9%)

2. Bouin-Hollande fixative

132

3. Ehrlich’s hematoxylin

4. Eosin

Procedure

The pancreas from untreated and parallel experimental groups

were blotted free of mucus, washed in physiological saline, cut into

pieces of desired size and fixed in Bouin-Hollande fixative for

72 hours. After fixation, the tissues were washed in 70% alcohol for

two or three days to remove the excess picric acid and dehydrated in

graded series of alcohol. The tissues were cleared using xylene. The

cleared tissues were infiltrated with molten paraffin at 58-60C

through three changes (20-30 min each) and finally embedded in

paraffin. 3-5µm thick sections of all the tissues were obtained using a

rotary microtome (Leica, Germany) and stained in Ehrlich’s

hematoxylin with eosin as the counter stain. The slides were

mounted using DPX mountant.

3.11.2. Light microscopic as well as transmission electron

microscopic studies adopting resin embedding

Plastic embedding was done according to Hayat (1981), using

Karnovsky’s fluid (Karnovsky, 1965) as the perfusate.

Reagents

1. Perfusion fluid

The perfusate (Karnovsky’s fluid) was prepared with the

following composition: glutaraldehyde 1% and paraformaldehyde 1%

in phosphate buffer 0.1M

133

2. Phosphate buffer (0.2M)

3. Washing buffer

Prepared by dissolving 5g of sucrose in 100ml of 0.1M

phosphate buffer.

4. Osmium tetroxide

A stock solution of 2% osmium tetroxide was prepared

5. Spurr’s mixture

Prepared by adding 8ml of 2-nonen-1-ylsuccinic anhydride

(NSA), 1ml of 4-vinylcyclohexane dioxide, 10ml of resin and 0.1ml of

2-dimethylaminoethanol (DMAE).

6. Toluidine blue (TBO)

Aqueous toluidine blue O solution was prepared by dissolving

0.5g toluidine blue in benzoate buffer at pH 4.4.

7. Uranyl acetate

Uranyl acetate was prepared by adding 10ml of 50% ethanol to

a saturated solution of uranyl acetate. After two min of centrifugation,

the excess uranyl acetate was allowed to settle and the clear

supernatant was used as the stain (Hess and Thurston, 1977).

8. Lead Citrate

One half pellet of sodium hydroxide was added to 12ml of

double distilled water and centrifuged. 50mg of lead citrate (Anala R)

was dissolved in it by thorough shaking followed by centrifugation.

134

Procedure

Perfusion fixation and embedding

For perfusion fixation, the procedure adopted was according to

Hess and Moore (1993). The rat was anesthetized using ether and

laid on its back in a shallow tray. A vacuum system was used to

remove the fluids accumulating into a holding container. Vascular

perfusion is generally carried out intracardially and offers a good

preservation of organs (Hess and Thurston, 1977). In the present

study, the transfusion set consisted of a bottle with polythene catheter

containing the perfusate suspended at about 150cm above the animal.

The ascending aorta was cannulated with the polythene catheter

through an incision in the left ventricle. Care was taken not to let in

air during this process. The right atrium was incised for the outflow

of the fixative. Initially the fixative was allowed to flow rapidly and

then the rate of flow was reduced to minimum. Following perfusion,

pancreas was cut into 1mm cubes and immersed in 2.5%

glutaraldehyde (primary fixative) overnight. Then the tissues were

rinsed in washing buffer and post-fixed in 1% osmium tetroxide

(OsO4) (secondary fixative) for 2-3 hours. Subsequently, the tissues

were washed thoroughly in washing buffer to remove excess OsO4.

Then the tissues were dehydrated gradually in ethyl alcohol and

dealcoholized using propylene oxide. Infiltration was carried out with

propylene oxide and Spurr’s mixture (Sigma, USA) at increasing

concentrations at room temperature, using a slow speed rotary

shaker. Embedding was done in a flat embedding mould with tissues

135

oriented to get cross sections. Semithin sections were obtained with

Reichert Jung (Austria) ultramicrotome and stained in TBO. Area was

chosen to obtain ultra-thin sections (silver to gray, 60nm–90nm) using

LKB-Bromma ultracut (Germany). Sections were picked in copper

grids and stained with uranyl acetate and lead citrate.

Leitz diaplan microscope (Leica, Germany) was used to obtain

light micrographs of TBO-stained semithin sections, while Philips

200C (Holland) transmission electron microscope (TEM) was used to

obtain electron micrographs at 2,000 to 70,000 X magnifications.

3.12. Statistical Analysis

All quantitative measurements were expressed as means ± SD

for control and experimental animals. The data were analyzed using

one way analysis of variance (ANOVA) on SPSS (Statistical Package for

Social Sciences) (Version 16.0) and the group means were compared

by Duncan’s Multiple Range Test (DMRT) (Duncan, 1957). The results

were considered statistically significant if the p value was less than

0.05.

3.13. DOCKING STUDIES

3.13.1. Protein databank

The Protein Data Bank (PDB) is a repository for the 3-D

structural data of large biological molecules, such as proteins and

nucleic acids. The data, typically obtained by X-ray crystallography or

NMR spectroscopy and submitted by biologists and biochemists from

136

around the world, are released into the public domain, and can be

accessed at no charge on the internet. The PDB is overseen by an

organization called the Worldwide Protein Data Bank, wwPDB.

The PDB is a key resource in areas of structural biology, such

as structural genomics. Most major scientific journals, and some

funding agencies, such as the NIH in the USA, now require scientists

to submit their structure data to the PDB. If the contents of the PDB

are thought of as primary data, then there are hundreds of derived

(i.e., secondary) databases that categorize the data differently. For

example, both SCOP and CATH categorize structures according to

type of structure and assumed evolutionary relations; GO categorize

structures based on genes (Tierney et al., 2002).

The PDB database is updated weekly. Likewise, the PDB Holdings

List is also updated weekly. As of 26 October, 2009, the breakdown of

current holdings was as follows:

Exp. Method Proteins

Nucleic Acids

Protein/NA Complexes

Other Total

X-ray 49273 1176 2273 17 52739

NMR 7065 874 150 6 8095

Electron

Microscopy 175 16 65 0 256

Hybrid 18 1 1 1 21

Other 116 4 4 13 137

Total 56647 2071 2493 37 61248

These data show that most structures are determined by X-ray

diffraction, but about 15% of structures are now determined by

137

protein NMR, and a few are even determined by cryo-electron

microscopy.

3.13.2 Chemsketch

The 3D structures of polysaccharide and catechin was

developed using Chemsketch software. It is a chemical intelligence in

a comprehensive drawing package provides a portal to an entire range

of analytical tools, and facilitates the transformation of structural or

analytical data into professional, easy-to-decipher reports or

presentations. ChemSketch is an advanced chemical drawing tool and

is the accepted interface for the industry's best NMR and molecular

property predictions, nomenclature, and analytical data handling

software. It contains tools for 2D structure cleaning, 3D optimization

and viewing, IUPAC International Chemical Identifier (InChI)

generation and conversion, drawing of polymers, organometallics, and

Markush structures—capabilities that are not even included in some

of the commercial packages from other software producers. Also

included is an IUPAC systematic naming capability for molecules with

fewer than 50 atoms and 3 rings.

3.13.3 Docking

Understanding the interactions between proteins and ligands is

crucial for the pharmaceutical and functional food industries. The

experimental structures of these protein/ligand complexes are usually

obtained, under highly expert control, by time-consuming techniques

such as X-ray crystallography or NMR. These techniques are therefore

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not suitable for routinely screening the possible interaction between

one receptor and thousands of ligands. To overcome this limitation,

computational algorithms (i.e. docking algorithms) have been

developed that uses the individual structures of the receptor and

ligand to predict the structure of their complex.

We used a new shape-based method, LigandFit, for accurately

docking ligands into protein active sites. The method employs a cavity

detection algorithm for detecting invaginations in the protein as

candidate active site regions. A shape comparison filter is combined

with a Monte Carlo conformational search for generating ligand poses

consistent with the active site shape. Candidate poses are minimized

in the context of the active site using a grid based method for

evaluating protein-ligand interaction energies. The method appears

quite promising, reproducing the X-ray structure ligand pose within

an RMSD of 2A. A high-throughput screening study was applied to the

thiamidine kinase receptor is also presented in which LigandFit,

when combined with LigScore, an internally developed scoring

function, yields very good hit rates for a ligand pool seeded with

known actives (Venkatachalam, et al., 2003).

Thus docking study of polysaccharide and catechin with

tyrosine kinase (1IRK) and Perroxisome–Proliferated-activated

receptor-gamma (1ZGY) was carried out by Ligand Fit of Discovery

studio (Version 2.1, Accelry’s Software Inc.). The software allows us to

virtually screen a database of compounds and predict the strongest

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binders based on various scoring functions. It explores the ways in

which these two molecules and the receptors fit together and dock to

each other well.

3.13.4. DOCKING PROTOCOL

3.13.4.1 Ligand Preparation

The three dimensional structures of Thiazolidinedione

(TZD)which was used as reference drug for docking studies was

downloaded in .sdf format from Pubchem database. Hydrogen Bonds

were added and the energy was minimized using CHARMm force field

in all the three compounds. Molecular weight, log P and number of

Hydrogen-bond donors and acceptors for the active principles were

noted. All the three molecules were satisfied Lipinski’s drug

properties.

3.13.4.2 Protein Selection

There are several PDB structures available for the same

receptors and the PDB structure with a good resolution of 2.00 when

compared to other structures was chosen for our study. The ligands

and crystallographic water molecules were removed from the protein;

and the chemistry of the protein was corrected for missing hydrogen.

Crystallographic disorders and unfilled valence atoms were corrected

using alternate conformations and valence monitor options. Following

the above steps of preparation, the protein was subjected to energy

minimization using the CHARMm force field. The active site of the

protein was first identified and it is defined as the binding site.