chapter 4 materials and methods -...

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CHAPTER 4

MATERIALS AND METHODS

This study was carried out in the Department of Anatomy, and animal house of

SRM Medical College Hospital and Research Centre, Potheri, Tamilnadu, India.

4.1 DRUGS AND CHEMICALS

Streptozotocin was purchased from Sisco research laboratory Mumbai.

Glibenclamide was obtained from Aventis pharma India Ltd. Various biochemical kits

were procured from Agappe diagnostics, Kerala. All other chemicals and reagents used in

this study were of analytical grade procured from Thermofischer scientific India Ltd,

Mumbai.

4.2 PLANT MATERIAL

4.2.1 COLLECTION AND AUTHENTICATION

Cynodon dactylon (L.) Pers. (fig.4.1) was collected from Kanniyakumari

district of Tamilnadu, India and authenticated by the Professor of Botany, Bharathiar

University, Coimbatore, Tamilnadu, India.

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Figure 4.1 Whole plant of Cynodon dactylon (L.)

4.2.2 EXTRACT PREPARATION

The whole plant of Cynodon dactylon was washed with tap water, air dried,

and grinded in a mechanical blender. The dried powder (100 g) of Cynodon dactylon was

extracted with distilled water in a soxhlet extractor (fig.4.2) and the resultant extract was

concentrated in a rotary vaccum evaporator (fig. 4.3). The concentrated dark extract was

stored in an air tight container. The percentage yield (recovery) of evaporated extract was

calculated as follows:

Yield (%) = (Extract + Container (g) – (Empty container (g)X 100

Sample Weight (g)

The percentage yields were expressed in terms of the air dried drug.

Figure 4.2 Soxhlet Extractor

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Figure 4.3 Rotary Vaccum Evaporator

4.2.3 PRELIMINARY PHYTOCHEMICAL SCREENING [103, 104]

Various chemical tests were carried out with different extracts of Cynodon

dactylon for the presence of Phytochemical constituents.

Test for alkaloids

Wagner’s test:

To 1 ml of the extract, a few drops of Wagner’s reagent were added and the

formation of a reddish brown precipitate indicates the presence of alkaloids.

Test for Flavonoids

Shinoda Test:

To 1 ml of the extract, magnesium turnings, 1-2 drops of concentrated

hydrochloric acid were added. Formation of pink color indicates the presence of

Flavonoids.

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Lead acetate test:

To 1 ml of the extract, few drops of 10% Lead acetate solution were added.

Appearance of yellow colour precipitate indicates the presence of flavonoids.

Test for Phenols and Tannins

Lead acetate test:

To 1 ml of the extract, few ml of 1% lead acetate solution was added and the

formation of precipitate indicates the presence of tannins and phenolic compounds.

Ferric chloride test:

To 1ml of the extract, few ml of 5% ferric chloride was added. The

development of dark bluish black color indicates the presence of tannins.

Sodium hydroxide test:

A small quantity of extract was dissolved in 0.5ml of 20% sulphuric acid

solution. Followed by addition of few drops of aqueous sodium hydroxide solution, it turns

blue, indicated the presence of phenols.

Test for steroids and sterols

Salkowski’s test:

The extract was dissolved in 2ml of chloroform and equal volume of

concentrated sulphuric acid was added along the sides of the test tube. The upper layer

turns red and lower layer turns yellow with green fluorescence, indicating the presence of

the steroids and sterols compound, in the extract.

Test for Saponins

Honey comb test:

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5 ml of the extract was taken in a test tube and few drops of 5% of sodium

bicarbonate solution were added. The mixture was shaken vigorously and kept for 3

minutes. Formation of honey comb like froth shows the presence of saponins.

Foam Test:

About 1 ml of the extract was diluted with 20ml distilled water and shaken well

in a graduated cylinder for 15 min. The formation of foam to a length of 1cm indicated the

presence of Saponins and steroids.

Test for Glycosides

Legal Test:

The extract was dissolved in pyridine and freshly prepared sodium

nitroprusside solution was added. The formation of pink to red color indicates the presence

of glycosides.

Test for Protein & amino acids

Biuret test:

To 1ml of extract, equal volume of 40% NaOH solution and two drops of 1%

copper sulphate solution were added. The appearance of violet colour indicates the

presence of protein.

Ninhydrin test:

To 1ml of extract, 2 drops of freshly prepared 0.2% ninhydrin reagent was

added and heated. The appearance of pink or purple colour indicates the presence of

proteins, peptides or amino acids.

Test for Carbohydrates

Fehling’s test:

Five ml of Fehling’s solution was added to 2 ml of the extract boiled in water

bath. The formation of yellow or red precipitate indicates the presence of reducing power.

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Benedict’s test:

Five ml of Benedict’s solution was added to 2 ml of the extract boiled in water

bath. The appearance of red or yellow or green precipitate indicates the presence of

reducing sugars.

4.2.3.1 DETERMINATION OF TOTAL PHENOLICS AND TANNINS [105]

Ten microlitre aliquots of the extracts (10mg/2ml) were taken in test tubes and

made up to the volume of 1 ml with distilled water. Then 0.5 ml of Folin-Ciocalteu Phenol

reagent (1.1 with water) and 2.5 ml of sodium carbonate solution (20%) were added

sequentially in each tube. Soon after vortexing the reaction mixture, the test tubes were

placed in dark for 40 min and the absorbance was recorded at 725 nm against the reagent

blank. The analysis was performed in triplicate and the results were expressed as Gallic

acid equivalents.

4.2.3.2 DETERMINATION OF TOTAL FLAVONOIDS CONTENT [106]

A 0.5ml aliquot of appropriately (10mg/2ml) diluted sample solution was

mixed with 2ml of distilled water and subsequently with 0.15ml of 5% NaNO2 solution.

After 6 min, 0.15ml of 10% AlCl3 solution was added and allowed to stand for 6 min, and

then 2ml of 4% NaOH solution was added to the mixture. Immediately, water was added to

bring the final volume to 5ml, and then the mixture was thoroughly mixed and allowed to

stand for another 15min. Absorbance of the mixture was determined at 510 nm versus

water blank. The analysis was performed in triplicate and the results were expressed as

Rutin equivalent.

4.2.4 INVITRO ANTI-OXIDANT ACTIVITY

4.2.4.1 DPPH RADICAL SCAVENGING ACTIVITY [107]

The antioxidant activity of the sample was determined in terms of hydrogen

donating or radical scavenging ability, using the stable radical 1, 1-Diphenyl-2-Picryl

hydrazyl (DPPH) .The sample extracts at various concentrations (200-1000 g) was taken

and the volume was adjusted to 100 l with methanol. 5 ml of 0.1 mM methanolic solution

of DPPH was added and allowed to stand for 20 min at 27 . The absorbance of the sample

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was measured at 517 nm. Percentage radical scavenging activity of the sample was

calculated as follows:

% DPPH radical scavenging activity = Control OD – Sample OD

Control OD

The analysis was performed in triplicate. The sample concentration providing

50% inhibition (IC50) under the assay condition was calculated from the graph of inhibition

percentage against sample concentration.

4.2.4.2 REDUCING POWER ASSAY [105]

200-1000 g of extract was taken in 1 ml of phosphate buffer and 5 ml of 0.2M

phosphate buffer (pH 6.6) was added. To this, 5ml of 1% potassium ferricyanide solution

was added and the mixture was incubated at 50 for 20 min. After the incubation, 5ml of

10% Trichloroacetic acid was added. The content was then centrifuged at 1000 rpm for 10

min. The upper layer of the supernatant (5ml) was mixed with 5 ml of distilled water and

0.5ml of 0.1% ferric chloride. The absorbance of the reaction mixture was read

spectroscopically at 700nm.

4.2.4.3 HYROXYL RADICAL SCAVENGING ACTIVITY [108]

Different concentrations of the extract (200-1000 g) were added with 1ml of

iron-EDTA solution (0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 ml of

EDTA solution (0.018%), and 1ml of dimethylsulfoxide (DMSO) (0.85% v/v in 0.1M

phosphate buffer, pH 7.4). The reaction was initiated by adding 0.5ml of ascorbic acid

(0.22%) and incubated at 80-90 for 15 min in a water bath. After incubation the reaction

was terminated by the addition of 1ml of ice-cold TCA (17.5% w/v). Three milliliters of

Nash reagent (75.0g of ammonium acetate, 3ml of glacial acetic acid and 2 ml of acetyl

acetone were mixed and raised to 1 L with distiller water) was added and left at room

temperature for 15 min. The intensity of the colour formed was measured spectroscopically

at 412 nm against reagent blank. The % hydroxyl radical scavenging activity was

calculated as follows:-

% Hydroxyl radical scavenging activity = Control OD – Sample OD

Control OD

x 100

x 100

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4.2.4.4 SUPEROXIDE RADICAL SCAVENGING ACTIVITY [109]

The assay was based on the capacity of the sample to inhibit farmazan

formation by scavenging the superoxide radicals generated in riboflavin-light-NBT system.

Each 3 ml reaction mixture contained 50mM sodium phosphate buffer (pH 7.6), 20 mg

riboflavin, 12mM EDTA, 0.1mg NBT and various concentrations (200-1000 g) of sample

extracts. Reaction was started by illuminating the reaction mixture with sample extract for

90 seconds. Immediately after illumination the absorbance was measured at 590 nm. The

entire reaction assembly was enclosed in a box lined with aluminium foil. Identical tubes

with reaction mixture kept in dark served as blank. The percentage inhibition of superoxide

anion generation was calculated as:

% Superoxide radical scavenging activity = Control OD – Sample OD

Control OD




4.2.4.5 LIPID PEROXIDATION INHIBITION ASSAY [110]

Goat liver was washed thoroughly in cold phosphate buffer saline (pH 7.4.) and

homogenized to give a 10% homogenate. The homogenate was filtered and centrifuged at

10000 rpm for 10 min and the supernatant used to carry out the assay. To 0.05mL of 10%

homogenate, 0.5mL of the sample (50-250 g) was added. To this, 0.05 mL of 0.07M

ferrous sulphate was added and incubated at room temperature for 30 min. To the

incubated solution, 1.5 ml of 20% acetic acid (pH 3.5) and 1.5 ml of 0.8% TCA (in 1%

SDS) were added. The tubes were incubated at 100 for 1 hr and cooled to room

temperature. About 5ml of Butanol was added and centrifuged at 3000 rpm for 10 min.

The upper layer was used to read the absorbance at 532 nm. The percentage inhibition was

calculated as follows:-

x 100

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% inhibition = Control OD – Sample OD

Control OD]




4.2.4.6 IN VITRO INHIBITION OF α-GLUCOSIDASE [111]

The enzyme α-glucosidase inhibitory activity was determined by premixing

α-glucosidase (0.07 Units) with 100-500μg /ml of extract. Then 3mM

p-nitrophenylglucopyranoside was added as a substrate. This reaction mixture was

incubated at 37 for 30 min and the reaction was terminated by addition of 2ml of sodium

carbonate. The α-glucosidase activity was determined by measuring the p-nitrophenyl

release from p-nitrophenylglucopyranoside at 400 nm. The % α-glucosidase inhibitory

activity is calculated by the following formula.

% Inhibition = Control OD – Sample OD

Control OD

The IC50 value was defined as the concentration of the sample extract to inhibit

50% of α-glucosidase activity under assay condition.

4.2.4.7 IN VITRO INHIBITION OF α-AMYLASE [111]

The α-Amylase (0.5 mg/ml) was premixed with extract at various

concentrations (100-500μg/ml) and starch as a substrate was added as a 0.5% starch

solution to start the reaction. The reaction was carried out at 37 for 5 min and terminated

by addition of 2 ml of DNS (3, 5-dinitrosalicylic acid) reagent. The reaction mixture was

heated for 15 min at 100 and diluted with 10ml of distilled water in an ice bath; α-

amylase activity was determined by measuring spectrum at 540 nm. The % α-amylase

inhibitory activity is calculated by the following formula.

% Inhibition = Control OD – Sample OD

Control OD

x 100

x 100

x 100

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The IC50value was defined as the concentration of the sample extract to inhibit

50% of α-amylase activity under assay condition.

4.2.5 HPTLC FINGER PRINTING

High performance thin layer chromatography (HPTLC) finger printing profile

of Cynodon dactylon aqueous extract, performed as per AHRF method with CAMAG

HPTLC instrument. HPTLC is a modern sophisticated, automated separation technique,

used to achieve precise, sensitive and significant separation of secondary metabolites both

quantitatively and qualitatively. It is a versatile tool for the evaluation of botanical

materials efficiently and cost effectively.

HPTLC is a chromatographic pattern of analysis of pharmacologically active

or chemically characteristic constituents present in the extract, provides a valuable tool for

identification, authentication and quality control of herbal drugs. It is also a diagnostic

method to find out the adulterants and to check the purity present in the extracts/herbal

drugs.

BASIC STEPS

Instrument CAMAG HPTLC

HPTLC Applicator CAMAG LINOMAT IV

HPTLC Scanner CAMAG TLC SCANNER II

Method As per AHRF method

Sample Cynodon dactylon aqueous extract

Mobile phase Toluene: Ethyl acetate: Acetonitrile:

Formic acid (3:6:1:0.2)

Stationary Phase TLC silica gel 60F 254, Merck

Volume Applied 20μl (As such)

Lamp Deuterium (254nm)

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4.3 EXPERIMENTAL ANIMALS

Adult male albino wistar rats (aged 10 weeks, weighing 150-200 g)

approximately were acclimatized and housed in the central animal house of SRM medical

college hospital and research centre, SRM university campus, Potheri, Tamilnadu. All

animals were kept in 12:12 hr light: dark cycle, at a room temperature of 22±2˚C. Rats

were fed with standard rat pellet supplied by Provimi animal nutrition India ltd, Bangalore,

India, were also allowed free access to water. Animal experimentation were carried out

under the supervision of on duty veterinary medical officer in accordance to the ethical

norms approved by the Institutional animal ethical committee (IAEC) of SRM Medical

College,Potheri,Tamilandu, India. (Ref: 45/IAEC/2011).

4.4 EXPERIMENTAL DIABETES INDUCTION

Animals were fasted overnight and diabetes was induced by single

intraperitoneal injection of streptozotocin (45mg/kg body weight) prepared in 0.1 M

Citrate buffer at pH 4.5. To overcome drug induced hypoglycemia, animals were allowed

to drink 5% glucose solution overnight. Citrate buffer alone was injected to control rats.

After 72 hours of STZ injection, (taken as 0th

day) fasting blood glucose levels of each

animal were analyzed. Animals with fasting blood glucose levels > 200 mg/dl were

considered as diabetic and taken for the study.

4.4.1 MECHANISM OF ACTION OF STREPTOZOTOCIN

Streptozotocin (STZ, 2-DEOXY-2-(3-(methyl-3-nitrosoureido) -

D-glucopyranose) is synthesized from Streptomycetes achromogenes and is used to induce

both insulin dependent and non-insulin dependent diabetes mellitus. The dose of STZ is

not narrow as in the case of alloxan. The most commonly used dose in adult rats is between

40 to 60 mg/kg body weight. Streptozotocin action in β- cells is accompanied by

characteristic alterations in the blood insulin and glucose concentrations. The changes in

blood glucose and insulin concentrations reflect abnormalities in β – cell function. STZ

impairs glucose oxidation and decreases insulin biosynthesis and secretion. STZ is taken

up by the pancreatic β –cells via glucose transporter GLUT 2. A reduced expression of

GLUT 2 has been found to prevent the diabetogenic action of STZ. Intracellular action of

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STZ results in changes of DNA pancreatic β – cells fragmentation. The main reason for the

STZ induced β – cell death is DNA alkylation.

STZ is a nitric oxide (NO) donor and NO was found to bring about the

destruction of pancreatic islet cells; it was proposed that this molecule contributes to STZ –

induced DNA damage. STZ increases the release of ROS that causes DNA damage, by

DNA alkylation. This in turn, activates the poly ADP ribose polymerase, resulting in

reduction in the intracellular NAD+, which results in ATP depletion, and there by causes

β – cell death.

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4.5 EXPERIMENTAL DESIGN

The rats were randomly divided into 5 groups of 6 rats in each group.

Group I : Normal control rats

Group II : Diabetic control rats.

Group III : Diabetic rats treated with Glibenclamide (5 mg/kg body

weight/rat/day) for 45 days, via Oral gavage.

Group IV : Normal rats administered with aqueous extract of Cynodon

dactylon (500 mg/kg body weight/rat/day) for 45 days, via Oral

gavage.

Group V : Diabetic rats treated with aqueous extract of Cynodon dactylon

(500 mg/kg body weight/rat/day) for 45 days, via oral gavage.

4.6 TOXICITY AND DOSAGE SELECTION

LD50 – Safe up to the dose of 2000mg/kg body weight [112].

Therapeutic Dosage of 500 mg/kg, body weight, selected based on studies of

Singh [98].

This dose was optimal for Hyperglycemia, as different doses of the extract did

not elicit any significant biochemical changes.

Duration of administration of the extract – 45 days.

4.7 ASSESSMENT OF PHYSIOLOGICAL PARAMETERS

4.7.1 GRAVIMETRY

The body weight of the experimental animals was measured with electronic

weighing balance.

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4.7.2 FOOD AND WATER INTAKE

The amount of food and water intake of experimental animals of all the groups

was recorded manually.

4.8 ESTIMATION OF BIOCHEMICAL PARAMETERS

4.8.1 BLOOD SAMPLE COLLECTION

Blood samples were collected from lateral tail vein of rats, for estimation of

blood glucose values every week with Accu-chek active Glucometer. Blood samples for

the estimation of other biochemical parameters were collected from Retro-orbital plexuses,

under light ether anesthesia on 0th

day, 21st day and 45

th day.

4.8.2 Estimation of Fasting Blood glucose [113]

The fasting blood glucose levels of the experimental animals were analyzed by

GOD-POD method with Agappe LyphoCHEK glucose kit.

PRINCIPLE

Enzymatic colorimetric determination of glucose according to the following

reaction.

Glucose Oxidase

Glucose + O2 Gluconic Acid + H2O2

Peroxidase

2 H2O2 + Phenol + 4-Aminoantipyrine Red Quinonimine +

4 H2O

PROCEDURE

Mode of Reaction is fixed time kinetic. Added 1000 ml of Reagent into 10ml

of sample mixed well and incubated for 10 minutes at 37 . Change in the absorbance of

standard and sample measured against blank Reagent.

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END POINT CALCULATION

Glucose concentration (mg/dL) = Absorbance of sample

× 100 Absorbance of standard

Where 100 is the standard concentration

4.8.3 ESTIMATION OF HbA1C [114]

HbA1C values estimated with micro column direct method with Agappe

diagnostic kit.

Principle

After preparing the hemolysate, the labile fraction is eliminated. HbA1C is

specifically eluted after wasting away the HbA1a+b fraction and is quantified by direct

photometric reading at 415nm.

Procedure

Hemolysate Preparation

Bring the column and reagent to room temperature (21 - 26 ). Pipette 50μl

blood and 200μl reagent 1 into a test tube and shake thoroughly and set aside for 10-15

minutes.

Column preparation

Remove the upper cap of the column and then snap the tip of the bottom. Using

the flat end of a pipette, push the upper disc down, just to touch the resin surface taking

care not to compress it. Allow the column to drain completely to waste.

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HbA1C Separation

Pipette 50μl hemolysate carefully on the upper filter.

Then add 200 μL reagent 2 on the upper filter.

Pipette 2 μL of reagent 2 carefully on the upper filter and let the column

drain to waste.

Place the column over a test tube and add 4ml Reagent 3 and collect the

elute (HbA1C fraction)

Then shake thoroughly and read the absorbance (A) at 415nm.

Reading of Hb Total

Pipette 12 ml reagent 3 and 50 μL hemolysate into a test tube. Shake well and

at 415 nm.

Calculation

% HbA1C = A HbA1C x 100

AHb Total 3

4.8.4 ESTIMATION OF PLASMA INSULIN [115]

Rat Insulin ELISA is a solid phase two-site enzyme immunoassay. It is based

on the direct sandwich technique in which two monoclonal antibodies are directed against

separate antigenic determinants on the insulin molecule. During incubation insulin in the

sample reacts with Peroxidase-conjugated anti-insulin antibodies and anti-insulin

antibodies bound to the micro titration well. A simple washing step removes unbound

enzyme labeled antibody. The bound conjugate is detected by reaction with 3, 3’, 5, 5’-

tetramethylbenzidine. The reaction is stopped by adding acid to give a colorimetric

endpoint that is read spectrophotometrically at 450nm.

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Material Required

• Pipettes for 25, 50, 100 and 200 μL (Repeating pipettes preferred for addition

of enzyme conjugate solution, Substrate TMB and Stop Solution.)

• Micro plate reader with 450 nm filter

• Wash device for micro plates

• Tube (10–100 mL) for preparation of enzyme conjugate solution

• 1000 mL/10 L bottle

• Redistilled water

• Plate shaker (The recommended velocity is 700-900 cycles per minute, orbital

movement)

Reagents

• 1 x Rat Insulin ELISA kit (10-1251-01) contains reagents.

• Coated Plate 1 Mouse monoclonal anti-insulin.

• Calibrators 1, 2, 3, 4, 5.

• Calibrator 0 1 vial 5 mL,

• Peroxidase conjugated mouse monoclonal.

Wash Buffer 21X 1 bottle 50 mL dilute with1000 mL Storage after dilution:

redistilled water to 2-8°C for 4 weeks. Make wash buffer 1X solution.

Substrate TMB 1 bottle- Colorless solution

Use 0.5 M H2 SO4 Preparation of enzyme conjugate 1X solution Prepare the

needed volume of enzyme conjugate solution by dilution of Enzyme Conjugate 11X (1+10)

in Enzyme Conjugate Buffer. When preparing enzyme conjugate 1X solution for the whole

plate, pour all of the Enzyme Conjugate Buffer into the Enzyme Conjugate 11X vial, Mix

gently.

Procedure

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Prepare a calibrator curve for each assay run. All reagents and samples must be

brought to room temperature before use.

Prepare enzyme conjugate 1X solution, wash buffer 1X solution and samples.

Prepare sufficient micro plate wells to accommodate Calibrators and samples

in duplicate.

Pipette 25 μL each of Calibrators and samples into appropriate wells.

Add 100 μL of enzyme conjugate 1X solution into each well.

Incubate on a plate shaker (700-900 rpm) for 2 hours at room temperature

(18-25°C).

Wash 6 times with 700 µL wash buffer 1X solution per well using an automatic

plate washer with overflow-wash function or manually, discard the reaction volume by

inverting the micro plate over a sink. Add 350 µL wash solution to each well. Discard the

wash solution, tap firmly several times against absorbent paper to remove excess liquid.

Repeat 5 times. Avoid prolonged soaking during washing procedure.

Add 200 μL Substrate TMB into each well.

Incubate 15 minutes at room temperature (18-25°C).

Add 50 μL Stop Solution to each well. Place the plate on the shaker for

approximately 5 seconds to ensure mixing

Read optical density at 450 nm and calculate results. Read within 30 minutes.

4.8.5 ORAL GLUCOSE TOLERANCE TEST (OGTT)

Prior to OGTT rats were fasted for 16 hours, distilled water (control), ACD

500 mg/kg body weight and the reference drug, glibenclamide ,at a dose of 5mg/kg body

weight were orally administered to groups of 6 rats each. Thirty minutes later, glucose

(3g/kg) was orally administered to each rat with a feeding syringe. Blood samples were

collected from the tail vein by tail milking at 0 min (just before the oral administration of

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glucose), 30, 60, 90,120 min after glucose load for the assay of glucose, with Accu-chek

Glucometer.

4.8.6 ESTIMATION OF LIPID PROFILE

4.8.6.1 TOTAL CHOLESTEROL (TC) [116]

Serum total cholesterol estimated with Agappe LiquiCHEK cholesterol kit, by

CHOD – PAP method.

Principle

Enzymatic colorimetric determination of total cholesterol according to the

following reactions.

Cholesterol ester + H2O Cholesterol + fatty acids

Cholesterol + O2 4 – Cholesten-3-one + H2O2

2H2O2+Phenol+4-Aminoantipyrine Red Quinone + 4 H2O

Procedure

Added 1000 μL of working reagent 1 into 10 μL of sample and mixed well,

incubated for 5 minutes at 37 . The absorbance of sample and standard measured against

the reagent blank.

CALCULATION

Cholesterol concentration(mg/dL) = Absorbance of sample

x 200

Absorbance of standard


4.8.6.2 SERUM TRIGLYCERIDES (TG) [117]

Cholesterol esterase

Cholesterol Oxidase

Peroxidase

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Enzymatic determination of triglycerides based on the following reactions.

Lipoprotein lipase

Triglycerides + H2O Glycerol + fatty acid

Glycerol kinase

Glycerol + ATP Glycerol – 3- Phosphate + ADP

Glycerol Phosphate Dehydrogenase

Glycerol – 3- Phosphate +O2 Dihydroxyacetone

Phosphate + H2O2

H2O2 + 4-Aminophenazone + p- Chlorophenol Quinone + H2O

Procedure

Add 1000 μL of reagent into three tubes labeled as blank, standard and sample,

then add 10 μL of standard to standard tube and sample to sample tube, mixed well and

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

against reagent blank at 505nm.

Calculation

Triglycerides in (mg /dL) =

Absorbance of sample × 200



4.8.6.3 HIGH DENSITY LIPOPROTEIN CHOLESTEROL (HDL-C) [118]

Serum HDL – C levels estimated with HDL cholesterol PPT – SET from Crest

Bio systems, Goa.

Peroxidase

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Principle

When the serum is reacted with the polyethylene glycol contained in the

precipitating reagent, all the VLDL and LDL are precipitated. The HDL remains in the

supernatant and is then assayed as a sample for cholesterol using cholesterol (CHOD/PAP)

reagent.

Procedure

Mixed 1ml of reagent 1 with 0.05ml of sample and incubated for 5 minutes at

37o

C, after this 0.1m) of reagent 2 was added, shaked well and again incubated for 5

minutes at 37o C. Measure the absorbance of the standard and test sample against the blank.

Calculation

HDL Cholesterol in mg/dL = Absorbance of sample

× 25 × 2


Where 25 is the standard concentration and 2 is the dilution factor due to the

deprotenization step.

4.8.6.4 LOW DENSITY LIPOPROTEIN CHOLESTEROL (LDL - C) [119]

Low-density lipoprotein cholesterol (LDL - C) was estimated by Friedwald

et al.,’s (1972) formula which states:

LDL cholesterol = TC – (VLDL + HDL cholesterol)

4.8.6.5 VERY LOW DENSITY LIPOPROTEIN CHOLESTEROL (VLDL - C)

[119]

Very low-density lipoprotein (VLDL) estimated by Friedwald et al.,’s (1972)

formula which states:

VLDL cholesterol = TG/5

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4.8.7 DETERMINATION OF SERUM GLUTAMATE PYRUVATE

TRANSAMINASE (SGPT) ACTIVITY [120]

Serum SGPT levels determined by modified IFCC method with SGPT kit,

from coral clinical systems, Goa.

Principle

SGPT catalyzes the transfer of amino group between L-Alanine and

α - Ketoglutarate to form NAD. The rate of oxidation of NADH to NAD is measured as a

decrease in absorbance which is proportional to the SGPT activity in the sample

SGPT

L-Alanine + α Ketoglutarate Pyruvate + L-Glutamate

LDH

Pyruvate + NADH + H+ Lactate + NAD

+

Procedure

Added 1000 μL of reagent to 100 μL of sample mixed well and incubated for 1

minute at 37oC. The initial absorbance is read at 340nm, after 1 minute and the reading

repeated after every 1,2 and 3 minutes, then mean absorbance change per minute ( A /

minute) calculated.

Calculation

SGPT activity in U/L at 37oC = A / min X 1746

Where 1746 is the factor.

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4.8.8 DETERMINATION OF SERUM GLUTAMATE OXALOACETATE

TRANSAMINASE (SGOT) ACTIVITY [121]

Serum SGOT levels determined by modified IFCC method with SGOT kit,

from Crest bio-Systems, Goa.

Principle

SGOT catalyzes the transfer of amino groups between L-Aspartate and α

Ketoglutarate to form oxaloacetate and glutamate. The oxaloacetate formed reacts with

NADH in the presence of malate Dehydrogenase to form NAD. The rate of oxidation of

NADH to NAD is measured as a decrease in absorbance is proportional to the SGOT

activity in the sample.

L. Aspartate + α Ketoglutarate oxaloacetate + L-Glutamate

Oxaloacetate + NADH + H+

Malate + NAD +

Procedure

Added 1000 μL of reagent to 100 μL of sample mixed well and incubated for 1

minute at 37oC. The initial absorbance is measured at 340nm, after 1minute and the

reading repeated after every 1, 2 and 3 minutes. Then the mean absorbance change per

minute ( A / minute) is calculated.

Calculation

SGOT activity in U/L at 37oC = A / minute X 1746


SGOT

MDH

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4.8.9 DETERMINATION OF ALKALINE PHOSPHATASE (ALP) LEVELS

[122]

Serum ALP levels determined with Liquizyme kit from Beacon diagnostics,

Gujarat.

Principle

Alkaline phosphatase in serum catalyses the hydrolysis of P. Nitrophenyl

phosphate to P.Nitrophenol and phosphate. The rate of formation of p.Nitrophenol is

measured as an increase in the absorbance which is proportional to the ALP activity in the

sample.

P – Nitrophenylphosphatase p-Nitrophenol + Phosphate

Procedure

Added 1000 μL of reagent to 20 μL of sample. Mixed well and read the initial

absorbance at 405nm after 1 minute. Absorbance reading repeated after every 1, 2 and 3

minutes. Then the mean absorbance change per minute ( A / min) Calculated.

Calculation

ALP activity in U/L = ( A / min. X 2764)


4.8.10 ESTIMATION OF TOTAL PROTEIN (TP) [123]

Serum total protein estimated by direct Biuret method using total protein kit,

from Agappe diagnostics, Kerala.

Principle

Colorimetric determination of total protein based on the principle of the Biuret

reaction (Copper salt in an alkaline medium). Protein in serum sample forms a blue colored

ALP

45

complex when treated with cupric ions in alkaline solution. The intensity of the blue colour

is proportional to the protein concentration

Procedure

Added 1000 µl of reagent to 20µl of sample mixed well and incubated for 10

minutes at 37oC. The absorbance of standard and sample against reagent blank is

measured at 546mm.

Calculation

Total protein concentration in (gm /dL) = Absorbance of sample

× 6 Absorbance of standard

Where 6 is the standard concentration.

4.8.11 ESTIMATION OF CREATININE [124]

Serum creatinine levels estimated by modified Jaffe’s method, with creatinine

kit from Agappe Diagnostics, Kerala.

Principle

Creatinine reacts with picric acid to produce a colored compounds creatinine

alkaline picrate. The change in absorbance is proportional to the creatinine concentration.

Procedure

Added 1000 µl of reagent to 100 µl of sample, mix well and absorbance is

read at 505mm after 60 seconds (T1) second reading (T2) taken after 60 seconds from the

first one.

Calculation

Creatinine concentration (mg / dl) = (T2 – T1) of sample

x 2

(T2 – T1) of standard


46

4.9 COLLECTION OF TISSUE SAMPLES

After 45 days of experiment, animals were sacrificed, following the guidelines

of animal ethical committee. The Pancreas and liver tissues were excised and fixed in 10%

neutral buffered formalin (NBF) solution for histological analysis. Parts of the liver tissues

were rinsed in ice cold phosphate buffered saline at pH 7.4.Tissues were then homogenized

with Remi homogenizer. The homogenate centrifuged at 3000 RPM for 10 minutes,

supernatant collected was used for the estimation of In vivo antioxidant activity.

4.10 IN VIVO ANTIOXIDANT ACTIVITY

4.10.1 ASSAY OF SUPEROXIDE DISMUTASE (SOD) [125]

Reagents:

1) Sodium pyrophosphate buffer (0.025M):1.115g in 100ml of distilled water.

2) Phenazonium Metho Sulphate (PMS) (186µM): 3mg in 10 ml of distilled water

(930µM).Then 1:5 dilutions were carried out to obtain 186µM.

3) Nitro Blue Tetrazolium (chloride) (NBT) (300µM):3mg in 10 ml of phosphate

buffer.

4) NADH (780µM):6mg in 10 ml of phosphate buffer.

Procedure

Superoxide dismutase was assayed by taking 0.05ml of spleen homogenate

followed by addition of 0.3ml of sodium pyrophosphate buffer (0.025M,PH 8.3), 0.025ml

of PMS (186µM) and 0.075ml of NBT (300µM in buffer of PH 8.3) The reaction was

started by addition of 0.075 ml of NADH (780µM in buffer of PH 8.3). After incubation at

300C for 90 seconds, the reaction was stopped by addition of 0.25ml glacial acetic acid.

Then the reaction mixture was stirred vigorously and shaken with 2.0ml of n-Butanol. The

mixture was allowed to stand for 10 minutes and centrifuged. 1.5ml of n-Butanol alone

was served as blank. The colour intensity of the chromogen was read at 560nm.

47

4.10.2 LIPID PEROXIDATION ASSAY (TBARS) [110]

Reagents:

1) Thiobarbituric Acid (TBA) (0.8%):0.8gms in 0.5N HCL

2) Butylated Hydroxyl Toluene (0.05%):0.05gms in methanol.

3) Saline (0.9%):0.9g in 100ml distilled water.

Procedure

The method involved heating of homogenized spleen sample with 0.8ml saline,

0.5ml of BHT and 3.5ml TBA reagent for 11/2 min in a boiling water bath. After cooling,

the solution was centrifuged at 2,000 rpm for 10 min and the precipitate obtained was

removed. The absorbance of the supernatant was determined at 532 nm using

spectrophotometer against a blank that contained all the reagents minus the biological

sample. The values

were expressed in mg/g tissue.

4.10.3 ASSAY OF GLUTATHIONE PERIOXIDASE (GPX) [126]

Reagents:

1. Sodium Azide (10mM): 16mg in 25ml of distilled water

2. GSH (2mM): 30.732mg in 50ml of distilled water.

3. H2O2 (1mM): 29µl in 1000ml of distilled water

4. 10% TCA: 10g in 100ml of distilled water

5. EDTA(0.4mM): 16mg in 100ml of distilled water

6. Tris HCL Buffer(0.4mM): 6.304g in 100ml of distilled water

7. DTNB (0.6mM): 12mg in 50ml PO4 buffer.

Procedure

The remaining glutathione (GSH) after the enzyme catalyzed reaction, was

complexed with 5, 5’dithiobis-(2-nitrobenzoic acid) (DTNB) that absorbs at a maximum

wavelength of 412 nm. To the tissue aliquot, 0.4 ml of sodium phosphate buffer (0.4 M,

48

pH 7.0), 0.1 ml of sodium Azide (10 mM), 0.2 ml of GSH (4 mM), 0.1 ml of H2O2

(2.5 mM) and water were added to make a final incubation volume of 2 ml. The contents

were then incubated for 10 min at 37 C and the reaction was terminated by the addition of

0.5 ml of 10% TCA. The tubes were centrifuged and to 1 ml of the supernatant, 3 ml of 0.3

M disodium hydrogen phosphate solution and 1 ml of 0.04% DTNB in 1% sodium citrate

were added. The colour developed was read at 412 nm. The standard was also run

simultaneously. Enzyme activity was expressed as g of GSH consumed/min/mg protein.

4.10.4 CATALASE ASSAY (CAT) [127]

Reagents

1) 0.01M phosphate buffer

2) 0.2M H2O2

3) Stock dichromate/acetic acid solution: This reagent was prepared by mixing

5% solution of potassium dichromate with glacial acetic acid.

4) Working dichromate/acetic acid solution: stock solution was diluted to 1:5

with water to make the working solution.

Procedure

CAT causes rapid decomposition of H2O2 to H2O. The method is based on the

fact that dichromate in acetic acid is reduced to chromic acetate when heated in the

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

chromic acetate thus formed was measured at 610 nm. The reaction mixture consisted of

0.5 ml of 0.2 mM H2O2, 1 ml of 10 mM sodium phosphate buffer (pH 7.0) and 0.4 ml

water. 0.2 ml of the diluted enzyme was added to initiate the reaction. To this, 2 ml of the

dichromate/acetic acid reagent (5% solution of potassium dichromate in water with glacial

acetic acid in the ratio of 1:3, diluted 1:5 with water) was added after 30 and 60 sec of

incubation. To the control tube, the enzyme was added after the addition of the acid

reagent. The tubes were then heated for 10 min and the color developed was read at 610

nm. The activity of CAT was expressed as mM of H2O2 consumed/min/mg protein.

49

4.11 HISTOPATHOLOGICAL STUDIES

4.11.1 HISTOPATHOLOGY OF PANCREAS AND LIVER WITH

HEMATOXYLIN & EOSIN (H&E) STAIN [128]

The formalin fixed pancreas and liver tissues were processed in automatic

tissue processor, sections of 5 micron thickness made with Leica rotary microtome, and

then stained with hematoxylin and eosin (H& E) for general histological examination. The

stained slides were then photomicrographed with APCAM-5 USB 2 digital camera

attached to a computer monitor, supplied by ADELTAVISION OPTEC India microscope

Ltd.

Procedure:

1. Deparaffinize sections, 2 changes of xylene, 10 minutes each.

2. Re-hydrate in 2 changes of absolute alcohol, 5 minutes each.

3. 95% alcohol for 2 minutes each and 70% alcohol for 2 minutes.

4. Wash briefly in distilled water.

5. Stain in Harris haematoxylin solution for 8 minutes.

6. Wash in running tap water for 5 minutes.

7. Differentiate in 1% acid alcohol for 30 seconds.

8. Wash running tap water for 1 minute.

9. Bluing in 0.2% ammonia water or saturated lithium carbonate solution for 30

seconds to 1 minute.


11. Rinse in 95% alcohol, 10 dips.

12. Counter stain in eosin for 30 seconds to 1 minute.

13. Dehydrate through 95% alcohol, 2 changes of absolute alcohol, 5 minutes each.

14. Clear in 2 changes of xylene, 5 minutes each.

15. Mount with xylene based mounting medium.

50

4.11.2 HISTOLOGICAL STUDY OF PANCREAS AND LIVER WITH

MASSON’S TRICHROME (MT) STAIN [129]

To demonstrate collagen fibres in the pancreas and liver, sections were stained

with Masson trichrome stain. The stained slides were then photomicrographed with

APCAM-5 USB 2 digital camera attached to a computer monitor, supplied by

ADELTAVISION OPTEC India microscope Ltd.

Reagent:

1. Weigert’s Iron Hematoxylin Solution.

2. Biebrich Scarlet-Acid Fuchsin Solution.

3. Phosphomolybdic-Phosphotungstic Acid Solution.

4. Aniline Blue Solution.

5. 1% Acetic Acid.

Procedure:

1. Deparaffinize and rehydrate through 100% alcohol, 95% alcohol and 70%

alcohol.

2. Wash in distilled water.

3. For Formalin fixed tissue, re-fix in Bouin’s solution for 1 hour at 56 C to

improve staining quality although this step is not absolutely necessary.

4. Rinse in running tap water for 5-10 minutes to remove the yellow colour.

5. Stain in Weigert’s iron hematoxylin working solution for 10 minutes.

6. Rinse in running warm tap water for 10 minutes.


8. Stain in Biebrich scarlet-acid fuchsin solution for 10-15 minutes. Solution can

be saved for future use.


51

10. Differentiate in Phosphomolybdic-Phosphotungstic acid solution for 10-50

minutes or until collagen is not red.

11. Transfer sections directly (without rinse) to aniline blue solution and stain for

5-10 minutes. Rinse briefly in distilled water and differentiate in 1% acetic acid

solution for 2-5 minutes.


13. Dehydrate very quickly through 95% ethyl alcohol, absolute ethyl alcohol

(these steps will wipe off Biebrich scarlet-acid Fuchsin staining) and clear in

xylene.

14. Mount with resinous mounting medium.

4.11.3 HISTOLOGICAL STUDY OF PANCREATIC ISLETS WITH

GOMORI’S CHROME ALUM HEMATOXYLIN STAIN [130]

To histologically demonstrate the β-cells in the islets of pancreas, pancreatic

tissue sections were stained with Gomori’s chrome alum hematoxylin stain, a beta cell

specific stain. The stained slides were then photomicrographed with APCAM-5 USB 2

digital camera attached to a computer monitor, supplied by ADELTAVISION OPTEC

India microscope Ltd.

Solutions:

Bouin’s Fluid.

Potassium Permanganate Solution.

5% Sodium Bisulfite Solution.

Chromium Hematoxylin Solution.

1% Acid Alcohol.

0.5% Phloxine B Solution.

5% Phosphototungstic Acid.

52

Staining procedure:

1. Treat in Bouin’s fluid for 12-24 hours.

2. Rinse in running tap water for 15 minutes.

3. Treat in Potassium permanganate for 1 minute.

4. Decolorize in sodium bisulfate.


6. Stain in chromium hematoxylin at 60º C foe 1-2 hours.

7. Rinse in acid alcohol for 1 minute.


9. Rinse in distilled water.

10. Treat in phosphotungstic acid for 1 minute.


12. Differentiate in 95% ethyl alcohol for approximately 1 minute.

13. Blow dry.

14. Mount.

4.11.4 HISTOLOGICAL STUDY OF LIVER WITH GORDON AND SWEETS

RETICULIN (GSR) STAIN [131]

To histologically demonstrate reticulin fibres in the liver, liver sections were

stained with Gordon and Sweets reticulin stain. The stained slides were then

photomicrographed with APCAM-5 USB 2 digital camera attached to a computer monitor,

supplied by ADELTAVISION OPTEC India microscope Ltd.

53

Reagents:

1. Acidified Potassium Permanganate XE “Potassium Permanganate: Acidified.

2. Silver Solution.

3. 2% aqueous oxalic acid.

4. 4% aqueous iron alum.

5. 10% formalin.

6. 2% Sodium thiosulphate.

7. Neutral – Acidified.

Procedure

1. Deparaffinize sections with xylene then take through alcohols to water.

2. Oxidase in acidified potassium permanganate for 3 minutes.


4. Decolourise with 2% oxalic acid for 1 min.


6. Mordant in 4% iron alum for 10 minutes.


8. Impregnate in ammoniacal silver solution for 11 seconds.

9. Rinse quickly in distilled water.

10. Immediately reduce with 10% aqueous formalin for 2 minutes.



13. Fix with 2% aqueous sodium thiosulphate (hypo) for 2 minutes.

14. Wash in water for 2 minutes.

15. Counter stain with neutral red (optional-see technical note 5) for 2 minutes.

16. Dehydrate, clear and mount.

54

4.11.5 HISTOCHEMICAL STUDY OF LIVER WITH PERIODIC ACID

SCHIFF (PAS) STAIN [132]

To histochemically demonstrate the glycogen contents in the liver sections,

sections were stained with Periodic acid Schiff stain. The stained slides were then

photomicrographed with APCAM-5 USB 2 digital camera attached to a computer monitor,

supplied by ADELTAVISION OPTEC India microscope Ltd.

Solutions and Reagent:

1. 0.5% Periodic Acid Solution.

2. Schiff Reagent.

3. Mayer’s Hematoxylin Solution.

Procedure:

1. Deparaffinize and hydrate to water.

2. Oxidize in 0.5% periodic acid solution for 5 minutes.


4. Place in Schiff reagent for 15 minutes (Sections become light pink colour

during this step).

5. Wash in lukewarm tap water for 5 minutes (Immediately sections turn dark

pink colour).

6. Counter stain in Mayer’s hematoxylin for 1 minute.

7. Wash in tap water for 5 minutes.

8. Dehydrate and cover slip using a synthetic mounting medium.

4.11.6 IMMUNOHISTOCHEMICAL STUDY OF PANCREATIC ISLETS

Immunohistochemical expression of Insulin in the β cells of pancreatic islets of

normal and experimental groups of animals, carried out with Primary monoclonal mouse

anti-insulin antibody, purchased from Biogenex, USA, Catalog no.MU029-UC.

55

Immunohistochemical procedure was done using Ultravision Quanto-Detection system,

from Thermofischer scientific, USA. The stained slides were then photomicrographed with

APCAM-5 USB 2 digital camera attached to a computer monitor, supplied by

ADELTAVISION OPTEC India microscope Ltd.

Procedure

Paraffin sections of 5 micron thickness were mounted on positively charged

slides and subjected to the immunohistochemical procedure, using an Ultravision Quanto-

Detection system (Thermofischer scientific, USA),following the manufacturer’s

instructions.

STEPS INVOLVED

1. Deparaffinize and rehydrate tissue sections.

2. Buffer wash step.

3. Endogenous Peroxidase activity was inhibited by 3%H2O2 in distilled water for

10 minutes.

4. Buffer wash step (sections were washed with PBS).

5. Non-specific background staining was blocked by incubating with Ultra V

block for 5 minutes, followed by blow step.

6. Sections were incubated with monoclonal mouse anti-insulin antibody (1:50)

for 30 minutes at room temperature, purchased from Biogenex, USA, Catalog

no.MU029-UC.

7. Sections were washed with PBS and then incubated for 10 minutes with

primary antibody amplifier Quanto.

8. Buffer wash step, and then incubated for 10 minutes with HRP Polymer

Quanto.

9. Once again sections were washed with PBS and distilled water.

10. Peroxidase was detected with DAB Quanto chromogen and DAB Quanto

substrate kit.

56

11. Sections were then washed with distilled water for 10 minutes and then

dehydrated.

12. Nuclei were stained with Harris hematoxylin, slides were mounted with DPX

and then photomicrographed.

4.12 STATISTICAL ANALYSIS

Statistical analysis was done with Statistical package for social sciences

software (SPSS) version 21.

Analysis 1

Repeated measures ANOVA was carried out to find the significant difference

within each group for each parameter. Sphericity assumption was verified for a p

value of >0.05 using Maulchy’s test. If the p value is less than 0.05, then

greenhouse geisser correction epsilon has been taken into account.

There is significant difference within the group 2, 3 and 5 for all parameters. There

is no significant difference within the group 1 for all parameters. There is no

significant difference within the group 4 for all parameters.

Analysis 2

One-way Analysis of Variance was carried to find the significant difference

between the groups which is followed by the Tukey’s Honestly Significant Difference

(HSD) Test for the Post hoc analysis.

chapter 4 materials and methods -...

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