Chapter 2 Materials and Methods

2.1 MATERIALS

2.1.1 Animals

Albino mice and Wistar rats were purchased from Small animal breeding station,

Kerala Agricultural University, Mannuthy, Thrissur and were kept for a week under

environmentally controlled conditions with free access to pelleted food (Sai Durga foods,

Bangalore) and water.

2.1.2 Plant materials used

The leaves of Lagerstromia speciosa L. (L. speciosa) and bark of Mangifera

indica L (M. indica) were collected from the premises of Amala Ayurvedic Hospital,

Thrissur, Kerala, India. The plant materials were identified by Dr. C.N. Sunil, Dept of

Botany, of S.N.M. College, Maliankara and was authenticated at Botanical Survey of

India, Coimbatore. The voucher specimens, (L. speciosa BSI No. 62373; M. indica BSI

No. 579) has been kept in Fr. Gabriel Herbarium, Amala Ayurvedic Hospital and

Research Centre, Thrissur.

2.1.3 Chemicals

TBA (Thiobarbituric acid) : Hi Media Laboratories, Mumbai, India. Streptozotocin : Sigma fine chemicals, St.Louis, USA. 1, 4-Bis (5-Phenyloxazol-zyl) Benzene Sisco Research (POPOP) : Laboratories, Mumbai, India. 2,5-Diphenyloxazole (PPO) : -do-

Folins Ciocalteu’s reagent : -do-

5’-5’ Dithiobis (2-nitro benzoic acid)(DTNB) : -do-

Reduced glutathione (GSH) : -do-

Ascorbic acid : -do-

2-Deoxy-D-ribose : -do-

1-Chloro 2,4 dinitro benzene (CDNB) : -do-

Nitroblue tetrazolium (NBT) : -do-

Crystal violet : Romali, Mumbai, India.

14C –glucose : BRIT, Mumbai, India

Acrylamide/Bis SRL Mumbai

Tris-HCl do

Tris- Buffer do

Hydrogen Peroxide do

Sodium Azide do

Ethidium Bromide do

DPPH do

DMSO do

Carrageenan do

Guanidinium Hydrochloride do

NBT do

SDS E-Merck, Germany

Haematoxylin do

Eosin do

β-mercapto ethanol BDH

Gel loading Dye Genei, Bangalore

Bromo phenol Blue Sigma Aldrich, USA

α –ketoglutarate do

Riboflavin do

2.1.5 Reagent Kits

Glucoseoxidase peroxidase kit : Span Diagnostics Ltd

Urea reagent kit : -do-

Glycohemoglobin (HbA1c) :Pointe Scientific. Inc, USA

Cyanmeth reagent : Agappe Diagnostic kit

2.1.6 Instruments Used

Inverted microscope : Leica, Germany.

Upright research microscope : Meiji, Japan.

Horizontal Laminar flow hood : Kemi,Ernakulam, India

Incubator : Beston, India

Spectrophotometer : Elico SL 159 and SL 177

High speed cooling centrifuge : Remi, Chennai, India.

ELISA-Reader : Awareness Technology, Gujarat, India. Micro tome : Lab Agencies,

Ernakulam, India.

Liquid scintillation counter : LKB Rack Beta, 1209 Wallac, Finland.

UV-trans illuminator : Genei, Bangalore

Deep freezer (–70 and –20oC) : Remi and Tropicana

Cold Lab : LKB, Bromma Electric balance : Contech, instrument company, Mumbai : Casbee, CAS weighing India, Guraigon

pH meter : Elico IL 120

Lyophilyser : Labconco, Labconco Corporation, Missouri

Water bath Shaker : Pooja Lab equipment, Mumbai

Rotating Water bath : Superfit, India

Homogenizer : York Scientific industries, Delhi

Electronic Oven : Amur Instrumentation, Kerala

2.1.8 Reagents

A. Phosphate buffered saline (PBS)

NaCl - 8.00g

KCl - 0.20g

KH2PO4 - 0.20g

Na2HPO4. 2H2O - 1.44g

Dissolved in distilled water, made up to 1000.0ml. pH was adjusted to 7.2 with

1N HCl. Sterilized by autoclaving at 15lbs for 15min.

PBS-EDTA was prepared by a adding 2 g of EDTA in 1 litre of PBS.

B.Scintillation fluid

PPO - 2.50g

POPOP - 0.25g

Naphthalene - 100.0g

These reagents were dissolved in 1,4- Dioxan in a final volume of one litre. Kept

in dark without exposing to light at any stage.

C. Krebs Henseleit bicarbonate buffer (KHB)

NaCl - 6.9 g

KCl -0.35 g

CaCl2 -0.28 g

MgSO4 .7H2O -1.28 g

NaHCO3 -2.1 g

KH2PO4 - 0.16 g

Glucose - 2.0 g

These ingredients were dissolved in distilled water in a final volume of one litre.

D. SDS- PAGE Buffer

Acrylamide 29.2 g

N N’-Bis- Methylene-Acylamide 0.8g

SDS 10g

Tris HCl (pH 8.8) 1.5 M

Tris HCl (pH6.8) 0.5 M

E. Harris Haematoxylin

Haematoxylin 5g

Ethanol 50 mL

Potash Alum 50 mg

Potassium Iodide 50 mg

Distilled water - 950 mL

Haematoxylin was dissolved in ethanol by the aid of gentle heat.The alum was dissolved

in distilled water by heating and stirring and kept overnight at 40c .Alcoholic

Haematoxylin was added to the alum solution. The mixture was cooledand potassium

iodide was added and filtered.

F. Eosin Solution

Eosine 500 mg

Ethanol 100mL

Eosin was dissolved in 5mL of ethanol and made up to 100mL with ethanol

G. Griess Reagent

Reagent 1 0.1%N(1-Napthyl ethylene diamino dihydrochloride)

Reagent 2 1% sulphanilamide in 2% orthophosphoric acid

Mix reagent 1 and 2 in 1:1 proportions

2.2 METHODS

2.2.1 Preparation of tissue homogenates

Liver, Kidney, brain and pancreas was excised and rinsed thoroughly in ice-cold

saline to remove the blood. They were then gently blotted between the folds of a filter

paper and weighed in an analytical balance. 10 % of homogenate was prepared in 0.05 M

phosphate buffer (pH 7) using a teflon homogeniser at 4oC. This homogenate was used

for the determination of in vivo antioxidant enzymes studies and in vitro lipid

peroxidation scavenging study.

2.2.2 Determination of tissue and blood superoxide dismutase (SOD) activity

Superoxide dismutase activity was determined according to the method of Mc

Cord and Fridovich (1969).

Principle

Illumination of riboflavin solution in the presence of EDTA causes a reduction of

riboflavin. It then re-oxidizes and simultaneously reduces oxygen to O2ˉ, which is

allowed to react with a detector molecule NBT, which was reduced to formazan blue. The

SOD in the sample will inhibit the formazan production.

Procedure-Tissue

0.01 ml of the homogenate was mixed with 0.2 ml of 0.1 M EDTA (containing

0.0015% NaCN), 0.1 ml of 1.5 mM NBT and phosphate buffer (67 mM, pH 7.8) in a

total volume of 2.65 ml. After adding 0.05 ml of riboflavin, the absorbance of the

solution was measured against distilled water at 560 nm. All the tubes were illuminated

with incandescent lamp uniformly for 15 min and absorbances of the blue color formed

were measured again. Percent of inhibition was calculated after comparing absorbance of

sample with the absorbance of control (the tube containing no enzyme activity). The

volume of the sample required to scavenge 50 % of the generated superoxide anion was

considered as 1 unit of enzyme activity and expressed in U/ mg protein.

Procedure-Blood

Blood SOD activity was determined according to the method of Mc Cord and

Fridovich (1969) after removing the haemoglobin by the method of Minami and

Yoshikawa (1979).

0.1 ml of the heparinised blood was haemolysed by 0.9 ml of cold water (4oC).

The haemolysate was treated with 0.25 ml of CHCl3 and 0.5 ml of ethanol with vigorous

mixing to remove the haemoglobin. The mixture was centrifuged at 15000 rpm for 60

min. The 0.025 ml of the clear supernatant was used for the SOD assay as described as in

the tissue SOD estimation procedure. The volume of the sample required to scavenge 50

% of the generated superoxide anion was considered as 1 unit of enzyme activity and was

expressed as U/g Hb.

2.2.3 Determination of tissue and blood catalase (CAT) activity

Tissue and blood catalase activity was determined according to the method of

Aebi, (1974).

Principle

Catalase catalyses the decomposition of H2O2. In the ultraviolet range H2O2

shows a continual increase in absorption with decreasing wavelength. The decomposition

of H2O2 can be followed directly by the decrease in extinction at 240 nm.

2H2O2 2 H2O + O2

Procedure-Tissue

0.1 ml of the tissue homogenate (approximately 0.1 mg protein) was mixed with

1.9 ml of the phosphate buffer (0.5 M, pH 7). After adding 1 ml of 30 mM H2O2 solution

in buffer, decrease in extinction was measured at 240 nm, at 1 min interval for 3 min. A

sample control was placed in the reference cuvette containing 0.1 ml of tissue

homogenate and 2.9 ml of the buffer. Activity of catalase was calculated using the molar

extinction coefficient of 43.6 cm-1.

mmoles of H2O2 decomposed/min/mg protein

or = ΔA/min x 1000 x 3 (U/mg protein) 43.6 x mg protein in sample

Procedure-Blood

Erythrocyte sediment was prepared from the heparinised blood and washed 3

times with isotonic saline. A stock haemolysate containing approximately 5 g. Hb/dl was

prepared by the addition of 4 parts by volume of distilled water. A 1:500 dilution of this

concentrated haemolysate with sodium-potassium phosphate buffer (0.05 M, pH 7) was

prepared immediately before the assay. Reference cuvette contained 1 ml of buffer and 2

ml of haemolysate and test cuvette contained 2 ml diluted haemolysate. The reaction was

started by addition of 1 ml of H2O2 (30 mM in the buffer) to the test cuvette, mixed well

and the decrease in extinction was measured at 240 nm for 30 sec. by 15 sec. interval.

Catalase activity was calculated using the formula and expressed as k/g Hb, where k is a

rate constant of 1st order reaction.

Catalase = 2.3 x (log E1─ log E2 ) x dil. factor 15 x g Hb/ml of blood (k/g Hb) = 0.153 x 1000 x (log E1- log E2 ) g Hb/ml of blood E1 is E240 at t=0 and E2 is E240 at t=15 sec.

2.2.4 Determination of tissue and blood glutathione (GSH)

Reduced glutathione in the tissue was determined according to the method of

Moron et al., (1979).

Principle

The acid soluble sulfhydryl groups (non-protein thiols of which more than 93% is

reduced glutathione) form a yellow colored complex with dithionitrobenzene (DTNB).

The absorbance of the colored complex was measured at 412 nm.

Procedure-Tissue

0.5 ml of the tissue homogenate was mixed with 0.1 ml of 25 % TCA and kept on

ice for few minutes. These were then subjected to centrifugation at 3000 g for few

minutes to settle the precipitate. 0.3 ml of the supernatant was mixed with 0.7 ml of 0.2

M sodium phosphate buffer (pH 8) and 2 ml of 0.6 mM DTNB (prepared in 0.2 M buffer,

pH 8). The yellow color obtained was measured after 10 min at 412 nm against a blank

which contained 0.1 ml of 5% TCA in place of the supernatant. A standard graph was

prepared using different concentrations (10-50 nmoles) of GSH in 0.3 ml of 5 % TCA.

The GSH content was calculated with the help of this standard graph and expressed as

nmol/mg protein.

Procedure-Blood

A 20 % haemolysate of heparinised blood was prepared in distilled water and

proceeded for the glutathione determination as described as in the tissue GSH estimation

procedure. The GSH level was expressed as nmoles/ml of blood.

2.2.5 Determination of tissue and blood glutathione peroxidase (GPx) activity

Glutathione peroxidase activity was determined according to the method of

Hafemann et al., (1974).

Principle

The activity of GPx was determined by measuring the decrease in GSH content

after incubating the sample in the presence of H2O2 and NaN3.

H2O2 + 2 GSH 2H2O + 2 GSSG

Procedure-Tissue

Tissue homogenate (approximately 0.5 mg protein) was incubated with 0.1 ml of

5mM GSH, 0.1 ml of 1. 25 mM H2O2, 0.1ml of 25 mM NaN3 and phosphate buffer (0.05

mM, pH 7) in a total volume of 2.5 ml at 37oC for 10 min. The reaction was stopped by

adding 2 ml of 1.65 % metaphosphoric acid (HPO3) and the reaction mixture was

centrifuged at 1500 rpm for 10 min. 2 ml of the supernatant was mixed with 2 ml 0.4 M

Na2HPO4 and 1ml of 1mM DTNB. The absorbance of the yellow colored complex was

measured at 412 nm after incubation for 10 min at 37oC against distilled water. A sample

without the tissue homogenate processed in the same way was kept as the non-enzymatic

reaction.

One unit of enzyme activity was defined as decrease in GSH 0.001/min after

subtraction of the decrease in GSH per minute for the non-enzymatic reaction and is

expressed as units/mg protein.

Procedure-Blood

0.02 ml of heparinised blood was treated with 0.1 ml of 5mM GSH, 0.1 ml of 1.

25 mM H2O2, 0.1ml of 25 mM NaN3 and phosphate buffer (0.05 mM, pH 7) in a total

volume of 2.5 ml at 37oC for 10 min. The reaction was stopped by adding 2 ml of 1.65 %

HPO3 and the reaction mixture was centrifuged at 1500 rpm for 10 min. 2 ml of

supernatant was used for the estimation according to the procedure given under tissue

GPx determination. The result was expressed as U/g Hb.

2.2.6 Determination of tissue and serum lipid peroxidation

The level of lipid peroxidation was measured as malondialdehyde (MDA)

according to the method of Ohkawa et al (1979).

Principle

The tissue malondialdehyde was allowed to react with TBA. The MDA-TBA

adduct formed during the reaction in acidic medium was extracted to the organic layer

and the absorbance was measured at 532 nm.

Procedure-Tissue

4 ml of reaction mixture containing 0.4 ml of the tissue homogenate, 1.5 ml of 0.8

% TBA, 1.5 ml of acetic acid (20 %, pH 3.5) and distilled water was kept for 1 h in a

boiling water bath at 95oC. After 1 h, the reaction mixture was removed from the water

bath, cooled and added 1 ml of distilled water. 5 ml of butanol: pyridine mixture (15:1)

was added to the reaction tube, mixed thoroughly and centrifuged at 3000 rpm for 10

min. Absorbance of the clear supernatant was measured at 532 nm against

butanol:pyridine mixture. The MDA was calculated with the help of a standard graph

made by using different concentrations (1-10 nmol) of 1'1'3'3'-tetramethoxypropane in 1

ml distilled water and is expressed as nmol of MDA/mg protein.

Procedure-Serum

To 0.5 ml serum, 2.5 ml of 20 % TCA was added and the tube is left to stand for

10 min at room temperature. After centrifugation at 3500 rev/min for 10 min, the

precipitate was suspended in 1 ml distilled water and estimated the TBARS by procedure

given under tissue lipid peroxidation determination. The result was expressed as nmol/ml

of serum.

2.2.7 Determination of serum glutamate oxaloacetate transaminase (SGOT) activity

SGOT activity was determined according to the method of Reitman and Frankle

(1957).

Principle

Serum containing glutamate oxaloacetate transaminase catalyses the reaction

between L-aspartate and -ketoglutarate, to form oxaloacetate and glutamate. The

unstable oxaloacetate is converted to pyruvate and reacts with 2,4,-

dinitrophenylhydrazine. The absorbance of the resultant brown colored

phenylhydrazone is measured at 505 nm under alkaline conditions.

Procedure

Reagents used were from Span diagnostic kit. 0.1 ml of serum was added to 0.5

ml of the buffered substrate (2 mM of -ketoglutarate and 100 mM L-aspartate in 100 ml

phosphate buffer 0.1M, pH 7.4) at 37oC and incubated for 60 min. After the incubation,

0.5 ml of dinitrophenylhydrazine (19.8 mg/dl 1 N HCl) was added, mixed well and kept

at room temperature for 20 min. 0.4 ml of NaOH was added and read the absorbance after

10 min at 505 nm using the reagent blank. A control tube containing buffered substrate

was treated with serum after the incubation at 37o C was also followed in the same

manner. The enzyme activity was calculated from calibration curve made from the

standard sodium pyruvate, 2 mM. The enzyme activity (U/ml) is converted to IU/l by

multiplying with 0.483.

2.2.8 Determination of serum glutamate pyruvate transaminase (SGPT) activity

SGPT activity was determined according to the method of Reitman and Frankle

(1957).

Principle

Serum containing glutamate pyruvate transaminase catalyses the reaction between

L-alanine and -ketoglutarate, to form pyruvate and glutamate. The pyruvate thus formed

was treated with 2,4,-dinitrophenylhydrazine. The absorbance of the resultant brown

colored phenylhydrazone is measured at 505nm under alkaline condition.

Procedure

Reagents used were from Span diagnostic kit. 0.1 ml of serum was added to 0.5

ml of the buffered substrate (2 mM of -ketoglutarate and 100 mM L-alanine in 100 ml

phosphate buffer 0.1M, pH 7.4) at 37oC and incubated for 30 min. After the incubation,

0.5 ml of dinitrophenylhydrazine (19.8 mg/dl 1 N HCl) was added, mixed well and kept

at room temperature for 20 min. 0.4 ml of NaOH was added and read the absorbance after

10 min at 505 nm using the reagent blank. A control tube containing buffered substrate

was treated with serum after the incubation at 37o C was also followed in the same

manner. The enzyme activity was calculated from the standard (sodium pyruvate, 2 mM)

calibration curve. The enzyme activity (U/ml) is converted to IU/l by multiplying with

0.483.

2.2.9 Determination of serum alkaline phosphatase (ALP) activity

Serum ALP activity was determined according to the method of Kind and King

(1954).

Principle

ALP in the serum reacts with disodium phenyl phosphate under alkaline pH 10

release phenol. Phenol reacts with 4-aminoantipyrene in the presence of alkaline

oxidizing agent to give a red colored complex, which is measured at 510 nm against

reagent blank.

Procedure

Reagents used were from Span diagnostic kit. 0.05 ml of serum was incubated

with 0.5 ml of the buffered substrate (1ml of 0.254 g of disodium phenyl phosphate

dihydrate/dl water mixed with 1ml of the carbonate buffer pH 10) and 1.54 ml of distilled

water at 37oC for 15 min. After the incubation, 2 ml chromogen (1ml of 0.6 g 4-

aminoantipyrene/dl water and 1ml of potassium ferricyanide 2.4 g/dl water) reagent were

added and optical density was measured at 510 nm. Phenol (10 mg %) was used as the

standard for the calibration curve. The activity (KA/dl) is converted to IU/l by

multiplying with 7.1.

Serum ALP (IU/l) = O.DT-O.DC x 10 x 7.1 O.DS

2.2.10 Determination of serum urea

Serum urea was determined according to the method of Marsh et al., as described

in Text book of Clinical Biochemistry, Varley (1980).

Principle

Urea on heating with diacetylmonoxime under acidic condition condenses with

diacetyl to form a pink colored diazine complex. The reaction was catalyzed by

thiosemicarbazide and Fe3+ ions. The absorbance of the complex was measured at 525

nm.

Procedure

Reagents used were from Span diagnostic kit. 5 ml of diluted urea reagent (1:5

with distilled water) was mixed with 0.02 ml serum and 0.5 ml of diacetylmonoxime.

Mixed well and kept in boiling water bath for 10 min. Cooled and absorbance was

measured at 525 nm against reagent blank. A standard solution of urea (30 mg%) was

treated in the same way.

Serum urea (mg/dl) = O.DT x 30 O.Ds

2.2.11 Determination of serum creatinine

Serum creatinine was determined according to method of Brod and Serota as

described in Text book of Clinical Biochemistry, Varley (1980).

Principle

Creatinine forms a yellow-orange compound in alkaline medium with picric acid.

The intensity of the color was measured at 500 nm. The concentration of the dyestuff

formed over a certain reaction time is a measure of the creatinine concentration.

Procedure

Reagents used were from Merk diagnostic kit. 0.2 ml of serum was mixed with

0.5 ml of buffer (313 mM NaOH and 12.5 mM phosphate, pH 8) and 0.5 ml of 8.73 mM

picric acid. The absorbance was measured immediately after 1 min (O.Dt1) and exactly

after 5 min (O.Dt2) at 500 nm. A standard creatinine solution (1 mg/dl) was treated in the

same way.

Creatinine concentration (mg/dl) = O.Dt2 —O.Dt1 O.Ds2—O.Ds1

2.2.12 Determination of protein

This assay relies on the formation of protein copper complex and reduction of

phosphomolybdate-phosphotungstate reagent (Folin Ciocaltau reagent) by tyrosine and

tryptophan residues of protein (Lowry et al., 1951).

Reagents

Solution A

Sodium potassium tartarate (2%) - 1ml

CuSO4 (1%) - 1ml

Na2CO3 (2% in 0.1N NaOH) - 98ml

Solution B

Folin’s phenol reagent(1N) - diluted 1:1 with distilled water

Procedure

20μl sample or different concentrations of BSA (150μg, 100μg, 50μg and 25μg,

0.0μg – as standard) were made up to 1.2ml with distilled water. To this, 6.0ml of

solution A was added and then incubated at RT for 10minutes. The reaction mixture was

vortex mixed and 300μl solution B was added and incubated further for 30minutes at RT.

Optical density of the blue colour developed was read at 660nm.

2.2.13 Determination of serum total protein-serum

Serum protein was determined by the method of Reinhold, as described in Text

book of Clinical Biochemistry, Varley (1980).

Principle

Protein reacted with cupric ions in alkaline medium to form a violet colored

complex. The intensity of the complex was measured at 530 nm.

Procedure

The reagents used were from Span Diagnostic kit. 1 ml of the working Biuret

reagent was mixed with 0.01 ml of serum and absorbance was measured at 530 nm

against reagent blank. 0.01 ml of the standard solution was treated in the same way.

Serum total protein (g/dl) = O.DT x 6 O.Ds

2.2.14 Determination of serum albumin

Serum protein was determined using bromocresol green (Dumas and Peters,

1979).

Principle

Albumin in serum bound with bromocresol green at pH 4.2 to form green colored

complex. The intensity of the color was measured at 640 nm.

Reagents used were from Ranbaxy Diagnostic kit. 0.01 ml of serum was mixed

with 1 ml of BCG reagent (Succinate buffer 75 mM pH 4.2 and Bromocresol green 0.14

g/l). The absorbance was measured at 628 nm against reagent blank. Human albumin (3.8

mg/dl) was used as the standard.

Albumin (g/dl) = O.DT x 3.8 O.DS

2.2.15 Determination of haemoglobin (Hb) in blood

Haemoglobin was determined according to the method of Drabkin and Austin

(1932).

Principle

Haemoglobin was treated with a reagent containing potassium ferricyanide,

potassium cyanide and potassium dihydrogenphosphate. The ferricyanide forms

methaemoglobin, which is converted to cyanmethaemoglobin by the cyanide. The

intensity of the color formed is measured at 546 nm against reagent blank. The optical

density is directly proportional to the amount of haemoglobin present in the blood.

Procedure

The reagents used were from Agappe Diagnostic kit. 0.02 ml of fresh whole blood

was mixed with 5 ml of the Cyanmeth Reagent. The optical density was measured at 546

nm against reagent blank after 5 min incubation at room temperature. The O.D of

standard solution corresponding to 60 mg/dl haemoglobin at 546 nm was read against

reagent blank used for calculating the concentration of haemoglobin in the blood.

Haemoglobin (g/dl) = OD of the test x 251 x Conc. of Std OD of the std 1000 2.2.16 Determination of Serum glucose

Principle

Glucose is oxidized by glucose oxidase to gluconic acid and hydrogen peroxide.

In a subsequent peroxidase catalyzed reaction, the oxygen liberated is accepted by the

chromogen system to give a red coloured quinoneamine compound.

Procedure

Serum was pipette out 20 µl and added to the test tube containing 1.5 ml of

working glucose reagent and 1.5 ml of distilled water. Mixed well and incubated at 37ºC

for 15 minutes. The absorbance was read at 505 nm against reagent blank.

Calculation

Absorbance of Test Concentration of glucose= ----------------------------- × 100 Absorbance of Standard

2.2.17 Estimation of Glucose 6-Phosphate dehydrogenase (Lohr and waller., 1974)

Principle Glucose 6-Phosphate Dehydrogenase is assayed by measuring the increase in

absorbance at 340 nm when NADP reduces to NADPH. This reaction takes place when

electrons are transferred from glucose 6 phosphate to NADP in the reaction catalysed by

Glucose 6-phosphate dehydrogenase.

Procedure

The tissue in EDTA saline (0.4g tissue in 4 mL EDTA saline) was homogenized and

centrifuged at 40 c for 20 minutes at 15000 rpm. 3 mL of reaction mixture containing 2.8

mL of buffer, 0.1 mL of tissue sample, 50 microlitre of NADP, 50 microlitre of Glucose

6-Phosphate. The initial reading was taken at 340 nm with reference cuvette containing

the complete assay mixture without NADP and continued for 7 minutes with 2 minutes

intervals.

2.2.18 Determination of alpha – Amylase

Principle

The direct amylase assay involves the use of a chromogenic substitute, 2 –

chloro-4 nitrophenol linked with maltotriose.

CNP-G3 amylase CNP+CNPG2+ Glucose + Maltotriose

amylase hydrolyses the 2-chloro-4-nitrophenol -D-maltotrioside (CNPG3) to release

2-chloro-4 nitrophenol (CNP) and form 2-chloro-4 nitrophenol - - D maltotrioside

(CNPG2), maltotriose (G3) and glucose. The rate of formation of the CNP can be

detected spectro photometrically at 405 nm to give a direct measurement of -amylase

activity in the sample. The reaction is not inhibited by endogenous factors.

Procedure

1 ml of the CNP-G3 reagent was taken and incubated at 370C for 5mts. Intestinal

tissue homogenate was mixed with this reagent and read at 405 nm at 1 minute interval

upto 5 minutes.

2.2.19 Determination of liver glycogen

Principle

The method originally, used by Pfluger and modified by Good et al. consists in

the digestion of the tissue in hot concentrated KOH, precipitation of the glycogen with

ethanol, hydrolysis of the glycogen with acid, and determination of the glucose in the

hydrolysate as reducing sugar (Hassid and Abraham, 1957)

Procedure

Approximately 1 g of animal tissue was digested with KOH (30%) by heating the

tube in a boiling water bath for about 20 to 30 minutes. When the tissue was dissolved,

0.5 ml of saturated sodium sulfate is added and the glycogen was precipitated by the

addition of 1.1 to 1.2 volume of 95 % ethanol. The contents are stirred with a stirring rod,

and the rod washed with a small quantity of 60% ethanol. The tube and contents are

heated again until the mixture begins to boil, then cooled and centrifuged at 3000 rpm.

The mother liquor was decanted, and the test tube was allowed to drain. The remaining

adhering alcohol may be expelled by heating the tube in a boiling water bath. The

precipitated glycogen was redissolved in 2 ml of distilled water and reprecipitated with

2.5 ml of 95% ethanol, the alcoholic supernatant liquid decanted, and the tube drained as

before. The precipitate was redissolved into 50 ml of water. 5 ml aliquot of the solution

containing an amount of carbohydrate equivalent to 15 to 150 μg of glucose was

transferred to a colorimetric tube. 5 ml of the glucose standard containing 100 μg of

hexose was introduced into a second tube. To a third tube 5 ml of distilled water was

added, which serves as a blank. The tubes are submerged in cold water, 10 ml of the

anthrone reagent was added to each test tube from a fast-flowing pipette, and the

reactants are mixed by swirling the tubes. The cold tubes are covered with glass marbles

and heated for ten minutes in a boiling water bath. They are then immediately cooled in

bath containing cold water and read in the spectrophotometer at 626 nm using the blank.

The amount of glycogen in the aliquot used was calculated.

Calculation

Microgram of glycogen in aliquot = 100 X U 1.11X S

Where U = the optical density of the unknown test solution

S = the optical density of the 100 glucose standard

1.11 = the factor determined by Morris for the conversion of

glucose to glycogen, with this equation.

2.2.20 Determination of Non-esterified Free Fatty Acid (NEFA)

Principle

Serum is extracted with a chloroform-heptane-methanol mixture in the presence of a

phosphate buffer to eliminate their from phospholipids and the extract is shaken with a

high density copper reagent at pH 8.1.The copper soaps remain in the copper organic

layer from this an aliquot is removed and the copper content is determined with diphenyl

carbazide.

Reagents required

1. Extraction solvent: chloroform,heptane and methanol (5:5:1) was prepared.

2. Phosphate buffer (pH6.4)

3. Stock copper solution (500mmol/l)

4. Triethanol amine solution (1mol/l)

5. Sodium hydroxide (1mol/l)

6. 1,5 diphenyl carbazide solution (4g/l)

7. Stock palmitic acid (2mmol/l)

Procedure

To 50µL serum, 1ml phosphate buffer, 6 ml extraction solvent is added .The tubes

were shaken vigorously shaken for 90 seconds and left undisturbed for 15 minutes and

then centrifuged at 4000rpm for 10 minutes. The buffer was carefully removed by suction

and5 ml extraction solvent settled at the bottom of the tubes was transferred to a

centrifuge tube to which 2ml of copper reagent was added, the tubes were shaken

vigorously for 5 minutes and then centrifuged at 3000 rpm for 5 minutes. 3ml of the

upper layer was transferred to a tube containing 0.5 ml of 1,5 diphenyl carbazide

solution, mixed, and reading was taken after 15 minutes at 550 nm.

Calculation

Serum NEFA (micro moles/l)= 500* reading of unknown/ reading of standard

2.2.21 Estimation of phospholipids (King and Wooten, 1965)

Principle

The phospholipids in plasma are quantitatively precipitated by TCA together with plasma

protein. An estimation of the total phosphate contained in the precipitate by the method

of Firke and Subbarao yields the value for phospholipid content of the plasma

Reagents

TCA 10%- 100g TCA dissolved in H2O and made up to 1 litre., Perchloric acid-60%,

Ammonium molybdate solution-5%

Reducing agent-50 mg ascorbic acid was dissolved in 25 ml D H2O and filtered and use

always fresh.

Stock standard phosphate solution- 2.194 g of pure KH2 PO4 was dissolved in water and

volume made up to 500 mL. This solution contains 1.0 mg Phosphorus/mL

Phosphate standard- Working standard is made up diluting 2 mL of stock standard

solution to 500 mL with water. The solution contains 0.02 mg phosphorus per 5 mL. The

solution is kept saturated with chloroform to prevent bacterial growth.

Procedure- 0.2 ml of plasma is measured into a centrifuge tube and diluted with 1ml of

water. 5 ml of 10% TCA is added slowly with swirling of the contents of the tube. The

precipitate is centrifuged and the supernatant is poured off. And the tube is drained for 15

minutes. 0.5 ml of perchloric acid is added and the tube is treated until the digestion is

completed. The cooled contents are diluted with 5 ml of H2O, 0.4 ml of 5% ammonium

molybdate and 0.2 ml of reducing agent are added to the test. A standard was prepared by

taking 5 ml of the standard phosphate solution, 0.4 ml of perchloric acid, 0.4 ml

molybdate solution and 0.2 ml of reducing agent. A blank was prepared as for the

standard except that 5 ml of 10% TCA was taken instead of standard phosphate solution.

2.2.22 Protein Carbonyl assay: Levene et al., (1990).

Carbonyl protein assessment in the10% brain homogenate was performed as described

by Levene et al (1990). An equal amount of (3 mg) were precipitated with 10%

trichloro acetic acid (TCA) and, after centrifugation, the pellet was treated with 1 mL of

2%(w/v) dinitrophenyl hydrazine (DNPH) in 2 N HCl or with 1 mL 2N HCl as a

control blank. Samples were incubated for 1 hour at room temperature with stirring at 5

minutes intervals. Next 50 % TCA was added and the precipitated proteins were

subsequently washed three times with ethyl acetate: ethanol (1:1) mixture and three

times with10% TCA and centrifuged at 3400g to remove DNPH. The final precipitate

was dissolved in 6M guanidine, pH 2.3. The absorbances of the DNPH treated samples

and controls were measured spectrophotometrically at 370nm. BSA is taken as the

standard.

2.2.23 Estimation of cholesterol (Allain et al, 1974)

Principle:

Commercially available cholesterol reagents commonly combine all of the

enzymes and other required components into a single photometric reagent. The reagents

contain cholesteryl ester hydroxylase to cleave cholesterol esters. The 3-OH group of

cholesterol is then oxidized to a ketone (cholest-4-en-3-one) and H2O2 in an oxygen-

requiring reaction catalyzed by cholesterol oxidase. H2O2 undergoes a peroxidase-

catalyzed reaction with phenol and 4-aminoantipyrine that forms a coloured dye

(Quinoneimine dye).

Reagents:

Reaction solution:

Phosphate buffer -50mmol/L

Phenol -24mmol/L

Sodium cholate -0.5mmol/L

Cholesterol esterase -200U/L

Cholesterol oxidase -250U/L

Peroxidase -1000U/L

4-aminoantipyrine -0.5mmol/L

Standard: Cholesterol -200mg/dL

Sample material; Serum or plasma.

Procedure:

To about I ml of the reaction solution, 10l of sample was added. Mixed well and

incubated for 5 minutes at 37oC for 5 minutes. The absorbance of the sample and

standard were taken against the reagent blank at 500nm.Concentration of Cholesterol =

(OD of sample/OD of standard) x 100

Blank

Standard

Sample

Reaction solution Distilled water Standard Sample

1ml 10l

- -

1ml

- 10l

-

1ml

- -

10l 2.2.24 Estimation of HDL.

Reagents:

Precipitating reagent

Reaction solution:

Phosphate buffer -50mmol/L

Phenol -24mmol/L

Sodium cholate -0.5mmol/L

Cholesterol esterase -200U/L

Cholesterol oxidase -250U/L

Peroxidase -1000U/L

4-aminoantipyrine -0.5mmol/L

Standard: Cholesterol-200mg/dL

Sample material; Serum or plasma.

Procedure:

Take o.5ml of serum or plasma and add 0.05ml of precipitating reagent, Mix well,

leave it at room temperature for 10 minutes, centrifuge the mixture at 3000rpm for 10

minutes. Take 0.02ml of the clear supernatant and add to the reaction solution. Mix well

and incubated for 5 minutes at 37oC and absorbance of sample and standard against

blank was measured at 510nm.

HDL cholesterol in mg% = (OD of sample / OD of Standard) x 100 x 1.1

Blank

Standard

Sample

Reaction solution Supernatant from step I Standard

1ml

- -

1ml

- 10l

1ml 20l

-

2.2.25 Estimation of Triglycerides (Jacobs and Van Denmark, 1960)

Principle:

Triglycerides incubated with lipoprotein lipase are hydrolyzed to free fatty

acids and glycerol. Glycerol kinase catalyzes the conversion of glycerol and ATP to

glycerol-3-phosphate and ADP.The glycerol-3-phosphate gets oxidized to dihydroxy

acetone phosphate by glycerol phosphate oxidase. Hydrogen peroxide (H2O2) formed in

this reaction with the help of peroxidase, reacts with chromogens.4-aminoantipyrine

3,5,dicholoro-2-hydroxybenzenesulphonic acid to give a red colored complex which is

read at 510nm (500-530nm).

Reagent:

1.Enzyme reagent

2.Diluent Buffer

3.Triglycerides Standard, 200mg/dl

Procedure:

Add 10ul of the sample to the working reagent and mix well. Incubate for 10

minutes at 370C.Measure the absorbance of the standard and sample against the reagent

blank at 510nm(490-520nm). The final color is stable for at least 30 minutes.

Concentration of Triglyceride (mg/dl) = (O.D of sample / O.D of standard) X 250

Blank

Standard

Sample

Reaction solution Standard Sample

1ml

- -

1ml 10l

-

1ml

- 10l

2.2.26 Estimation of lactate dehydrogenase (Pesce, 1984)

Principle: LDH catalyses the conversion of pyruate to lactate with simultaneous oxidation

of reduced NADH to NAD. Therate of decrease in absorbance due to formation of NAD

is measured at 340nm and is proportional to the LDH activity in the sample.

Procedure:

10µl of sample was added to the 1ml reaction mixture containing 50mM buffer, 0.60mM

pyruate and 0.18mM NADH. Mix well and read the absorbance after 60sec. repeat the

reading four times after every 30sec ie upto 120 seconds at 340nm.

Calculation:

LDH activity (IU/L) : ΔA/minute x kinetic factor.

ΔA/minute : mean absorbance change per minute.

kinetic factor: 16030

2.2.27 Estimation of Creatine Phospho kinase (Moss and Hendeson, 1999)

Principle:

N-Acetyl-L-Cystine (NAC) reactivates Creatine kinase (CK) molecules by reducing

oxidized sulfydryl compound to ensure full catalytic of CK. CK catalyses the conversion

of creatine phosphokinase to creatine with simultaneous conversion of ADP (Adenosine

diphosphate) to ATP thus produced is used to form glucose 6 phosphate in a reaction

catalysed by hexokinase. glucose 6 phosphate produced is further oxidized to

phosphogluconate , whereby NADP is reduced to NADPH in the reaction catalysed by

glucose 6 phosphate dehydrogenase. The rate of increase in absorbance due to formation

of NADPH is measured at 340nm and is proportional to the CK activity in the sample.

Procedure:

10µl of sample was added to the 1ml reaction mixture containing 100mM imidazole

buffer,30mM Creatine phosphate; 20mM D-glucose; 5mM AMP; 2mM ADP;20mM

NAC; 2mM NADP; >2.5KU hexokinase; 1.5KU glucose 6 phosphate dehydrogenase.

Mix well and read the absorbance after 180sec. repeat the reading thrice after every 60sec

ie upto 180 seconds at 340nm.

Calculation:

LDH activity (IU/L) : ΔA/minute x kinetic factor.

ΔA/minute : mean absorbance change per minute.

kinetic factor: 8199

2.2.28 Histopathological analysis.

The tissue as soon as they are removed was fixed in 10% neutral buffered

formalin for at least 24h. The tissues were dehydrated in alcohol series, cleaned in xylene

and embedded in paraffin. About 5-6μm thick sections were taken using a microtome and

the wax ribbon was affixed on a clean glass slide by gentle heating. The slides were then

deparaffinated and stained with hematoxylin and eosin and mounted with a cover slip

using DPX. These were then observed under the microscope for any histological changes

(Culling, 1976).

Gomori’s Method of staining for pancreatic Islet cells

Fixation: Bouin’s or 10% buffered neutral formalin. If formalin fixed, the paraffin

sections should be mordanted in Bouin’s solution for 16 hours before staining. For these

paraffin section is cut into 6microns.

Staining procedure:

1. Deparaffinze and hydrate to distilled water

2. Mordant in Bouin’s solution for 16 hours

3. Wash in tap water for 15’to remove picric acid.

4.Wash in KMnO4 solution for 1’.

1. Differentiate in sodium bisulfite solution.

2. Wash well in tap water.

3. Chromium hematoxylin solution for 10’ or less.

4. Check under microscope and stain until β-cell stand out deep blue

5. Differentiate in acid alcohol solution for 1’

6. Wash in tap water until the section is clear blue.

7. Keep in phloxine B solution for 5’

8. Rinse in distilled water

9. Keep in phosphotungstic acid solution for 1’.

10. Wash in tap water for 5’.(section should regain its red colour)

11. Differentiate in 95% alcohol (If the section is too red) and if alpha cells do not

stand out clear enough rinse in 80% alcohol for 15’ to 20’

12. Dehydrate in absolute alcohol

13. Clear in Xylene

14. Mount with permount or histoclad.

2.3 Statistical analysis

Students unpaired ‘t’ test by Lutz, 1978, was used for the statistical evaluation of

the data. Determined the statistical significance between two values in the control (X) and

treated (Y) group,‘t’ value was calculated using the equation.

_ _ X - Y t= --------------------------- √[(1/nx) + (1/ny)] _ _ Where X and Y are the means of the two samples X and Y, nx and ny are the

sample size S was found out using the equation √ (nx – 1) Sx2 + ( ny – 1) Sy2 S= -------------------------------------- Nx + ny – 2 Where Sx and Sy are the standard deviation of the two samples. By knoing the ‘t’

value and degree of freedom (nx + ny -2), statistical significance was deduce from ‘t’

distribution table.

Some data were also analyzed by one-way analysis of variance (ANOVA) using

MSTAT-C, soft ware package, UK. If found significant pair wise comparison of ethyl

acetate, methanol and aqueous extract treated groups with the control group was done by

Dunnett’s-t test (Cochran and Cox, 1957).

Critical difference (c.d) = t x √ 2 x EMS/ r

Where t is the Dunnett’s t value for P<0.01 or P<0.05, EMS is the error mean

square value and r is the number of observations/group. Value is found to be significant if

the difference between average value of the control group and the treated group was

greater than the c.d.

For comparison of normal with control group critical difference (lsd) was used.

P< 0.05 was found to be significant.