CHAPTER 4 MATERIALS AND METHODS -...
Transcript of CHAPTER 4 MATERIALS AND METHODS -...
21
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.
22
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
23
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.
24
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:
25
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.
26
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
27
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
28
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.
4.2.4.4 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
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.
4.2.4.5 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
29
% inhibition = 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.
4.2.4.6 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
30
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)
31
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
32
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.
33
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.
34
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.
35
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.
36
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.
37
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
38
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
39
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
Where 200 is the standard concentration
4.8.6.2 SERUM TRIGLYCERIDES (TG) [117]
Cholesterol esterase
Cholesterol Oxidase
Peroxidase
40
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
Absorbance of standard
Where 200 is the standard concentration
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
41
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
Absorbance of standard
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
42
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.
43
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
Where 1746 is the factor.
SGOT
MDH
44
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)
Where 2764 is the factor.
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
Where 2 is the standard concentration
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.
10. Wash in running tap water for 5 minutes.
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.
7. Wash in distilled water.
8. Stain in Biebrich scarlet-acid fuchsin solution for 10-15 minutes. Solution can
be saved for future use.
9. Wash in distilled water.
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.
12. Wash in distilled water.
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.
5. Rinse in running tap water for 10 minutes.
6. Stain in chromium hematoxylin at 60º C foe 1-2 hours.
7. Rinse in acid alcohol for 1 minute.
8. Rinse in running tap water for 5 minutes.
9. Rinse in distilled water.
10. Treat in phosphotungstic acid for 1 minute.
11. Rinse in running tap water for 5 minutes.
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.
3. Rinse in distilled water.
4. Decolourise with 2% oxalic acid for 1 min.
5. Rinse in distilled water.
6. Mordant in 4% iron alum for 10 minutes.
7. Rinse in distilled water.
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.
11. Wash in running tap water for 2 minutes.
12. Rinse in distilled water.
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.
3. Rinse in distilled water.
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.