Embed Size (px)
Citation preview
CHAPTER-3: MATERIALS AND METHODS
3.1. PLAN OF STUDY
Collection of raw materials for the study.
Phytochemical analysis and HPTLC fingerprint of DIA-2 and its component
herbs.
In vitro antioxidant and anti-diabetic assay.
Institutional Animal Ethics Committee (IAEC) Approval
Determining the cytotoxic dose level for in vitro studies using 3T3-L1 adipocytes
Determining the effect of DIA-2 and its component herbs on glucose uptake in
3T3-L1 adipocytes and expression of GLUT-4 protein.
Determining the effect of DIA-2 and its component herbs on adipogenesis and
expression of adiopocyte specific transcriptional factors.
Determining the effect of DIA-2 and component herbs on oxidative stress in
H2O2-treated 3T3-L1 adipocytes.
Studying the effect of DIA-2 treatment on body weight, plasma biochemistry
(glucose, insulin, triglyceride and total cholesterol), and antioxidant status from
target tissues of high fat/STZ treated rat.
Acute and repeated oral toxicity studies of DIA-2.
3.2. MATERIALS
3.2.1. Herbal extract
Authentic standardized extract of Allium sativum (ASE) and Lagerstroemia
speciosa (LSE) were obtained from M/s. Amsar Pvt. Ltd, Indore, India and M/s. K.
Patel Phyto Extractions Ltd, Mumbai, India, respectively. ASE is an aqueous
extract of dried bulbs Allium sativum and LSE is 40% methanolic extract of dried
leaves of Lagerstroemia speciosa. Both ASE and LSE were supplied in the powder
form by the dealer and claimed to contain 1.1 % alliin w/w and 1.28 % w/w
corosolic acid, respectively. The certificate of analysis is enclosed (: Annexure)
3.2.2. Chemicals and Drugs
Ammonium molybdate, Nicotinamide Adenine Dinucleotide (Reduced)
Disodium Salt (NADH), Adenosine-5'-triphosphate (ATP), L-Ascorbic acid,
Anthrone, 2,4-Dinitrophenylhydrazine(DNPH), 5,5’-dithiobis- (2-nitrobenzoic acid),
Gallic acid, α –glucosidase, Glucose-6 Phosphate, Glutathione reduced (GSH),
Guanidine HCL, Ninhydrin, Naphthyl ethylene diamine, Nitro Blue Tetrazolium,
Phenazonium Metho Sulphate, Ponceau S, Sulphanilamide, Sodium nitroprusside
and Sodium dodecyl sulphate (SDS) were obtained from M/s. Sisco Research
Laboratories Pvt. Ltd., Mumbai, Maharashtra, India.
Butylated Hydroxl Toluene (BHT), 2, 2’-diphyenyl-2-picrylhydrazyl, Dialysis
tube, Folins-Ciocalteau’s reagent, Periodic acid, α-tocopherol, Tetra-sodium
pyrophosphate and Thiobarbutaric acid, Acrylamide Tris Buffer, Nitrocellulose
membrane and Whatman filter papers were obtained from M/s. Himedia
Laboratories, Mumbai, Maharashtra, India.
Haematoxylin and Eosin were obtained from Merk India Ltd, Mumbai,
Maharashtra, India.
Quercitin, Sorbitol dehydrogenase, Fetal bovine serum, Penicillin
Streptomycin Gentamycin, Amphotericin B, 1-(4, 5-Dimethylthiazol-2-yl)-3, 5-
diphenylformazan Thiazolyl blue formazan powder (MTT), Insulin, Dexamethasone
(DEX), (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) [HEPES], Aprotinin,
Leupeptin, IGEPAL CA-630 (IGEPAL), Tetramethylethylenediamine (TEMED), Oil
Red O, Phenylmethanesulfonylfluoride (PMSF), TRIzol Reagent, 2', 7’-
dichlorfluorescein-diacetate [DCF-DA] were obtained from M/s. Sigma Aldrich
(USA).
2- Deoxy-D-3[H] glucose was obtained from M/s. Amersham Pharmacia
Biotech (U.K.) Dulbecco’s minimum essential medium, Isobutylmethyl xanthine
(IBMX) and other cell culture solutions and supplements were purchased from Life
Technologies Inc. (U.S.A.). Dulbecco’s Modified Eagle Medium (DMEM) was
obtained from GIBCO, BRL (U.S.A.).
Cell culture plates, flasks and 96-well plates were obtained from M/s.
Tarsons products Pvt. Ltd., Kolkata, India.
Rosiglitazone and Miglitol was a kind gift from Dr. Reddy’s Laboratories,
Hyderabad, India and Orchid Chemicals & Pharmaceuticals Limited Chennai, India,
respectively.
3.2.3. CELL LINES
3T3-L1 mouse fibroblasts (preadipocyte) were procured from National
Centre for Cell Science, Pune, India.
3.2.4. BIOCHEMICAL REAGENTS AND KITS
All biochemical kits were obtained from M/s. Accurex Biomedical Pvt.Ltd
Mumbai, India. 2, 2’- azino-di [3-ethylbenzthiazoline sulphonate] (ABTS) assay kit
was obtained from Cayman Chemical Company, Michigan, USA.
3.3. METHODS
3.3.1. Phytochemical screening of ASE and LSE
Phytochemicals are the individual chemicals from which plants are made.
The test solution of extracts (ASE and LSE) were prepared by dissolving by 10 mg
of extract in 100 mL of 80 % v/v methanol and used for various qualitative
phytochemical analysis described herein. The different chemical constituents tested
for ASE and LSE includes flavonoids, phenolic compounds, tannins (Raman 2006),
reducing sugars, steroids and triterpenoid glycosides, anthroquinone glycosides,
saponin glycosides and alkaloids (Shah 2010)
3.3.1.1 Qualitative Phytochemical Analysis
Test for flavonoids
Test solution was treated with 1 mL of 10 % sodium hydroxide. The
appearance of a yellow colour which turns colourless on addition of a few drops of
dilute hydrochloric acid. This indicates the presence of flavonoids.
Test for Phenolic compounds
To the test solution few drops of alcoholic ferric chloride solution were
added, a bluish green or bluish black precipitate indicates the presence of phenols.
Test for Tannins
To the test solution, a few drops of 1% lead acetate solution were added.
Formation of yellow precipitate indicates the presence of tannins.
Test for Reducing Sugars
Test solution was heated with equal volumes of Fehling’s solution A and B.
Formation of a red precipitate of cuprous oxide indicates the presence of reducing
sugars.
Test for Steroids & Triterpenoid Glycosides
5 mL of test solution was evaporated to dryness and extracted with
chloroform (CHCl3), few drops of acetic anhydride was added followed by
concentrated sulphuric from side wall of the test tube to the CHCl3 extract.
Formation of violet to blue coloured ring at the junction of the two liquids, indicated
the presence of steroid moiety.
Test for Anthroquinone Glycosides
To the test solution, 5 mL of dilute hydrochloric acid was added and boiled
on a water bath for 10 min and filtered. Filtrate was extracted with benzene and
equal amount of ammonia solution was added and shaked well. Formation of pink
or red colour in ammonia layer indicated the presence of anthraquinone moiety.
Test for Saponin Glycosides
To the test solution, 20 mL of water was added and shaken for few minutes;
formation frothing which persists for 60 s indicated the presence of saponins.
Test for Alkaloids
To the test solution, Dragendoff’s reagent (potassium bismuth iodide) was
added and the formation of orangish red colour indicates the presence of alkaloid.
3.3.1.2. Quantitative Phytochemical Analysis
Total Phenol Content
Total phenol content was determined using the methods described earlier
(Mcdonald et al., 2001).
Reagents
1. Folins-Ciocalteau’s reagent: one part of commercially available Folins-
Ciocalteau’s reagent (FCR) was mixed with nine parts of distilled water before
use.
2. Sodium carbonate (7.5%)
3. Stock gallic acid solution: 1 mg/mL stock solution of gallic acid was prepared
using methanol.
4. Working standard: 100 μg/mL working standard solution was prepared by
making up 1 mL of stock gallic acid solution to 10 mL with methanol. This
solution is used for the estimation.
5. Preparation of extract: 100 g/mL of ASE and 500 g/mL of LSE was prepared
in 80% methanol and was used for estimation.
Procedure
Briefly, to 1 mL of the extract [(ASE (100 g/mL) or LSE (500 g/mL) or
gallic acid], 5 mL of Folin-Ciocalteau’s Reagent (FCR) and 4 mL of sodium
carbonate (7.5%) was added. The mixture was allowed to stand for 15 min at 37°C,
after which absorbance was measured at 765 nm using spectrophotometer. A
calibration curve was plotted using gallic acid (8-40 g/mL) as standard. The
amount of total phenols was expressed as gallic acid equivalent (GAE) in
microgram per milligram of extract from a calibration curve.
Total Flavonoid Content
Total flavonoid content was determined using the methods reported earlier
(Chang et al., 2002).
Reagents
1. Sodium acetate (1 M)
2. Aluminium chloride (10 %)
3. Stock Quercetin solution: 1 mg/mL stock solution of quercetin was prepared
using methanol.
4. Working standard: 1 mL of the stock solution was diluted to 10 mL with
methanol. One mL of this solution contains 100 μg quercitin
5. Preparation of extract: 1000 g/mL of ASE and LSE was prepared using 80%
methanol and was used for estimation.
Procedure
Briefly, 500 L of the extract (1000 g/mL) or quercetin was made up to 4.5
mL of methanol. 100 L of aluminium chloride (10% w/v), 100 µL of sodium acetate
(1 M) and 2.8 mL of distilled water were added. All the above reagents excluding
the extract or quercetin served as blank. The tubes were incubated at room
temperature for 30 min. The absorbance was measured at 415 nm. A calibration
curve was constructed using quercetin (8 - 40 g/mL). The total of amount
flavonoids was expressed as quercetin equivalent (QE) in microgram per milligram
of extract from a calibration curve.
Tannin Content
Tannin content was determined as per the method of Schanderl 1970.
Reagents
1. Folins-Ciocalteau’s reagent: one part of commercially available Folins-
Ciocalteau’s reagent was mixed with two part of distilled water before use.
2. Sodium carbonate (35%)
3. Stock tannin solution: 1 mg/mL stock solution of tannin was prepared using
methanol.
4. Working standard: 1 mL of the stock solution was made up to 25 mL with
methanol in a standard flask. One mL of this solution contains 40 μg tannins.
5. Preparation of extract: 200 g/mL of ASE and 1000 g/mL of LSE was
prepared using 80% v/v methanol and was used for the estimation.
Procedure
Briefly, to 1 mL of the extract [ASE (200 g/mL) or LSE (1000 g/mL)], 1 mL
of distilled water was added, followed by 0.5 mL of Folin’s phenol reagent and 5 mL
of sodium carbonate (35 %) were added. After incubation at room temperature for 5
minutes, absorbance was measured at 640 nm against blank. The tannin content
was expressed as gallic acid equivalent (GAE) in microgram (µg) per milligram of
extract from a calibration curve.
3.3.2. HPTLC fingerprint of ASE and LSE.
Sample Preparation
80% v/v methanol (MeOH) solution was prepared. 100 mg of test substance
was weighed and transfered into a 10 mL volumetric flask. The contents were
dissolved with 5 mL of 80% MeOH; and sonicated until it got dissolved. The volume
was made up to 10 mL with 80% MeOH. The contents were sonicated for another
10 minutes and filtered using whatman filter paper and this solution was stored in 5
mL glass vials and used for HPTLC analysis.
HPTLC-Photo-densitometry conditions and instrumentation
Instrument : CAMAG HPTLC (Switzerland)
Applicator : CAMAG linomat V
Syringe : Hamilton syringe (100 μL)
Scanner : CAMAG TLC scanner 3
Photo-documentation : CAMAG Reprostar 3 (winCATS software version
1.3.4.)
Layer
Stationary phase : Pre coated silica gel 60 F254 on aluminium plates
(Merck KGaA, Darmstadt, Germany)
Plate thickness : 0.2 mm
Plate size : 100 x 100 mm
Mobile phase
ASE : chloroform: methanol: acetic acid: water
(64:32:12:8)
LSE : chloroform: ethanol: acetic acid: water (60:30:10:5)
Solvent ratio : Vol/ Vol (v/v)
Environmental Condition
Temperature : 25±2°C
Relative Humidity : 55–65%.
Sample application
Application rate : 10 s μL–1
Table speed : 10 mm s-1
Distance from starting : 15 mm
Distance from bottom : 10 mm
Volume applied : 2.5 – 10 μL
Band length : 10 mm
Distance between tracks : 10 mm
Development
Developing chamber : Twin trough glass chamber (20 x10 cm)
Developing solvent : 5 mL mobile phase/through
Chamber saturation time : 1 h
Developing mode : Ascending mode
Development distance : 80 mm
Detection reagent : Nil
Plate Drying : hair dryer for 5 min
Detection
Scanning wavelength : 254 nm and 366 nm
Slit dimensions : 5 mm × 0.45 mm
Documentation
Calculation of Rf = Distance travelled by solute component/Distance
travelled by solvent front.
Detection Reagent Preparation (0.2% Ninhydrin Solution)
200 mg of ninhydrin was weighed and dissolved in a small volume (~1 mL)
of acetone; to this, 1 mL of pyridine was added, and the volume was made up to
100 mL with acetone.
3.3.2.1. HPTLC fingerprint of DIA-2
After formulation of DIA-2, the HPTLC chromatogram for DIA-2 was also
developed. The method was developed similar to that of its individual ingredients
as described in section 3.3.2. The solvent system used to develop HPTLC finger
print of DIA-2 was chloroform: methanol: acetonitrile (80:10:10)
3.3.3. IN VITRO STUDIES
3.3.3.1. Anti-oxidant activity of ASE and LSE
Oxidative stress has been anticipated to play an important role in the
pathogenesis of diabetic complications. Numerous antioxidant treatments have
shown beneficial effects in improving both hyperglycemia and hyperglycemia
induced oxidative stress. Here we investigated the antioxidant effect of ASE and
LSE.
Reducing Power
In reducing power assay, ascorbic acid is oxidized to dehydroascorbic acid by
catalytic action of copper and reducing property of thiourea, the later also prevents
the interference of plant extract from non-ascorbic acid chromogens. The ketone
groups of dehydroascorbic acid reacts with 2, 4-Dinitrophenylhydrazine (DNPH),
undergoes rearrangement in acidic conditions (85 % sulphuric acid) to form a red
colored complex which was read at 540 nm. DHA, on the other hand, is a gentle
oxidizing agent, which can accept hydrogen atoms to reform AA. If the extracts are
capable of donating or transfering hydrogen atom to DHA and reforming it to AA,
it’s said to possess better reducing activity. The reducing power of the extracts was
evaluated by the method of Oyaizu 1986.
Reagents
1. DTC Reagent: 0.4 g of thiourea, 0.05 g copper sulphate and 3.0 g of 2, 4
Dinitrophenylhydrazine was weighed and dissolved in 100 mL of 9 N H2SO4
2. 85% Sulphuric acid.
3. Stock ascorbic acid solutions: 10 mg of Ascorbic acid was weighed and
dissolved in 10 mL of 5 % TCA. (Concentration: 1 mg/mL).
4. Working standard: 1 mL of the stock solution was diluted to 10 mL with 5%
TCA. (1 mL of this solution contains 100 µg ascorbic acid)
5. Preparation of extract: 10 mg of extract was weighed and dissolved in 10 mL of
respective solvents and this was used for estimation.
Procedure
To 1 mL of prepared extract/ ascorbic acid, 0.1 mL of DTC reagent was
added and incubated at 37ºC for 3 hours. After incubation, 1.25 mL of 85 % H2SO4
was added under ice-cold condition. The mixture was kept at room temperature for
30 minutes. The absorbance was measured at 540 nm against a blank using
UV/Visible spectrophotometer (Perkin Elmer, Lambda 25, USA). The experiments
were repeated in triplicates. The content of Vitamin C was calculated using a
standard graph and the reducing power of the extracts was expressed as the
number of gram equivalents of ascorbic acid (gram equivalent/ 1 g of vitamin C).
Total Antioxidant Capacity
The total antioxidant capacity assay is a quantitative assay and is based on
the reduction of molybdate-VI [Mo (VI)] to molybdate- VI [Mo (V)] by the extract and
subsequent formation of a green phosphate/Mo (V) complex at acid pH, which was
measured spectrophotometrically at 695 nm. The total α tocopherol content of
extract was determined by the method of Prieto et al., 1999.
Reagents
1. α-Tocopherol reagent: 28 mM Sodium Phosphate and 4 mM Ammonium
molybdate were weighed and dissolved in 100 mL of 0.6 M Sulfuric acid.
2. Standard α-Tocopherol solution: 1 mg/mL solution of α-Tocopherol was
prepared using ethanol.
3. Preparation of extract: 10 mg of extract was weighed and dissolved in 10 mL of
respective solvents and this was used for estimation.
Procedure
An aliquot of prepared extract/α-tocopherol was combined with α-tocopherol
reagent solution. In case of blank, solvent was used in place of the sample. The
tubes were capped and incubated in a boiling water bath at 95oC for 60-90 min.
Samples were cooled to room temperature, the absorbance of each was
measured at 695 nm against the blank in a Perkin Elmer UV/visible
spectrophotometer (Lambda 25,USA). The experiments were repeated in
triplicates. The content of α-tocopherol was calculated using a standard graph and
the total antioxidant activity of the extracts was expressed as the number of gram
equivalents of vitamin E (gram equivalent/ 1 g of vitamin E).
Nitric oxide scavenging assay
Nitric oxide (NO) turnover is important for proper endothelial function to
maintain a healthy vascular system. NO is synthesized by endothelial nitric
oxide synthase (eNOS), which plays a key role in the regulation of endothelial
function. NO has a dual role where low concentrations are essential for normal
homeostasis but high levels have harmful effects leading to cellular damage.
Peroxynitrite is a reactive oxidant that is produced from the reaction of nitric oxide
with superoxide anion and impairs endothelial function through multiple
mechanisms leading to vascular dysfunction. During diabetes mellitus there is
reduced bioavailability of NO due to increased reactive oxygen species production,
secondary to hyperglycemia. The inhibition of reactive oxygen species pathways in
conjunction with the nitric oxide (NO) pathway that leads to oxidative stress will
delay the progression of diabetic complications. The control of nitrosative stress
caused by nitric oxide free radical and oxidative stress could be as useful non-
pharmacological tool to delay the progression of diabetic complications. In vitro
nitric oxide-scavenging activity was performed using the method of Green et al.,
1982.
Reagents
1. Sodium nitroprusside (5 mM)
2. Greiss reagent: To 1 gm sulphanilamide dissolved in few mL of water, 2 mL of
orthophosphoric acid was added. Then 0.1% naphthyl ethylene diamine was
added and the volume was made up to 100 mL with water.
3. Preparation of extract: Various concentrations (1.5 –1,000 μg/mL) of ASE and
LSE were prepared in 80% methanol and similar range of standard vitamin E
was prepared. 1 mL of each concentration of the extract/standard was used for
the estimation.
Procedure
5 mM sodium nitroprusside in phosphate buffered saline was mixed with 3
mL of different concentrations (1.5–1,000 μg/mL) of the extracts (ASE, LSE) and
incubated at 25ºC for 150 min. The samples from the above were allowed to react
with Greiss’ reagent. The absorbance of the chromophore formed during the
diazotization of nitrite with sulphanilamide and subsequent coupling with
naphthylethylenediamine was read at 546 nm. The percentage of nitric oxide
radical–scavenging activity of the test sample was calculated, and the results were
compared with standard Vitamin E. The experiments were repeated three times.
The percentage inhibitions of free radicals were calculated by the formula:
(%) Inhibition = [(Abs. of control − Abs. of test)/Abs. of control)] × 100.
DPPH radical scavenging assay
The approach of scavenging the stable DPPH radicals is a widely used
method to evaluate the hydrogen donating capacity of antioxidants present in a
compound or medicinal plants, since it involves a relatively short time compared to
other methods. In vitro DPPH* radical scavenging potential of ASE, LSE and
Vitamin E was analysed by the method of Koleva et al., 2002.
Reagents
1. Absolute alcohol
2. 2,2’-diphenyl-2-picrylhydrazyl (DPPH*,150 µM) in ethanol
3. Preparation of extract: 100 mg of extract/standard Vitamin E was weighed and
dissolved in 1 mL of respective solvents. Further it was diluted (1:2) to get
various concentrations ranging from 1.5-1000 µg/10µL. 10 µL of each
concentration of the extract / standard Vitamin E was used for estimation.
Procedure
About 10 μL of each concentration (1.5–1,000 μg/10 μL) of test extract was
added to 190 μL DPPH solution. After vortexing, the mixture was incubated for 20
min at 37ºC. The control blank contained the solvent without extract. The decrease
in absorbance of the test extract was measured at 517 nm. The percentage
inhibition of the test extracts against DPPH was calculated and compared with
Vitamin E, a standard antioxidant.
The percentage inhibitions of free radicals were calculated by the formula:
(%) Inhibition = [(Abs. of control − Abs. of test)/Abs. of control)] × 100.
Lipid peroxidation inhibitory assay
In vitro lipid peroxidation inhibition assay was carried out by the method of
Ohkawa et al., 1979.
Reagents
1. Ferrous sulphate (25 M)
2. Ascorbate (100 M)
3. Potassium dihydrogen phosphate (10mM):
4. 5% trichloro acetic acid (TCA)
5. 0.375 % Thiobarbituric acid (TBA) in 0.5N HCl.
6. 10% liver homogenate: Fresh chick liver was homogenised with 10% KCl and
then the 10 % liver homogenate was used for the assay.
7. Preparation of extract /standard vitamin E: 10mg of extract/standard Vitamin E
was weighed and dissolved in 10 mL of respective solvents. Further it was
diluted (1:2) to get various concentrations ranging from 1.5-1000 µg / 1000 µL
concentration. 1 mL of each concentration of the extract /standard vitamin E
was used for estimation.
Procedure
The test system contained 1 mL of tissue homogenate with extracts (1.5–1,
000 μg/mL). In the control system, to 1 mL of tissue homogenate, the lipid
peroxidation was initiated by the addition of 0.1 mL of FeSO4 (25 μM), 0.1 mL of
ascorbate (100 mM), and 0.1 mL of KH2PO4 (10 μM), and the volume was made up
to 3 mL with distilled water and incubated at 37◦C for 1 h. Then, 1 mL of 5% TCA
and 1 mL of TBA were added to this reaction mixture, and the tubes were boiled for
30 min in a boiling water bath. This was centrifuged at 3,500 rpm for 10 min. In the
test system, the homogenate was incubated with different concentrations
(1.5–1000 μg/mL) of extracts (ASE, LSE). The extent of inhibition of lipid
peroxidation was evaluated by the estimation of thiobarbituric acid reactive
substance level by measuring the absorbance at 532 nm. The percentage inhibition
of the test extracts against lipid peroxidation was calculated and compared with
Vitamin E, a standard antioxidant.
The percentage inhibition of lipid peroxidation was calculated by the formula.
(%) Inhibition = [(Abs. of control- Abs. of test)/ Abs. of control)] x 100
3.3.3.2. Anti-oxidant activity of DIA-2 and component herbs.
Oxidative stress plays a role in the pathogenesis of diabetic complications.
The antioxidant and antidiabetic activity of ASE and LSE were investigated using
methods described in section 3.3.3.1. However, whether a combination of these
two herbs (DIA-2) could scavenge the reactive oxygen species remains uncertain.
Super oxide radical formation is considered as a main source of reactive oxygen
species and its increased activity is supposed to be involved in the development of
diabetic complications.
Super oxide radical scavenging assay
Reagents
1. Sodium pyrophosphate buffer (0.025 M):1.115 g in 100 mL of distilled water.
2. Phenazonium Metho Sulphate (PMS) (186 µM): 3mg in 10 mL of distilled
(930 µM).Then 1:5 dilutions were carried out to obtain 186 µM.
3. Nitro Blue Tetrazolium chloride (NBT) (300 µM): 3mg in 10 mL of phosphate
buffer.
4. NADH (780 µM):6mg in 10 mL of phosphate buffer.
5. ASE, LSE, DIA-2, Vitamin-C of varying concentrations (2-1000 μg/mL) were
prepared in respective solvents.
Procedure
Super oxide radical scavenging assay was carried out as per the method
described by Kakkar et al., 1984. 0.05 mL of extracts and DIA-2 of concentrations
ranging from 2-1000 μg/mL were added to test tubes, followed by 0.3 mL of sodium
pyrophosphate buffer (0.025 M, pH 8.3), 0.025 mL of PMS (186 µM) and 0.075 mL
of NBT (300 µM in buffer of pH 8.3) The reaction was initiated by addition of 0.075
mL of NADH (780 µM in buffer of pH 8.3). After incubation at 30°C for 90 seconds,
the reaction was stopped by addition of 0.25 mL glacial acetic acid. Then the
reaction mixture was stirred vigorously and shaken with 2.0 mL of n-butanol. The
mixture was allowed to stand for 10 minutes and centrifuged. 1.5 mL of n-butanol
alone served as blank. Ascorbic acid served as the positive control. The colour
intensity of the chromogen was read at 560 nm. Results were expressed as
percentage of inhibition of superoxide radicals. The percentage scavenging activity
was calculated using the formula: [1-(Abstest/Absblank)] x 100, where Abstest -Optical
density obtained in presence of test compounds and Absblank-Optical density
obtained in absence of test compounds.
ABTS radical scavenging assay of DIA-2
The ABTS radical scavenging assay was used to measure the antioxidant
capacity of DIA-2 and the activity was compared with standard vitamin E.
Reagents
1. Hydrogen peroxide (441 μM)
2. ABTS*+ (2, 2’- azino-di [3-ethylbenzthiazoline sulphonate] 2 mM
3. Preparation of extract / standard: 100 mg of extract and standard Trolox was
weighed separately and dissolved in 1 mL of respective solvents. Further it was
diluted (1:2) to get various concentrations ranging from 1.5-1000 µg / 10 µL
concentrations. 10 µL of each concentration of the extract / standard was used
for estimation.
Procedure
About 10 μL of test solution (extract/standard) in various concentrations
(1.5-1000 μg) was added to 10 μL of methmyoglobin and 150 μL of ABTS*+ .The
reaction was initiated by adding 40 μL of H2O2, shaken well and the amount of
ABTS formed was measured at 690 nm. The percentage inhibition was calculated
and compared with Trolox. The experiments were repeated in triplicates. The
percentage of scavenging potential of the extract/standard against ABTS*+ was
calculated by the formula below and the results were computed. The IC50 value was
determined as the concentration of the test mixture that gave 50% reduction in the
absorbance from a control blank.
(%) Inhibition = [(Abs. of control- Abs. of test)/ Abs. of control)] x 100
3.3.3.3. Anti-diabetic assay of DIA-2 and component herbs.
The in vitro anti-diabetic assays were carried out for ASE and LSE. After
obtaining the cytotoxicity data of ASE, LSE and their combinations (1:1 and 1:2
mixture of ASE and LSE), The results showed 1:1 mixture of ASE and LSE (DIA-2)
to be least cytotoxic compared to 1:2 mixture of ASE and LSE. Hence in vitro anti-
diabetic activities of DIA-2 were also performed.
α-glucosidase inhibitory assay
The α-glucosidase inhibitory assay was carried out by the previously
described method (Li et al., 2005).
Reagents
1. α -glucosidase solution (0.6 U/mL)
2. 37 mM sucrose
3. Preparation of extract/Standard: 10 mg extract / standard was weighed and
dissolved in 2 mL of respective solvents. Further, it was diluted (1:2) to get
various concentrations ranging from 1.5-1000 µg/ 200 µL. 0.2 mL of each
concentration of the extract / standard was used for estimation.
Procedure
200 μL of α-glucosidase solution (0.6 U/mL) was pre-incubated with 200 μL
of various concentrations (1-1000 μg/mL) of extract dissolved in ethanol: water
mixture (8:2) or vehicle control for 5 min. The reaction was initiated by adding 200
μL of the substrate (37 mM sucrose) and terminated after 30 min incubation at
37°C by heating at 90-100°C in a water bath. The formation of glucose was
determined by glucose-peroxidase (GOD-POD) method at 546 nm using star-
21plus biochemistry auto analyser (Rapid Diagnostic Pvt. Ltd, Delhi, India). The
inhibitory effect of extracts on -glucosidase was determined by the rate of
decrease in the amount of glucose liberated from molecules of substrate after
incubation with the enzyme. The IC50 values (concentration in μg/mL required for
50 % inhibition of α-glucosidase activity under the assay conditions specified.) were
determined by non-linear regression analysis using GraphPad Prism (Version 5.0).
The values are mean of triplicate incubations expressed as mean ± SEM. Miglitol
(1 to 1000 μg/mL) was used as positive control and -glucosidase inhibition was
carried out under the same assay conditions specified above.
The α - glucosidase inhibitory activity was calculated as follows.
(%) Inhibition = [(Abs. of control- Abs. of test)/ Abs. of control)] x 100
Inhibition of sorbitol accumulation
Heparinized rat blood samples were collected and immediately centrifuged
at 1500 rpm at 4°C for 10 min. The plasma was separated and erythrocytes were
washed three times with cold isotonic saline and centrifuged at 1500 rpm for 15
min. 200 μL of washed erythrocytes was added to 1 mL of Hank’s balanced salt
solution [0.137 M NaCl; 5.4 mM KCl; 0.25 mM Na2HPO4; 0.44 mM KH2PO4; 1.3 mM
CaCl2; 1.0 mM MgSO4; 4.2 mM NaHCO3] (pH 7.4) containing various
concentrations of ASE, LSE, DIA-2 or ascorbic acid (5- 100 μg/mL). All samples
were incubated at 37°C for 3 h at room temperature. The erythrocytes were
centrifuged at 3000 rpm at 4°C for 10 min and the supernatant was removed; then
the cells were washed three times with cold isotonic saline and centrifuged again.
The protein was precipitated with 2 mL of cold 6% perchloric acid and the
supernatant was neutralized with cold 1M K2CO3. The supernatant solution was
stored at –20°C until analysis for sorbitol content. Sorbitol content was analyzed as
described earlier (Malone et al., 1980; Clements et al., 1969). In brief, the reaction
mixture contained the appropriate protein-free supernatant, 50 mM glycine buffer
(pH 9.4), 0.2 mM NAD+, and 1.28 units of sorbitol dehydrogenase. The mixture
was incubated at 37°C for 30 min, and the relative fluorescence due to NADH was
measured by a fluorescence spectrometer (Perkin Elmer, LS-45, UK) at an
excitation wavelength of 366 nm and an emission wavelength of 452 nm. The
experiments were performed in triplicates (Nandhini et al., 2004). The percentage
inhibition of the test extracts against erythrocyte sorbitol accumulation was
calculated and compared with Vitamin C, a standard antioxidant.
Inhibition of protein glycation
Glycation reaction was initiated by addition of equal volumes of high
concentration glucose solution and a known concentration of albumin solution. To
prevent microbial contamination, sodium azide (0.1%) was added to the reaction
mixture prepared in 0.01 M phosphate buffer (pH=7.4).Various concentrations
(10-300 μg/mL) of ASE, LSE and DIA-2 were added in triplicates and incubated at
room temperature. The samples were dialyzed in phosphate buffer (the dialyzed
bag was prepared in 10 mM Ethylene Diamine Tetracetic Acid (EDTA) before this
process) on day 7. The extent of albumin glycation was measured as thiobarbituric
acid reactive substances (TBARS) content (Parker et al., 1981). In brief, 1 mL 20%
trichloroacetic acid (TCA) was added to above solution and then centrifuged for 10
minutes at 3000 rpm and the supernatant was discarded. 1 mL phosphate buffer
with above specification and 0.5 mL 0.3 N oxalic acid were added to the precipitate
and placed in boiling water bath for 30 min. The mixture was allowed to cool at
room temperature, 0.5 mL 40% TCA was added to each sample. After
centrifugation for 10 min at 3000 rpm, the supernatant was separated and 0.5 mL
5% M TBA was added to 1 mL of supernatant solution, then the whole was set in
40°C water bath for half an hour. At the end, the absorbance of the sample was
measured in 443 nm (Perkin Elmer, l25, Norwalk, CA, USA).
3.3.3.4. Cytotoxicity assay of ASE, LSE and its mixture
Cell Culture and Maintenance
3T3-L1 preadipocytes were grown in T25 tissue culture flask containing
Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal
bovine serum (FBS) and penicillin/streptomycin (100 IU, 100 mg/mL, respectively)
in a humidified atmosphere of 5% CO2 at 37°C. Preconfluent 3T3-L1 cells were
seeded in 96-well plates at a density of 8,000 cells/200 μL/well. Based on the
percentage confluence, the cells were trypsinized and sub-cultured. During the
process, the medium was replaced with fresh medium every 2-3 days. The cells
were examined for morphology using an inverted microscope (Motic AE30, Hong
Kong). The optimal cell concentration for 3T3 L1 adipocytes/well was optimised.
Measurement of cell viability
Cell viability experiment was carried out in 96 well micro titre plates as per
the earlier described method (Mossman 1983). Optimal concentration of cells were
treated with different concentrations (ranging from 1×100 ρg -1×108 ρg) of individual
extracts or their combination (1:1 and 1:2 combination of ASE and LSE) after 24 h
following plating and incubated at 37 C for one day. At 20 h following extract
exposure, the cells were incubated in a humidified atmosphere of 5% CO2 at 37°C
with 5 mg/mL MTT for 4 h. At the end of the experiment, the medium was removed,
and the insoluble formazan product was dissolved in dimethyl sulphoxide (DMSO)
(200 μL) and kept at least 15 min in the dark. MTT reduction was quantified by
measuring the absorbance at 570 nm and 630 nm in spectrophotometer (Multiskan,
Thermo scientific, Rockford, IL, U.S.A).
The percentage growth inhibition was calculated using the formula below:
% cell growth inhibition= 100-{(At-Ab)/ (Ac-Ab)} x100.
Where, At= Absorbance value of test, Ab= Absorbance value of blank,
Ac=Absorbance value of control. Cytotoxicity was expressed as IC50 value. The
IC50 value is the concentration of the extracts that cause 50 % inhibition or cell
death and was obtained by plotting the percentage inhibition versus concentration
of the extracts.
Measurement of cytotoxicity
For the LDH assay, the 3T3-L1 cells were plated in 96-microtiter plates.
Twenty four hour following plating, optimal concentration of cells was exposed to
varying concentrations of ASE, LSE and their combination (1:1 and 1:2 combination
of ASE and LSE). After 24 h of treatment, the cells were then treated with a cell
lysis solution (DMSO) for 30 minutes at room temperature to lyse. Release of LDH
in supernatant was assessed by using a LDH kit according to the procedures
(Merck Ltd, Mumbai, India) mentioned in the kit insert. The intensity of the colour is
proportionate to LDH activity. The absorbance was determined at 490 nm with 96-
well plate ELISA reader (Multiskan, Thermo Scientific, Rockford, IL, USA). The
percent LDH release was calculated as follows: (LDH in culture supernatant/LDH in
culture supernatant + LDH in cell lysate) × 100.
3.3.3.5. Glucose uptake assay and GLUT-4 protein expression of DIA-2
Glucose is fundamental to the metabolism of mammalian cells. Its passage
across cell membranes is mediated by a family of transporters termed glucose
transporters or GLUTS. In adipose and muscle tissue, insulin stimulates a rapid
and dramatic increase in glucose uptake, which is largely due to the redistribution
of the insulin-inducible glucose transporter, GLUT4. In response to insulin, GLUT4
is quickly shuttled from an intracellular storage site to the plasma membrane, where
it binds glucose. The effect of ASE, LSE and DIA-2 on glucose uptake and GLUT-4
expression was assessed In vitro using 3T3-L1 adipocytes.
Glucose Uptake Assay
3T3-L1 preadipocytes [obtained from National Centre For Cell Science
(NCCS), Pune, India] were cultured in Dulbecco’s Modified Eagle Medium (DMEM)
with 10% Fetal bovine serum (FBS) and supplemented with penicillin (120
units/mL), streptomycin (75 μg/mL), gentamycin (160 μg/mL) and amphotericin B (3
μg/mL) in 5% CO2 environment. 3T3-L1 preadipocytes grown in 24 well plates were
induced by the differentiation medium (combination of IBMX, DEX and insulin in
DMEM medium with 10% FBS) to differentiate into adipocytes. The extent of
differentiation was established by observing multinucleation of cells. 3T3-L1
adipocytes grown in 24-well plate were subjected to glucose uptake as per
standard methods (Muthusamy et al., 2008). After differentiation, the medium was
replaced using DMEM with 10% FBS containing 1 mg/mL of insulin for 2 days
followed by serum starvation for 5 h. After the induction, the cells were incubated
with various concentrations of ASE, LSE and DIA2 for 24 hours and stimulated with
insulin (100 nM) for 20 min. After experimental incubation, cells were rinsed once
with Krebs Ringer Phosphate HEPES (KRPH) solution (118 mM NaCl, 5 mM KCl,
1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4 and 30mM HEPES - pH 7.4) and
were subsequently incubated for 20 min in KRPH solution containing 0.5 μCi/mL 2-
Deoxy-D-3[H]glucose (2-DOG). The uptake was terminated by aspiration of media.
Cells were washed thrice with ice-cold KRPH solution and lysed in 0.1% Sodium
dodecyl sulphate (SDS). The lysates were transferred to 96-well plate with glass
fiber paper and air dried overnight. This plate was used to measure the cell-
associated radioactivity by liquid scintillation counting. All the assays were
performed in triplicates for concordance. Results were expressed as % glucose
uptake with respect to solvent control. Rosiglitazone (50 μM) was used as the
positive control.
GLUT-4 PROTEIN EXPRESSION
Reagents
Separating gel buffer
Double distilled (DD) water - 4 mL
30% Acrylamide - 3.3 mL
1.5M Tris Buffer - 2.5 mL (pH: 8.8)
10% SDS - 100 μL
10% Ammonium persulfate (APS) - 100 μL
TEMED - 4 μL
Stacking gel buffer
D.D water - 2.7 mL
30% Acrylamide - 670 μL
1M Tris Buffer - 0.5 mL (pH: 6.8)
10% SDS - 40 μL
10% APS - 40 μL
TEMED - 4 μL
Cell lysis Buffer
Totx Buffer - 975 μL
Aprotinin - 5 μL (10 mg/mL)
PMSF - 20 μL (1.74 mg/mL)
Alkaline phosphate buffer
1M Tris Buffer - 2.5 mL (pH 9.5)
4M NaCl - 625 μL
1M MgCl2 - 125 μL
Made up to 25mL with double distilled water
Procedure
1. Cells were grown in petriplates and varying concentration of extract (ASE and
LSE) or DIA-2 (1:1 mixture of ASE and LSE) was added and incubated for 24
hours.
2. The cells were transferred to microfuge tubes and washed with PBS at 14000
rpm for 30 seconds.
3. The cells were resuspended in 40µl lysis buffer [1% IGEPAL, 150mM NaCl,
50mM Tris, pH 7.5, 1mM EDTA, 1mM PMSF (phenylmethanesulfonylfluoride),
21 mg/mL Aprotinin, 10mg/mL Leupeptin).
4. Samples were centrifuged at 14000 g at 4oC for 30 min and supernatants were
collected and stored at -20oC.
5. Protein was estimated by Bradford method (Bradford 1976) at 640 nm and 100
mg protein of each sample was loaded on a 10% polyacrylamide gel.
6. Gel was run at 20 mA for 1hour. After run was over, the gel was kept in
Transfer buffer for 10 minutes. Nitrocellulose membrane and Whatman filter
papers were cut to the same size of the gel and incubated in transfer buffer for
10 minutes prior to the transfer.
7. The membrane was placed on the filter papers, and the gel was carefully
layered over it. Filter papers were placed on top of the gel, eliminating air
bubbles and transfer was set up at 120 mA for 90 minutes.
8. Stained with ponceau reagent (to check the protein transfer), washed with PBS
until the color disappears and blocking reagent (5% Skimmed Milk) was added
and blocked overnight at 4oC.
9. The blot was washed with tween PBS thrice and with PBS thrice.
10. Incubated with primary antibody (GLUT4 mouse antibody, Cell Signaling
Technology, Inc. USA) at appropriate dilution in 5% BSA in PBS 1 mL and 4 mL
of PBS for at least 1-3 hr at room temperature (RT).
11. The blot was washed with Tween with PBS (2mL in 2.5 litre PBS) for 5 to 6
times.
12. The blot was incubated with secondary antibody (ALP conjugated mouse
antibody) at appropriate dilution in 5% BSA in PBS 1 mL and 4 mL of PBS for
at least 1.30 hr at RT.
13. Wash the blot with Tween with PBS (2mL in 2.5 litre PBS) for 5 to 6 times.
14. 25 mL of developing reagent was prepared and blot was rinsed with 5 mL, 10
mL was added and incubated for 10 mins, then the remaining reagent was
added to the blot and 33 μL of BCIP and 66 μL of NBT were added and mixed
well, kept in dark for some time and the bands were observed.
3.3.3.6. IN VITRO ADIPOCYTE DIFFERENTIATION ASSAY
Experiments were performed in 24-well plates with cells seeded at a density
of 50,000 cells/mL and allowed to attach overnight and then differentiated as
described below. 24 hours prior to differentiation, cells were treated with various
concentration of ASE, LSE. DIA-2 and rosiglitazone. Cells were grown in
Dulbecco’s minimum essential medium (DMEM) supplemented with 10% FBS and
penicillin/streptomycin (100 IU, 100m g/mL, respectively) at 37 °C in 5% CO2. To
induce differentiation, 2-day postconfluent 3T3-L1 preadipocytes (day 0) were
stimulated for 24h by adding 51.8 mM 3-isobutyl-1-methylxanthine, 0.25 μM
dexamethasone, and 0.1% insulin (MDI) to the DMEM/10% FBS culture medium.
Subsequently, on day three, the MDI medium was replaced with DMEM/10% FBS
containing 1 μM insulin. On day 4, the MDI medium was replaced with DMEM/10%
FBS and refreshed for 2 days until analysis was performed on days 6~7.
Experimental values were mean of triplicates.
Oil Red O staining
The formation of oil droplets in treated and untreated differentiated 3T3 cells
were analysed by Oil Red O staining as per the earlier reported method (Lillie
1976) with slight modifications. After differentiation of the 3T3-L1 preadipocytes, the
media was removed; the cells were washed once with phosphate-buffered saline
(PBS), and then fixed for at least 1h with pre-chilled 10% formaldehyde in PBS.
Cells were stained with Oil Red O solution (a mixture of three parts of 0.5% (w/v)
Oil Red O in isopropanol and two parts of water) for 2 h at room temperature
followed by washing with PBS twice, ethanol once and water twice. Cells were kept
in water and photographed using inverted epifluorescence microscope (Motic,
China)
3.3.3.7. Optimisation of polymerase chain reaction (PCR) for the mRNA
expression of PPAR-γ, SREBP-1c, Leptin and β-actin
Polymerase Chain Reaction was performed using Prime RT-PCR premix
(2X) master mix (Genet Bio, Korea). β-actin served as housekeeping gene. PPAR-
, SREBP and Leptin primers were also used. The annealing temperature for each
primer was optimized.
-Actin
Forward primer : 5’-GACATGGAGAAAATCTGGCA-3’
Annealing Temperature : 55.30C
Reverse primer : 5’-AATGTCACGCACGATTTCCC-3’
Annealing Temperature : 55.30C
The primers at a concentration of 20 mol/ L were used for cDNA synthesis
Procedure
The isolated RNA was used for optimization of the annealing temperature.
The steps are as follows
Step: 1 Conversion of mRNA to cDNA (Reverse transcription):
i) Temperature 42 0C for a period of 30 sec
ii) Temperature 94 0C for a period of 5 min
Step: 2: PCR
i) Denaturation: Temperature 94 0C for a period of 1 min
ii) Annealing: For optimizing the annealing temperature of β-actin, gradient PCR
was ran setting temperature 500C as the lowest with gradient increase of 50C. The
temperature at each well was 450C, 45.10C, 45.70C, 46.60C, 47.70C, 49.00C,
50.40C, 51.80C, 53.00C, 54.10C, 55.00C and 55.40C.
iii) Synthesis: Temperature 720C for a period of 1 min. The cycle was repeated for
35 times from Step 2 for multifold increase of cDNA copies.
SREBP
Forward primer : 5’-ACCCTGGTGAGTGGAGGGACCATCTTG-3’
Annealing temperature : 71.00C
Reverse primer : 5’-CTTTGCTTCAGTGCCACCACCAGGTCTTT-3’
Annealing Temperature : 68.10C
The primers at a concentration of 20 mol/μL were used for cDNA synthesis
Procedure
The isolated RNA was used for optimization of the annealing temperature.
The steps are as follows
Step: 1 Conversion of mRNA to cDNA (Reverse transcription) :
i) Temperature 420C for a period of 30 sec
ii) Temperature 940C for a period of 5 min
Step: 2 PCR
i) Denaturation: Temperature 940C for a period of 1 min
ii) Annealing: For optimizing the annealing temperature of SREBP, gradient PCR
was ran setting temperature 68.10C as the lowest with gradient increase of 50C.
The temperature at each well was 63.10C, 63.50C, 64.20C, 65.20C, 66.50C, 67.80C,
68.20C, 70.50C, 71.70C, 72.60C and 73.30C.
iii) Synthesis: Temperature 720C for a period of 1 min. The cycle was repeated for
35 times from Step 2 for multifold increase of cDNA copies.
Leptin
Forward primer : 5’-CCCCATTCTGAGTTTGTCCA-3’
Annealing Temperature : 57.30C
Reverse primer : 5’-GCTGAAGAACTAGGTGAGAG-3’
Annealing temperature : 57.30C
The primers at a concentration of 20 mol/ L were used for cDNA synthesis
Procedure
The isolated RNA was used for optimization of the annealing temperature.
The steps are as follows
Step: 1 Conversion of mRNA to cDNA (Reverse transcription):
i) Temperature 42 0C for a period of 30 seconds
ii) Temperature 94 0C for a period of 5 minutes
Step: 2 PCR
i) Denaturation: Temperature 94 0C for a period of 1 minute
ii) Annealing: For optimizing the annealing temperature of leptin, gradient PCR
was ran setting temperature 52.30C as the lowest with gradient increase of 50C
The temperature at each well was 47.30C, 47.50C, 48.00C, 48.90C, 50.00C, 51.30C,
52.70C, 54.10C, 55.30C, 56.40C, 57.30C and 57.70C.
iii) Synthesis: Temperature 720C for a period of 1 minute. The cycle was repeated
for 35 times from Step 2 for multifold increase of cDNA copies.
PPAR
Forward primer : 5’-GAGCTGACCCAATGGTTGCTG-3’
Annealing Temperature : 61.80C
Reverse primer : 5’-GCTTCAATCGGATGGTTCTTC-3’
Annealing Temperature : 57.90C
The primers at a concentration of 20 mol/ l were used for cDNA synthesis
Procedure
The isolated RNA was used for optimization of the annealing temperature.
The steps are as follows
Step: 1 Conversion of mRNA to cDNA (Reverse transcription):
i) Temperature 42 0C for a period of 30 sec
ii) Temperature 94 0C for a period of 5 min
Step: 2 PCR
i) Denaturation: Temperature 94 0C for a period of 1 min
ii) Annealing: For optimizing the annealing temperature of PPARγ, gradient PCR
was ran setting temperature 52.90C as the lowest with gradient increase of 50C.
The temperature at each well was 47.90C, 48.10C, 48.60C, 49.50C, 50.60C, 51.90C,
53.30C, 54.70C, 56.00C, 57.00C, 57.90C and 58.30C.
iii) Synthesis: Temperature 720C for a period of 1 minute. The cycle was repeated
for 35 times from Step 2 for multifold increase of cDNA copies.
Isolation of mRNA and RT-PCR
Reverse transcriptase (RT) - PCR was performed to determine the level of
mRNA expression of SREBP, Leptin and PPARγ. Briefly, on day 6 after
differentiation, total RNA was extracted from cultured cells using TRIzol Reagent
(Sigma, USA). After homogenization, the tubes were incubated for 10 minutes and
centrifuged at 1000 rpm for 5 min. 200 L of chloroform was added to the
supernatant, allowed to incubate for 5 min at room temperature and centrifuged at
12000 rcf for 20 min. Then 500 L of isopropyl alcohol was added to the
supernatant to precipitate the total RNA and centrifuged at 12000 rcf for 15 min
following the incubation period of 10 min. The supernatant was decanted carefully;
the pellet was washed three times with 75% ethanol, centrifuged at 12000 rcf for 15
min and the pellet was allowed to air dry. The pellet was resuspended in 20 L of
RNase free water and stored in -80 C until use. The isolated RNA was allowed to
undergo reverse transcription and polymerization reaction to get cDNA using PCR
master cycler gradient. The formed cDNA was loaded in agarose gel, allowed to
run the electrophoresis at 80V for 30 min and the gene expression was analyzed
using the bands formed. 200 nanograms of RNA were used for reverse
transcription polymerase chain reaction (RT-PCR) according to the manufacturer’s
instructions (Genet Bio, Korea). The following sequence was performed for each
PCR reaction: 42◦C for 30s, 94◦C for 5min (1 cycle); 94 ◦C for 1min, 52.3◦C (leptin),
51.3◦C (PPARγ) and 68.2◦C (SREBP) for 1min, and 72 ◦C for 1 min (with 35
cycles); and a final extension phase at 74 ◦C for 10 min.
3.3.3.8. Effect of ASE, LSE and DIA-2 on ROS production in 3T3-L1
adipocytes
Intracellular ROS scavenging activity was carried out using DCF-DA as
described in earlier reported methods (Armstrong and Whiteman 2007). Briefly,
3T3-L1 adipocyte cell was seeded on 48-well plate at 10,000 cells/well and allowed
to attach overnight. Twenty four hours after plating, the cells were treated with
varying concentrations (10 and 100 μg/mL) of the extracts and incubated at 37°C.
One hour later, 100 µM of H2O2 was added to the plate. After 1 h, 10 µM of 2',
7’dichlorfluorescein diacetate (DCF-DA) solution was added and incubated for 1 h.
The effect of extract on the extent of ROS production was visually observed and
the cell images were captured using moticam digital camera fitted to inverted
fluorescent microscope (Motic AE30, Hong Kong).
3.3.4. IN VIVO STUIDES
Animals
Sprague-Dawley rats, weighing (120 to 150 g at the start of the study) were
obtained from central animal facility, Sri Ramachandra University, Chennai, India.
Animals were housed in colony cages (5-6/cage) and were kept under laboratory
standard conditions with temperature (22±2°C), 12-h light/12-h dark cycle and
relative humidity 40–60%. They had free access to Nutrilab rodent pellet feed
(Tetragon Chemie Pvt Ltd, Banglore, India) and purified water ad libitum prior to the
dietary manipulation. The study protocol was approved by the Institutional Animal
Ethics Committee (XIth IAEC/ SRMC & RI / 61/19/12/2006).
3.3.4.1. Acute oral toxicity
Acute oral toxicity study was performed according to the Organisation for
Economic Co operation and Development (OECD) test guideline 423: acute toxic
class (ATC) method. Female Sprague Dawely rats (120-150 g) were used for the
study and they were acclimatized for a period of 7 days before the start of the
experiment. The experimental animals were divided into two groups (n=3/group).
Group I served as control and received 0.5% carboxy methyl cellulose (CMC) as
vehicle (10 mL/kg, p.o) and Group II received DIA-2 (2000 mg/kg, p.o) prepared in
0.5% CMC. Mortality, clinical signs such as convulsion, abnormal respiration,
piloerection, changes in skin and fur, locomotor activity, tremors, salivation,
diarrhoea and lethargy were recorded at timely intervals for 24 h, with special
attention given during first 4 h following drug administration and once a day for 14
days thereafter. Gross pathological changes were observed and recorded at the
end of the study.
3.3.4.2. Antidiabetic activity of DIA-2
The anti-diabetic activity of DIA-2 was initially screened at two dose levels
(250 and 500 mg/kg body weight).The major parameters like body weight, plasma
glucose, insulin, triglycerides and total cholesterol and histopathological changes of
major organs (Pancreas, liver, kidney, brain) were analyzed. Based on these
results, two lower dose levels (62.5 and 125 mg/kg bodyweight) were tested. The
results showed significant change in the major parameters assessed earlier.
Additional parameters were performed at the later tested dose levels (62.5 and 125
mg/kg bodyweight).
3.3.4.2.1. Induction of Type 2 Diabetes in Rats
The rats were allocated to two dietary regimens consisting of 10 and 80
animals by feeding either normal pellet diet (NPD) or high fat diet (HFD) ad libitum,
respectively for the initial period of four weeks. The composition of the high fat diet
is given in the Table-2. After 4 weeks of dietary manipulation, HFD animals with ~3
fold total cholesterol (TC) and ~4 fold triglycerides (TG) levels increase were
defined as hyperlipidemic, and were injected with low dose (35 mg/kg, i.p) of
streptozotocin (STZ) (freshly prepared in ice cold citrate buffer pH 4.5). NPD
animals were injected with citrate buffer (1mL/kg, i.p). 72 h after STZ injection, the
Table-2: Composition of High fat diet (HFD)
Ingredients Percentage
Wheat 23.4
Yellow corn 23.4
Milk powder 19.5
Calcium chloride 1.0
Crude coconut oil 15.6
Pork lard 15.6
Vitamin B12 0.5
Common salt 1.0
rats with the fasting glucose level ≥250 mg/dl were considered diabetic and
selected for the anti-diabetic study. The rats were kept on their respective diets until
the end of the study.
3.3.4.2.2. Treatment and Groups
Treatment was scheduled once a day for 14 consecutive days. Group 1
kept on NPD while groups 2-7 on HFD throughout the study period.
Group 1: NPD + citrate buffer (1 mL/kg, i.p) + received 0.5% Carboxy Methyl
Cellulose. [CMC] (10 mL/kg, p.o); Normoglycemic (NG)
Group 2: HFD + STZ (35 mg/kg, i.p) + received 0.5% CMC (10 mL/kg, p.o);
Hyperglycemic (DG)
Group 3: HFD + STZ + RG (8 mg/kg, p.o) (RG)
Group 4-7: HFD + STZ + DIA-2 at 62.5, 125, 250 and 500 mg/kg, p.o,
respectively.
Weekly body weight, fasting plasma glucose (FPG), fasting plasma insulin
(FPI), total cholesterol (TC) and triglycerides (TG) were measured in all the
experimental animals to ascertain the role of DIA-2 in type II diabetic state as per
the manufacturer instructions mentioned in the kit (Merck, Mumbai, India). Rat
Insulin ELISA kit was obtained from DRG Instruments GmbH, (Marbourg,
Germany). The FPG, FPI, TC and TG were measured to access the effect of DIA-2
at various doses on blood sugar and lipid metabolism. Only doses showing
therapeutic effect were subjected to further biochemical analysis.
3.3.4.2.3. Liver and Kidney function test
After 14 days of treatment, blood was collected by sino-orbital puncture
under general anaesthesia using EDTA as anticoagulant. The plasma separated
was used for the determination of plasma biomarkers alanine transaminase (ALT),
aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein,
albumin, BUN and creatinine of hepatocellular, renal injury. The estimations were
performed by using the standard procedures of commercial diagnostic kit (Accurex
Biomedical Pvt.Ltd. Mumbai, India) on star-21 plus biochemistry auto analyser
(Rapid Diagnostic Pvt. Ltd, Delhi, India)
3.3.4.2.4. Preparation of tissue homogenate for biochemical estimations
After blood collection, all the animals from each group were sacrificed by
cervical dislocation under general anaesthesia. The tissues (brain, heart,liver,
kidney, pancreas) were harvested from all the experimental groups, washed in ice
cold normal saline and stored at ultra low deep freezer -800C (Thermo Fisher
Scientific, USA) until they are processed for biochemical estimation. 10% tissue
homogenate was prepared with ice cold 10% KCl and centrifuged at 1000 rpm for
15 min. The supernatant was used as the source of enzyme. The total protein
content was estimated (Lowry et al., 1951).
3.3.4.2.5. Activity of glycolytic enzymes
Hexokinase
Reagents:
1. Substrate - glucose (0.005 M): 45 mg in 50 mL of distilled water.
2. Tris HCL (0.01 M) pH: 8: 1.576 g in 100 mL of distilled water.
3. ATP (0.72 mM): 3.968 mg in 10 mL of distilled water.
4. KCL (0.1 M): 74.5 mg in 10 mL of distilled water.
5. Sodium fluoride (0.5M): 210 mg in 10 mL of distilled water.
6. Potassium dihydrogen phosphate (0.02 M): 27 mg in 10 mL of distilled water.
7. 10 % TCA: 10 g in 100 mL of distilled water.
8. Magnesium chloride (0.05 M): 101 mg in 10 mL of distilled water.
9. Anthrone (0.2%): 0.2 g in 100 mL of ice cold 95% sulfuric acid.
Procedure
Hexokinase activity was assayed in the liver tissue as per the earlier
reported methods (Brandstrup et al., 1957). Briefly, the incubation mixture
containing 2.5 mL of tris HCl buffer (0.01 M, pH 8), 1 mL of glucose (0.005 M) as
substrate, 0.5 mL of ATP (0.72 M), 0.1 mL of MgCl2 (0.05 M), 0.1 mL of Sodium
fluoride (0.5 M), 0.4 mL of KH2PO4 (0.02M) and 0.4 mL of KCl (0.1M) were pre-
incubated at 37°C for 15 min. 0.1 mL of supernatant was added to the incubation
mixture and incubated further at 37°C for 30 min. The reaction was immediately
arrested by the addition of 10% TCA. The control reaction rate was correspondingly
assessed by adding 0.1 mL of supernatant only after the arresting reaction and
the protein precipitate was removed by centrifugation at 3500 rpm for 10 min.
Aliquots of supernatant were taken and made up to 1 mL with distilled water
followed by addition of 4 mL of anthrone. The absorbance was read at 630 nm
using spectrophotometer (Multiskan, Thermo scientific, U.S.A). The results are
expressed in nanomoles of glucose consumed/min/mg of protein.
3.3.4.2.6. Activity of gluconeogenic enzymes
Glucose-6 Phosphatase
Glucose-6 Phosphatase (G-6-Pase) in liver tissue was assayed as per the
reported method (Koide and Oda, 1959).
Reagents:
1. Citrate Buffer(0.1M) pH 6.5
2. Glucose-6 Phosphate-substrate (0.01M)
3. 10 % TCA
4. ANSA
5. Ammonium molybdate
Procedure
Briefly, 0.3 mL of citrate buffer (0.1 M pH 6.5) was followed by the addition
of 0.5 mL of Glucose-6-Phosphate (0.01 M) as substrate. To the test, 0.2 mL of
supernatant was added and further incubated at 37°C for 1 h. The reaction was
immediately arrested by the addition of 10% TCA. The control reaction rate was
correspondingly assessed by adding 0.2 mL of supernatant only after the arresting
step. The precipitate was removed by centrifugation at 3500 rpm for 10 min.
Aliquots of supernatant were used to determine the activity glucose-6-phosphatase
by measuring the amount of inorganic phosphate (Pi) liberated from glucose
6-phosphate according to earlier methods (Fiske and Subbarow, 1925) at 640 nm
using a spectrophotometer (Multiskan, Thermo scientific, U.S.A). The activity of
G-6-pase was expressed in nanomoles of Pi liberated/min/mg of protein.
Fructose 1, 6 diphosphatase
Reagents
1. Tris HCL (0.01M) pH: 8: 1.576 g in 100 mL of distilled water.
2. Substrate - Fructose 1, 6 diphosphate (0.005 M): 0.010151 mg in 5 mL of
distilled water.
3. Magnesium chloride (0.1M): 0.203 g in 10 mL of distilled water.
4. KCL: 74.5 mg in 10 mL of distilled water.
5. K.EDTA (0.001M): 4.0447 mg in 10 mL of distilled water.
Procedure
Fructose 1, 6 diphosphatase activity in liver tissue was assayed as per
earlier methods (Gancedo and Gancedo, 1971). Briefly, 1.2 mL of citrate buffer
(0.1 M pH 6.5) was followed by the addition of 0.1 mL of fructose 1, 6 diphosphate
(0.005 M) as substrate, 250 μL of MgCl2 (0.1 M) ,0.1 mL of KCl and 0.25 mL of K.
EDTA (0.001 M) were added subsequently. To the test, 0.1 mL of supernatant was
added and further incubated at 37°C for 15 min. The reaction was immediately
arrested by the addition of 10% TCA. The control reaction rate was correspondingly
assessed by adding 0.1 mL of supernatant only after the arresting step. The
precipitate was removed by centrifugation at 3500 rpm for 10 min. Aliquots of
supernatant were used to determine the activity fructose 1, 6 diphosphatase by
measuring the amount of Pi liberated from fructose 1, 6 diphosphate (Fiske and
Subbarow, 1925) at 640 nm using a spectrophotometer (Multiskan, Thermo
scientific, U.S.A). The activity of F-1, 6-BPase was expressed in nanomoles of Pi
liberated/min/mg of protein.
3.3.4.2.7. Glycogen content
Reagents
1. 80% hot ethanol
2. 52% Perchloric acid
Procedure
The hepatic glycogen content was estimated as per the earlier methods
(Morales et al., 1973). Briefly, to 100 mg liver add 400 µL of 80% hot ethanol. This
was centrifuged and washed for about 2-3 times. The residue obtained was dried in
a sand bath for 2 - 3 min. To the dried residue, 2 mL of distilled water and 2.5 mL of
52% perchloric acid was added and incubated at 0º for 20 min. The precipitate was
removed by centrifugation at 3500 rpm for 10 min. Aliquots of supernatant were
made up to 1 mL with distilled water and 4 mL of anthrone was added and boiled
for 5 min. The amount of glucose liberated was measured at 630 nm using
spectrophotometer (Multiskan, Thermo scientific, U.S.A). The glycogen content was
expressed as nM of glucose/g of tissue.
3.3.4.2.8. Tissue Oxidant and Antioxidant status
The tissue oxidant/antioxidant status was measured in the tissues of brain,
heart, liver, kidney and pancreas.
Assay of lipid peroxides
Reagents
1. Thiobarbituric acid (TBA) (0.8%): 0.8 gm in 0.5 N HCL
2. Butylated hydroxyl toluene (0.05%): 0.05 gm in methanol.
3. Saline (0.9%): 0.9 g in 100mL distilled water.
Procedure
The amount of lipid peroxides was estimated according to the earlier
method (Ohkawa et al., 1979). Briefly, 0.2 mL of tissue homogenate was made up
to 1 mL with normal saline, 0.5 mL of BHT (0.05%) and 3.5 mL TBA (0.8%) reagent
ware added and heated for 90 min in a boiling water bath. After cooling, the
solution was centrifuged at 3500 rpm for 10 min and the precipitate obtained was
discarded. The absorbance of the supernatant was determined at 532 nm using
spectrophotometer (Multiskan, Thermo scientific, U.S.A). The level of lipid
peroxidation was expressed in terms of nanomoles of malondialdehyde (MDA)
equivalents/g of tissue.
Super oxide dismutase
Reagents
1. Sodium pyrophosphate buffer (0.025 M):1.115 g in 100 mL of distilled water.
2. Phenazonium Metho Sulphate (PMS) (186 µM): 3 mg in 10 mL of distilled (930
µM).Then 1:5 dilutions were carried out to obtain 186 µM.
3. Nitro Blue Tetrazolium chloride (NBT) (300µM): 3 mg in 10 mL of phosphate
buffer.
4. NADH (780 µM): 6 mg in 10 mL of phosphate buffer.
Procedure
Super oxide dismutase was assayed by the method used earlier (Kakkar et
al., 1984). Briefly, to 0.05 mL of supernatant, 0.3 mL of sodium pyrophosphate
buffer (0.025 M), 0.025 mL of PMS (186 μM) and 0.075 mL of NBT (300 μM) was
added. The reaction was started by addition of 0.075 mL of NADH (780 μM). After
incubation at 30οC for 90 seconds, the reaction was immediately stopped by
addition of 0.25 mL glacial acetic acid. Then the reaction mixture was stirred
vigorously and shaken with 2.0 mL of n-butanol. The mixture was allowed to stand
for 10 min and centrifuged. The colour intensity in the butanol layer was read at
560nm (Multiskan, Thermo scientific, U.S.A). The activity of SOD was expressed as
units/min/mg of protein. One unit of SOD activity is defined as the enzyme reaction,
which gives 50% inhibition of NBT reduction in one minute under the specified
assay conditions.
Glutathione peroxidase
Reagents
1. Sodium Azide (10 mM): 16 mg in 25 mL of distilled water
2. GSH (2 mM): 30.732 mg in 50 mL of distilled water.
3. H2O2 (1 mM): 29 µl in 1000 mL of distilled water
4. 10% TCA: 10 g in 100 mL of distilled water
5. K.EDTA(0.4 mM): 16 mg in 100 mL of distilled water
6. Tris HCL Buffer(0.4 mM): 6.304 g in 100 mL of distilled water
7. DTNB (0.6 mM): 12 mg in 50 mL PO4 buffer.
Procedure
The activity of glutathione peroxidase (GPx) was assayed as per the earlier
methods (Rotruck et al., 1973). Briefly, to a reaction mixture containing 200 µL of
tris HCl buffer (0.4 mM), 200 µL K.EDTA (0.4 mM) and 100 µL of sodium azide (10
mM); 200 µL of supernatant was added and mixed well. Thereafter, 200 µL of
reduced glutathione (2 mM) solution followed by 0.1 mL H2O2 (1 mM) were added.
The overall reaction was arrested by adding 0.5 mL of 10% TCA. The non-
enzymatic reaction rate was correspondingly assessed by replacing the enzyme
sample by buffer. The precipitate was removed by centrifugation at 4000 rpm for 10
min. The remaining reduced glutathione in the supernatant was determined by
adding 1.0 mL of DTNB (0.6 mM) and the absorbance was read at 412 nm using
spectrophotometer (Multiskan, Thermo scientific, U.S.A). The results are expressed
in micromoles of GSH consumed/min/mg of protein.
Reduced Glutathione
Reagents
1. 5% TCA: 5 g in 100 mL of distilled water.
2. Phosphate buffer (pH:8) 0.2 M
3. DTNB (0.6 mM): 12 mg in 50 mL PO4 buffer.
Procedure
The assay was performed as per the earlier methods (Moren et al., 1979).
Briefly, 0.25 mL of supernatant was added to equal volume of ice cold 5% TCA to
precipitate the protein present in the tissue. The precipitate was removed by
centrifugation at 4000 rpm for 10 min. To 1 mL aliquot of supernatant, 0.25 mL of
phosphate buffer (pH 8.0) and 0.5 mL of DTNB (0.6 mM) was added and mixed
well. The absorbance was read at 412 nm using spectrophotometer (Multiskan,
Thermo scientific, U.S.A). The results are expressed in nanomoles of GSH/ mg of
protein.
3.3.4.2.9. Protein Carbonyl Content
Reagents
1 2 N HCL (0.01 M):16.8 mL in l00 mL of distilled water.
2 Guanidine HCL (6 M): 5.7318 g in 10 mL of distilled water.
3 10 % TCA: 10 g in 100 mL of distilled water
4 20 % TCA:20 g in 100 mL of distilled water
5 DNPH (0.2%):0.2 g in 2N HCL.
6 Ethanol : Ethyl acetate (1:1 v/v)
Procedure
Liver protein carbonyl content was determined as described earlier (Levine
et al., 1990) with minor modifications. Protein carbonyl content was assayed by
taking 50 µL of tissue homogenate followed by the addition of 800 µL of 2, 4-
Dinitrophenylhydrazine (0.2%) to the test sample and 2N HCl to the control. This
mixture was incubated in dark at room temperature for 1 h. Tubes were vortexed
for every 15 min. Reaction was immediately arrested by the addition of 1mL of 20%
TCA. Tubes were incubated in ice for 5min. The supernatant was removed by
centrifugation at 10,000 × g for 10 min. The pellet was resuspended in 1 mL of 10%
TCA and the tubes were again placed in ice and incubated for 5 min. Supernatant
was removed by centrifugation at 10,000 × g for 10 min. Pellet was resuspended in
1 mL of ethanol:ethylacetate (1:1) and washed for 3 times. The pellet obtained was
suspended in guanidine hydrochloride and centrifuged at 10,000 × g for 10
min at 4°C. Then, 1 mL of the supernatant was taken and the OD was read at 360
nm using spectrophotometer (Perkin Elmer, l25, Norwalk, CA, USA). Guanidine
HCl reagent serves as blank. Results were expressed as μmoles/g tissue.
3.3.4.2.10. Na+ K+- ATPase Activity
Na+ K+- ATPase was estimated in the tissues (brain, heart, liver, kidney and
pancreas) by the method of Bonting (1970).
Reagents
1. Tris HCL (184 mM) pH: 7.5.
2. KCl (50 mM)
3. Sodium EDTA (1 mM)
4. NaCl (600 mM)
5. 10 % TCA
6. ATP (40 mM)
7. 15% sodium meta bisulphate
8. 20% sodium sulphate
9. Ammonium molybdate (2.5% in 5N Sulphuric acid)
10. ANSA (0.1%)
Procedure
Na+K+ATPase was assayed by taking 250 µl of Tris HCl buffer followed by
the addition of 50µl of 600 mM NaCl, 50 µL of 50 mM KCl, along with 50 µL of 1
mM Na EDTA and 50 µL of 80 mM ATP. The reaction mixture was pre-incubated at
37°C for 10 mins. 25 µL of 10% tissue homogenate was added to the test alone
and tubes were allowed to incubate at 37°C for 1h. After 1 h, the reaction was
immediately arrested by the addition of 10% TCA. The control reaction rate was
correspondingly assessed by adding 25 µL of 10% homogenate only after arresting
the reaction. The precipitate was removed by centrifugation at 3500 rpm for 10
minutes. To 50 µL of the supernatant, 1075 µL of distilled water, 125 µL of
Ammonium molybdate and 50 µL of ANSA were added and incubated for 10 mins
at 37°C.The intensity of blue colour was read at 640 nm using an UV/Visible
spectrophotometer (Perkin Elmer, Lambda 25, USA) against a blank that contained
all the reagents minus the supernatant. The results are expressed in nanomoles of
phosphorus liberated /min/mg protein.
3.3.4.2.11. Target organ histopathology
At necropsy, the liver, kidney, pancreas and brain from all the experimental
animals from the respective treatment groups were dissected and fixed in 10%
neutral buffered formalin for 48 h. Thereafter the liver tissues were processed for
paraffin embedding, sectioned at 4 μm thickness using the microtome (Leica
RM2125RT, Germany). The sections were stained with haematoxylin and eosin
(H&E) stain for general histopathological evaluation. For demonstration of liver
glycogen, liver tissues were stained with Periodic acid Schiff (PAS) stain (Bancroft
and Gamble 2008). The H&E and PAS stained liver sections were examined under
light microscope (Motic, B1 Series, Hong Kong). The PAS staining was rated by the
percentage of hepatocytes staining positive. The PAS staining of kidney sections
were also performed.
3.3.4.3. Repeated dose 28-day oral toxicity study
The study was performed as per the Organisation for Economic Co-
operation and Development (OECD) test guideline 407 (Adopted: 3 October 2008).
Both sexes of young healthy adult SD rats ranging from 140-160g were
acclimatized individually in sterile polypropylene cages for 7 days. Our earlier study
on acute oral toxicity studies indicated that acute oral LD50 of DIA-2 was greater
than 2000 mg/kg in female SD rats and in another study using high fat
(HFD)/streptozotocin (STZ) induced diabetic rat, DIA-2 was found to exhibit anti-
diabetic activity without exhibiting any toxic signs as evidenced from the
histopathological finding till the dose range of 125 mg/kg body weight. DIA-2 at 250
and 500 mg/kg body weight did not show any protection after 14 days of treatment
in HFD/STZ induced diabetic rats. On the basis of these results, three doses of
62.5 mg/kg (Low dose), 125 mg/kg (Mid dose) and 250 mg/kg (High dose) were
selected for the study. All the animals were randomized accordingly with their body
weights into 4 groups of 10 animals each (5 male, 5 female/each group). Group I
served as control which received vehicle at 10mL/kg body weight orally. Group II, III
and IV received DIA-2 at 62.5 mg/kg (Low dose), 125 mg/kg (Mid dose) and 250
mg/kg (High dose), p.o, respectively, for a period of 28 days. The experimental
animals received their respective treatments orally twice daily.
3.3.4.3.1. Study design
Clinical observations which include general behaviour, respiratory pattern,
cardiovascular signs, motor activities, reflexes, changes in skin, fur and mortality
signs were observed for all the experimental animals continuously at 30 min, 1h, 2h
and 4h after the administration of the doses and thereafter once a day for 28 days
preferably at the same time (1 hour after the dose administration). Body weights
were taken on 0th, 7th, 14th, 21st and 28th days of dose administration. Individual
doses were calculated based on the most recent weekly body weights and were
adjusted each week to maintain the targeted dose level. Doses were administered
to all groups at a constant dose volume of 10 mL/kg. Feed and water consumption
was monitored and recorded throughout the study and was expressed as 7 day
cumulative data.
On the last day of dose administration all the animals were kept for
overnight fasting (water allowed). The overnight fasted animals were anaesthetized
under general anaesthesia using isoflourane, blood samples were collected by
retro-orbital puncture in heparinised (for haematological and biochemical analysis).
Blood smear was prepared from the EDTA containing blood sample, air dried and
stained (Hemacolor rapid staining of blood smear, E.Merck, Mumbai, India) and
differential leukocyte count (DLC) performed. Haematological analysis of the blood
samples includes analysis of haemoglobin (HGB), red blood cell count (RBC), white
blood cell count (WBC), platelet count and hematocrit. These parameters were
measured using automated haematology analyzer (Model PE 6000 Rapid
Diagnostics Pvt Ltd, New Delhi, India).
The heparinised tube containing blood samples were centrifuged
immediately in refrigerated centrifuge (Model 5810 R, Eppendorf AG, Germany) at
1500 x g for 15 min. The plasma thus collected was analysed for serum glutamic
pyruvic transaminase (SGPT); serum glutamic oxaloacetic transaminase (SGOT);
alkaline phosphatase (ALP), lactate dehydrogenase (LDH), glucose, triglyceride,
cholesterol, total bilirubin, creatinine, urea, protein and albumin levels by using
biochemical kits (Accurex Biomedical Pvt. Ltd, Thane, India) in semi automated
biochemical analyzer (Model: Star 21 Plus, Rapid Diagnostics Pvt Ltd, New Delhi,
India).
3.3.4.3.2. Histopathology
All the experimental animals were sacrificed by cervical dislocation under
general anaesthesia for gross pathological examination of all major internal organs.
Organs like brain, heart, liver, spleen, kidneys, adrenals, lung and testis/ovaries
were dissected immediately after euthanasia, blotted free from blood and weighed.
The other organs including skin, eyes, brain,lungs, trachea,salivary gland,
esophagus, thyroid gland,parathyroid gland, heart, liver, spleen, stomach, intestine,
kidneys, adrenals, urinary bladder, uterus, bone, skeletal muscle,sciatic nerve,
testis/ovaries, epididymis, prostate and seminal vesiclels were collected from
control and high dose groups and when appropriate from the low dose groups. All
the specimens were fixed in 10% neutral buffered formalin for 48 h. Thereafter the
fixed tissues were processed for paraffin embedding, sectioned at 4 μm thickness
using the microtome (Leica RM2125RT, Germany). The sections were stained with
haematoxylin and eosin (H&E) stain for general histopathological evaluation.
