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Chapter - 3
In Vivo Antidiabetic Activity Studies
Education is what survives when what
has been learnt has been forgotten.
- B. E. Skinner
Chapter – 3 : In vivo antidiabetic activity studies Introduction
75
Introduction
Diabetes mellitus is a leading metabolic disorder worldwide, caused by
inherited and / or acquired deficiency in production of insulin in β cells of pancreas,
or by the desensitization of insulin receptors for insulin. Such a deficiency results in
the increased concentration of glucose in the blood which results in secondary
complications affecting eyes, kidneys, nerves and arteries (Ismail, 2009).
Experimental evidences suggest the involvement of free radicals in the pathogenesis
of diabetes (Matteucci and Giampietro, 2000) and more importantly in the
development of diabetic complications (Lipinski, 2001).
The number of people suffering from diabetes all over the world has increased
and the disease now kills more people than AIDS. Based on a report by Shaw et al.
(2010), diabetes among adults is expected to increase from 285 million in the year
2010 to 439 million or more by the year 2030. Several reports indicate that annual rate
of DM will increase in future worldwide, especially in India. It has been proposed that
approximately 87 million Indians will be affected by DM by the year 2030 (Shaw
et al., 2010). Out of the two types of diabetes, the incidence of non-insulin dependent
diabetes mellitus (NIDDM) is much higher than the insulin dependent diabetes
mellitus (IDDM) (Hussain, 2002).
In conventional therapy, IDDM is treated with exogenous insulin and NIDDM
with oral hypoglycemic agents (Jarald et al., 2008). Though, different types of oral
agents are available in the treatment of NIDDM, in the last few years there has been
an exponential growth in the use of herbal medicines for diabetes and these drugs are
gaining popularity both in developed and developing countries because of their
natural origin and less side effects (Grover et al., 2002b).
A systematic review of literature on the research work on A. paniculata,
T. cordifolia and T. foenum-graecum, have revealed that very few studies have been
carried out to show their in vivo antidiabetic activity. Majority of the studies on A.
paniculata have been carried out using the aerial parts. Antidiabetic activity studies
on T. cordifolia have been carried out using only root and stem. Earlier in vivo studies
Chapter – 3 : In vivo antidiabetic activity studies Introduction
76
on T. foenum-graecum have shown only the use of seeds. Hence, we have carried out
in vivo antidiabetic activity studies on these plants by taking only the leaves. In vitro
screening of different solvent leaf extracts for antioxidant activities played a strategic
role in the selection of the suitable solvent extract for the antidiabetic study. The
ethanol leaf extract of these plants exhibited highest in vitro antioxidant activity. This
provides deep insight into the use of extracts for therapeutic purpose and totalitarian
approach emphasized in traditional medicine. Hence, ethanol extract was selected for
carrying out the in vivo antidiabetic activity studies. The ethanol leaf extract of all the
three plants were investigated for their efficacy and safety in rats. The extracts were
tested for an array of biochemical parameters in rats like blood glucose levels, serum
urea levels, serum and urine creatinine levels and antioxidant enzyme activities in
liver. Histopathological studies of islet cells of pancreas in normal and streptozotocin
induced diabetic rats were also carried out. Details of the work carried out are
presented in this chapter.
Chapter – 3 : In vivo antidiabetic activity studies Material and Methods
77
Material and Methods
Plant material
Fresh and healthy leaves of A. paniculata and T. cordifolia were obtained from
local growers of Mysore. Trigonella foenum-graecum leaves were obtained from the
local market, Mysore. The sample specimen was identified based on the taxonomical
characteristics. The leaves were washed thoroughly in distilled water and the surface
water was removed by air drying under shade. The leaves were subsequently dried in
a hot air oven at 40°C for 48 h, powdered to 100-120 mesh in an apex grinder [Apex
Constructions, London] and used for extraction.
Chemicals
Ethanol was of AR grade from Merck (Mumbai, India). Streptozotocin was
from Sisco Research Laboratories (Mumbai, India). Sodium carbonate, copper
sulphate, phenol reagent, sodium potassium tartarate, formaldehyde, sodium
phosphate and hydrogen peroxide were from SD Fine Chemicals (Mumbai, India).
Creatinine and urea estimation kits were from Anamol Laboratories (Tarapur, India).
Superoxide dismutase and glutathione peroxidase estimation kits were from
RANDOX Laboratories (UK).
Preparation of solvent extract
Extraction was carried out according to the method of Okigbo et al. (2005).
Fifty grams each of the powdered material was extracted initially with 300 ml of
chloroform, hexane, methanol and ethanol separately for 24 h at 23 ± 2°C. The extract
was filtered with sterile Whatman No. 1 filter paper into a clean conical flask. Second
extraction was carried out with same amount of solvent for another 24 h at 23 ± 2°C
and filtered. The extracts were later pooled and transferred into the sample holder of
the rotary flash evaporator [Buchi Rotavapor R-124, Switzerland] for the evaporation
of the solvents. The evaporated extracts so obtained were preserved at 4°C in airtight
bottles until further use. The suspensions of the extracts were prepared by dissolving
the weighed amounts in freshly prepared 1% gum arabic solution.
Chapter – 3 : In vivo antidiabetic activity studies Material and Methods
78
Animals
Adult healthy Albino rats of Wistar strain of either sex weighing 120-160 g
with no prior drug treatment were used in the present study. Animals were maintained
at 22 ± 2°C with 12 h light and dark cycle. The animals were fed on a standard pellet
diet and had free access to water throughout the experiment. Animals that are
described as fasting were deprived of food for at least 16 h but were allowed free
access to drinking water. Animal study was performed in Central Animal Facility, JSS
Medical College, Mysore, with due permission from Institutional Animal Ethics
Committee (R.No. JSS/MC/IEC/5064/CPCSEA).
Experimental design
The animals were randomly divided into the 15 groups with six animals in
each group.
Group A saline or normal control
Group B and Group C normal controls fed with 250 mg/kg b.w.
and 500 mg/kg b.w. of A. paniculata leaf
extract respectively
Group B1 and Group C1 normal controls fed with 250 mg/kg b.w.
and 500 mg/kg b.w. of T.cordifolia leaf
extract respectively
Group B2 and Group C2 normal controls fed with 250 mg/kg b.w.
and 500 mg/kg b.w. of T. foenum-graecum
leaf extract respectively
Group D diabetic control
Group E diabetic rats treated with glibenclamide
(0.5 mg/kg b.w.)
Group F and Group G diabetic rats treated with 250 mg/kg b.w.
and 500 mg/kg b.w. of A. paniculata leaf
extract respectively
Group F1 and Group G1 diabetic rats treated with 250 mg/kg b.w.
and 500 mg/kg b.w. of T. cordofolia leaf
extract respectively
Group F2 and Group G2 diabetic rats treated with 250 mg/kg b.w.
and 500 mg/kg b.w. of T. foenum-graecum
leaf extract respectively
Chapter – 3 : In vivo antidiabetic activity studies Material and Methods
79
Saline, leaf extract and the standard drug were administered orally using a
mouth gauge. During the study period, body weight, food and water intake of rats
were monitored regularly.
Induction of diabetes
After initial determination of 16 h fasting blood glucose levels (blood drawn
through the tail vein puncture) animals were given single intraperitoneal injection of
streptozotocin at a dose of 45 mg/kg b.w. freshly dissolved in cold 0.9% saline
(Venkateswaran and Pari, 2003). Following injection, animals were carefully
observed for first 24 h for evidence of allergic reaction, behavioural changes and
convulsions. Fasting blood glucose levels were recorded after 5 days. Animals that
developed stable hyperglycemia with fasting blood glucose levels more than 200 mg/dl
were selected for the study.
Blood collection
Rats were fasted overnight; blood was drawn from the tail by tail vein
puncture during the experimental period and from cardiac puncture at the end of the
experimental period (after 28 days) under anaesthesia. Serum was separated by
centrifuging the collected blood samples at 10,000 rpm for 10 min at 4°C and used for
the estimation of creatinine and urea.
Urine collection
Urine from normal control rats, diabetic and diabetic treated rats were
collected under a layer of toluene by keeping the rats in metabolic cages for 24 h.
Collected urine samples were filtered through filter paper, centrifuged and stored at
4°C until further analysis.
Preparation of liver tissue homogenate
After 28 days, rats were dissected under anaesthesia. Liver was rinsed in ice
cold distilled water followed by chilled 0.9% saline. About 1 g of liver tissue was
homogenized in 10 ml of 10 mM phosphate buffered saline (pH 7.4) using REMI
homogenizer fitted with a teflon plunger (REMI Laboratory Instruments, Mumbai,
India). The homogenate was centrifuged at 10,000 rpm at 4°C for 15 min and the
supernatant was used for the determination of antioxidant liver enzymes viz.,
superoxide dismutase, catalase and glutathione peroxidase.
Chapter – 3 : In vivo antidiabetic activity studies Material and Methods
80
Analytical Methods
Blood glucose estimation
Blood samples were collected by tail vein puncture at weekly intervals for a
period of 28 days. Fasting blood glucose was measured by glucose oxidase-peroxidase
(GOD-POD) method (Trinder, 1969) in mg/dl using a digital glucometer (Braun
OmnitestR
EZ, Germany).
Protein estimation
Protein estimation was carried to the method of Lowry et al. (1951). The blue
colour developed by the reduction of phosphomolybdic-phosphotungstic components
in the Folin-Ciocalteau reagent by the aminoacids tyrosine and tryptophan present in
the protein is measured at 660 nm.
Procedure
Reagent A – 2% sodium carbonate in 0.1 N sodium hydroxide
Reagent B – 0.5% copper sulphate in 1% potassium sodium tartarate
Reagent C – 50 ml of reagent A and 1 ml of reagent B mixed prior to use
Reagent D – 1 ml of Folin-Ciocalteau phenol reagent diluted with 2.5 ml of distilled water
To 1 ml of the sample containing 50-200 µg of protein, 5 ml of reagent C was
added and mixed thoroughly and allowed to stand at room temperature for 10 min. To
this, 0.5 ml of reagent D was added, vortexed and allowed to stand at room
temperature for 30 min and absorbance was measured at 660 nm in a
spectrophotometer. Standard BSA (0-200 µg/ml) was treated similarly and a standard
graph was plotted.
Creatinine estimation
Creatinine estimation was carried out according to the method of Folin and
Wu (1919) in both serum and urine samples using creatinine estimation kit according
to manufacturer’s instruction. Creatinine when treated with alkaline picrate solution
yields a bright orange red complex. Intensity of the colour at 520 nm corresponds to
the concentration of creatinine in the sample.
Chapter – 3 : In vivo antidiabetic activity studies Material and Methods
81
Procedure
For urine: To 10 µl of the urine sample, 90 µl of distilled water was added. To this,
600 µl of picric acid reagent provided in the kit was added and mixed thoroughly
followed by the addition of 2 ml of 0.2 N sodium hydroxide. The mixture was
allowed to stand at room temperature for 15 min. The colour intensity developed was
measured at 520 nm. Standard creatinine (10 mg/dl concentration) provided in the kit
was also treated similarly. Creatinine levels were expressed as mg/day and calculated
according to manufacturer’s instruction as below:
Urine creatinine = Absorbance of sample / Absorbance of standard × Dilution factor
For serum: To 200 µl of serum sample, 1.2 ml of picric acid reagent was added,
vortexed and centrifuged at 2000 rpm for 10 min. To 700 µl of supernatant, 400 µl of
0.2 N sodium hydroxide was added and allowed to stand at room temperature for 15 min.
Colour intensity developed was measured at 520 nm. Standard creatinine (10 mg/dl
concentration) was also treated similarly. Creatinine levels were expressed as mg/dl
and calculated according to manufacturer’s instructions as below:
Serum creatinine = Absorbance of sample / Absorbance of standard × 2
Serum urea estimation
Serum urea was estimated according to enzymatic UV-kinetic method of
Talke and Schubert (1965). In the presence of urease, urea is hydrolyzed to ammonia
ion and carbon dioxide. In the presence of glutamate dehydrogenase (GLDH) the
formed ammonium ion reacts with α-ketoglutarate and NADH to form glutamate and
NAD+. An equimolar quantity of NADH undergoes oxidation during the reaction and
therefore the decrease in absorbance at 340 nm due to NADH oxidation is directly
proportional to the urea concentration in the sample. Urea was estimated according to
manufacturer’s instruction.
Procedure
Reagent R1 – 50 mM/l of tris buffer (pH 7.7) containing 10 KU/l of urease and 900
IU/l of glutamate dehydrogenase
Reagent R2 – 50 mM/l of tris buffer (pH 7.7) containing 0.25 mM/l of NADH and 10
mM/l of α-ketoglutarate
Chapter – 3 : In vivo antidiabetic activity studies Material and Methods
82
Reagent R1 and reagent R2 were mixed in the ratio of 4:1 to prepare the
desired volume of working reagent. To 10 µl of serum sample 1 ml of working
reagent was added and allowed to react for 30 sec. The absorbance at 340 nm was
read at 30th
and 60th
second. Standard urea (42.8 mg/dl) provided in the kit was also
treated similarly. Urea concentrations were expressed as mg/dl and calculated
according to the manufacturer’s instructions as below:
Urea (mg/dl) = Absorbance of sample / Absorbance of standard × 42.8
Liver antioxidant enzyme activity assay
Estimation of superoxide dismutase (SOD)
The activity of SOD was assayed according to the method of Woolliams et al.
(1983) in a Daytona analyser (RANDOX Laboratories Ltd., United Kingdom) using
RANDOX SOD kit. This method employs xanthine and xanthine oxidase to generate
superoxide radicals which reacts with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-
phenyltetrazolium chloride (I.N.T.) to form a red formazan dye. The SOD activity is
then measured by the degree of inhibition of this reaction. One unit of SOD is that
which causes a 50% inhibition of the rate of reduction of I.N.T. under the conditions
of the assay.
Procedure
Reagent R1 – 0.05 mM/l of xanthine and 0.025 mM/l of I.N.T. in 20 ml of buffer
[CAPS (40 mM/l, pH 10.2) + EDTA (0.94 mM/l)]
Reagent R2 – 80 U/l of xanthine oxidase in 10 ml of redistilled water
Subsequent dilutions of the standard (4.48 U/ml) were prepared with 0.01
mM/l phosphate buffer (pH 7) to produce a standard curve. To 5.6 µl of diluted
standard, 189 µl of reagent R1 and 28 µl of reagent R2 were added. The initial
absorbance after 30 sec and final absorbance after 3 min was taken at 505 nm.
Suitably diluted samples were treated similarly. SOD units/mg protein was calculated
according to manufacturer’s instruction as below:
SOD units/ml = SOD units/ml from standard curve × dilution factor
SOD units/mg protein = SOD units/ml mg protein/ml
Chapter – 3 : In vivo antidiabetic activity studies Material and Methods
83
Estimation of catalase
The activity of catalase was assayed according to the method of Luck (1971).
The UV absorption of hydrogen peroxide (H2O2) solution can be easily measured
between 230 and 250 nm. On decomposition of H2O2 by catalase, the absorption
decreases with time. The enzyme activity can be arrived at from this decrease.
Procedure
Before the assay, the standard H2O2 (30% w/v) dilution was set with
phosphate buffer (10 mM, pH 7.0) so that at 240 nm for 2 min, the absorbance is 0.50.
To the reaction mixture consisting of 1 ml H2O2 in phosphate buffer, 50 µl of sample
was added and change in absorbance at 240 nm for 2 min was monitored. Results
were expressed as units/min/mg protein.
Specific activity = ΔA / min / ε of H2O2 × mg of protein
Estimation of glutathione peroxidase (GSH-Px)
The activity of glutathione peroxidase was assayed according to the method of
Paglia and Valentine (1967) in a Daytona analyser using glutathione peroxidase kit.
Glutathione peroxidase catalyses the oxidation of glutathione by cumene
hydroperoxide. In the presence of glutathione reductase and NADPH the oxidised
glutathione is immediately converted to the reduced form with a concomitant
oxidation of NADPH to NADP+. The decrease in absorbance at 340 nm is measured.
Procedure
Reagent R1 – 4 nM/l of glutathione, 0.5 U/l gluthione reductase and 0.34 mM/l
NADPH in 6.5 ml of buffer (0.05 M/l phosphate buffer of pH 7.2 +
4.23 mM/l of EDTA)
Reagent R2 – 10 µl of 0.18 mM/l cumene hydroperoxide in 10 ml of saline
In this assay, 20 µl of sample was mixed with 1 ml of reagent R1 and 40 µl of
reagent R2 and mixed well. The initial absorbance of sample and reagent blank was
read after 1 min and again after 1 and 2 min at a wavelength of 340 nm. The reagent
blank value was subtracted from that of the sample. Glutathione peroxidase units/mg
protein was calculated according to the manufacturer’s instruction as below:
GSH-Px Units / l of homogenate = 8412 × ΔA 340 nm / minute
GSH-Px Units / mg protein = U / l homogenate / mg protein
Chapter – 3 : In vivo antidiabetic activity studies Material and Methods
84
Histopathological studies
At the end of the study, the rats were sacrificed, whole pancreas from each
animal was removed, washed in normal saline and fixed in 10% formalin, embedded
in paraffin and sections of 3-5 µm thickness were cut and routinely stained with basic
dye haematoxylin and acidic dye eosin to differentiate the nucleus and cytoplasm
(Kiernan, 2008). The sections were studied at 10× and 40× magnifications for the islet
cell characteristics using a binocular compound microscope.
Statistical analysis
All the values of fasting blood sugar, biochemical estimations, body weight,
and food and water intake were expressed as mean ± standard error of mean (SEM).
Statistical difference was evaluated by using one way analysis of variance (ANOVA)
followed by Turkey’s test. Data were considered statistically significant at P value ≤ 0.05.
Statistical analysis was performed using Graph Pad statistical software.
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
85
Results and discussion
Effect of leaf extracts on blood glucose level
Administration of streptozotocin produced 68.7% increase in fasting blood
glucose levels of diabetic control rats compared to the normal control rats and the
increased glucose levels in group D rats was maintained over a period of four weeks.
Administration of A. paniculata ethanol leaf extract to streptozotocin induced diabetic
rats for four weeks produced a significant blood glucose reduction (Table 3.1).
Reduction in blood glucose was observed from the first week by both extract and
glibenclamide. At the end of 4th
week, 500 mg/kg b.w. of extract produced 29.2%
blood glucose reduction in group G rats. Similar to group G rats, there was a lowering
of 25.2% blood glucose in the rats treated with 250 mg/kg b.w. of the extract. Among
the two doses of the extracts used, 500 mg/kg b.w. of the extract showed greater
reduction in blood glucose level which was comparable to glibenclamide.
Table 3.1 : Effect of ethanol extract of Andrographis paniculata leaves on glucose
levels in streptozotocin induced diabetic rats (n=6)
Groups Fasting blood glucose levels (mg/dl)
Day 0 Day 7 Day 14 Day 21 Day 28
Group A
Normal control rats
(0.9% NaCl)
77.0 ± 1.82a
78.25 ± 0.95a
77.75± 2.21a
79.50 ± 2.64a
79.25 ± 1.70a
Group D
STZ induced
diabetic control
246.25 ± 9.53b
284.0 ± 5.47c
309.50 ± 8.73d
319.75 ± 7.80c
319.50 ± 8.66c
Group E
STZ+ glibenclamide
(0.5 mg/kg b.w.)
248.50 ± 13.52b
222.2 ± 7.27b
190.0 ± 4.54b
178.25 ± 6.80b
157.75 ± 8.95b
Group F
STZ+extract
(250 mg/kg b.w.)
235.75 ± 10.65b
219.50 ± 7.41b 205.50 ± 6.24
c 189.75 ± 10.24
b 176.25 ± 9.06
b
Group G
STZ+extract
(500 mg/kg b.w.)
233.0 ± 12.03b
216.75 ± 8.65b
198.50 ± 8.18bc
179.75 ± 10.07b 163.25 ± 12.33
b
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
86
The effect of two different doses of ethanol leaf extracts of T. cordifolia on the
fasting blood glucose levels of diabetic rats is given in Table 3.2. In the diabetic rats,
the ethanol extract at a dose of 500 mg/kg b.w. produced a 20.7% fall in the blood
glucose levels at the end of the experimental period. The glibenclamide treated
diabetic rats showed greater reduction in the blood glucose level than the extract
treated rats. Eventhough, there was a reduction in blood glucose levels with 250
mg/kg b.w. of the extract, the decrease was much lower when compared to group G1
and group E animals.
Table 3.2 : Effect of ethanol extract of Tinospora cordifolia leaves on glucose
levels in streptozotocin induced diabetic rats (n=6)
Groups Fasting blood glucose levels (mg/dl)
Day 0 Day 7 Day 14 Day 21 Day 28
Group A
Normal control rats
(0.9% NaCl)
77.0 ± 1.82a
78.25 ± 0.95a
77.75± 2.21a
79.50 ± 2.64a
79.25 ± 1.70a
Group D
STZ induced diabetic
control
246.25 ± 9.53b
284.0 ± 5.47d
309.50 ± 8.73e
319.75 ± 7.80e
319.50 ± 8.66d
Group E
STZ+ glibenclamide
(0.5 mg/kg b.w.)
248.50 ± 13.52b
222.2 ± 7.27b
190.0 ± 4.54b
178.25 ± 6.80b
157.75 ± 8.95b
Group F1
STZ+extract
(250 mg/kg b.w.)
249.75 ± 13.74b
241.0 ± 9.05c
231.75 ± 6.80d
224.25 ± 8.88d
207.50 ± 6.65c
Group G1
STZ+extract
(500 mg/kg b.w.)
244.0 ± 13.63b
226.50 ± 4.65b
215.0 ± 5.71c
205.50 ± 5.68c
193.25 ± 6.44c
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
The antihyperglycemic effect of T. foenum-graecum ethanol leaf extract on the
fasting blood sugar levels of diabetic rats is shown in Table 3.3. Daily treatment of
ethanol extract of T. foenum-graecum leaves at a concentration of 250 mg/kg b.w. and
500 mg/kg b.w. for a period of 28 days led to a dose dependent fall in blood glucose
levels (23.5% and 31.2% respectively). The glucose lowering effect of the extract
seems to reach a maximum after 25 days of treatment and remains almost constant
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
87
during the next 4-5 days. The effect was more pronounced with animals receiving
500 mg/kg b.w. of extract which can be compared to that of glibenclamide. The
fasting blood glucose levels in normal control animals were found to be stable all
throughout the experimental period.
Table 3.3 : Effect of ethanol extract of Trigonella foenum-graecum leaves on
glucose levels in streptozotocin induced diabetic rats (n=6)
Groups Fasting blood glucose levels (mg/dl)
Day 0 Day 7 Day 14 Day 21 Day 28
Group A
Normal control rats
(0.9% NaCl)
77.0 ± 1.82a
78.25 ± 0.95a
77.75± 2.21a
79.50 ± 2.64a
79.25 ± 1.70a
Group D
STZ induced diabetic
control
246.25 ± 9.53b
284.0 ± 5.47c
309.50 ± 8.73d
319.75 ± 7.80d
319.50 ± 8.66d
Group E
STZ+ glibenclamide
(0.5 mg/kg b.w.)
248.50 ± 13.52b
222.2 ± 7.27b
190.0 ± 4.54b
178.25 ± 6.80b
157.75 ± 8.95b
Group F2
STZ+extract
(250 mg/kg b.w.)
248.25 ± 10.65b
240.0 ± 12.27b
226.50 ± 8.58c
208.5 ± 7.41c
189.75 ± 6.39c
Group G2
STZ+extract
(500 mg/kg b.w.)
243.25 ± 12.03b
229.0 ± 11.40b
204.0 ± 6.21b
184.50 ± 5.44b
167.25 ± 7.80b
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
Diabetes mellitus, characterized by hyperglycemia, is the most common and
serious metabolic disorder that is considered to be one of the five leading causes of
death in the world (Rahimi et al., 2005). Streptozotocin induced diabetes produced a
significant increase in blood glucose levels in the diabetic rats. It has been postulated
but, is still debated that the fasting hyperglycemia in NIDDM arises from the hepatic
overproduction of glucose (De-Fronzo, 1992). However, studies by Wi et al. (1998)
suggested that the post absorptive hyperglycemia in STZ induced diabetic rats is
mainly due to the decreased peripheral glucose clearance, while increased hepatic
glucose output might also be a contributing factor at a very high STZ dose.
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
88
In case of A. paniculata leaf extract, there was a greater reduction in the blood
glucose at a concentration of 500 mg/kg b.w. when compared to 250 mg/kg b.w. This
shows that the effect of ethanol extract was dose dependent. Earlier investigations on
the antihyperglycemic activity of the organic extracts of A. paniculata whole plant by
various authors also substantiate the results of the present study (Zhang and Tan,
2000a; Zhang and Tan, 2000b; Husen et al., 2004). In the previous studies on
A. paniculata, aqueous extract has been shown to reduce the blood glucose level only
at higher doses (Hossain et al., 2007; Fasola et al., 2010). Moreover, our results on
leaf extract also indicate a prolonged duration of antidiabetic action in a chronic study
which could be due to multiple sites of action possessed by the active principle of
A. paniculata. The antidiabetic effect of the extract may be due to the presence of one
or more than one antihyperglycemic principle and their synergistic properties
(Mukherjee et al., 2006) as A. paniculata contains polyphenols, flavonoids and
diterpenoids as the major bioactive components (Rao et al., 2004; Xu et al., 2010). In
this study also we have shown the presence of polyphenols, flavonoids, tannins,
terpenoids and glycosides in the ethanol leaf extract. The possible mode of action may
be the extra-pancreatic action such as increased glucose uptake (Yu et al., 2003; Rao,
2006) or α-glucosidase inhibition (Subramanian et al., 2008) or mediation of
β-endomorphin (Yu et al., 2008).
Tinospora cordifolia is a widely used plant in folk and Ayurvedic systems of
medicine. Many researchers have shown the antidiabetic activity of its stem and root
extracts (Wadood et al., 1992; Grover et al., 2001; Rathi et al., 2002; Prince and
Menon, 2003). Due to this reason the leaf extract of the plant was evaluated and the
data also confirmed the traditional indications. Earlier investigations on the
antidiabetic activity of T. cordifolia root aqueous extract has shown a significant
reduction in blood glucose in alloxan diabetic rats at a concentration of 2.5 g/kg b.w.
(Prince and Menon, 2000). Various workers have shown potent antidiabetic activity
of T. cordifolia stem extract in reducing the blood sugar level in STZ induced diabetic
rats (Rajalakshmi et al., 2009; Puranik et al., 2010). There are reports on the
antihyperglycemic effects of Tinospora sinensis (Pimpriker et al., 2009) and
Tinospora crispa (Noor and Ashcroft, 1989). The results of the present study on the
leaf extract of T. cordifolia substantiate the earlier work on the antidiabetic activity of
stem and root extracts. The ethanol extract of T. cordifolia also produced significant
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
89
but less antihyperglycemic activity in comparison with that of ethanol extracts of
A. paniculata and T. foenum-graecum at a concentration of 500 mg/kg b.w. which
may be attributed to the presence of lower concentration of active principle in the
extract or may be due to the lower dose used. The antidiabetic effect of T. cordifolia
leaf extract may be due to the presence of diterpene lactones, steroids, phenols and
aliphatic compounds (Singh et al., 2003) or other constituents present in the extract
which could act synergistically or independently to show the antidiabetic action. The
possible mechanism by which the ethanol extract brings about its hypoglycemic
action is by potentiating the insulin effect of plasma by increasing either the
pancreatic secretion of insulin from β cells of islets of langerhans or its release from
bound insulin (Prince and Menon, 2000).
Treatment with ethanol leaf extract of T. foenum-graecum showed significant
decrease in blood glucose levels which was comparable to glibenclamide. Decrease in
blood glucose levels was found to be more effective with 500 mg/kg b.w. which
shows its dose dependent effects. Glibenclamide showed rapid normalization of blood
glucose due to its insulin releasing effects (Nima and Jagruti, 2010). Earlier studies by
various workers have shown the antihyperglycemic activity of T. foenum-graecum
seeds in a dose dependent manner in experimentally induced diabetic rats (Ali et al.,
1995; Vats et al., 2002; Xue et al., 2007). A study by Abdel-Barry et al. (1997)
showed that the intraperitoneal administration of ethanol leaf extract of T. foenum-
graecum at a concentration of 0.8 g/kg b.w. caused a reduction in blood glucose
concentration in alloxan diabetic rats. From the results of the present study it is
evident that the antihyperglycemic activity of the ethanol extract can be achieved at a
lower concentration (0.5 g/kg b.w.) when given orally. The dietary fibre fraction of
fenugreek seeds has been shown to reduce the elevated blood glucose level of type 2
diabetic rats (Hannan et al., 2003). As the leaves also contain the dietary fibre the
possible constituent for the antihyperglycemic effect may be in part the dietary fibre
or compounds other than that. From the phytochemical analysis, it was shown that the
ethanol leaf extract contains phenols, polysterols, saponins, cardiac glycosides and
flavonoids. So, the antidiabetic activity of the plant extract may involve one or more
compounds which decrease blood glucose suggesting that the natural constituents
could act synergistically to induce a hypoglycemic effect (Marles and Farnsworth,
1995; Roy et al., 2005). The hypoglycemic effect of fenugreek seed has been
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
90
evidenced with polar solvents in diabetic animals (Zia et al., 2001; Moorthy et al.,
2010). Similarly, our study has shown the hypoglycemic effect of fenugreek leaves in
a polar solvent like ethanol. The probable mechanism, by which the plant extract
lowered the blood glucose levels in diabetic rats, may be by increasing glycogenesis,
inhibiting gluconeogenesis in the liver, inhibiting the absorption of glucose from the
intestine or stimulation of insulin secretion (Mowla et al., 2009).
Effect of leaf extract on body weight
Prior to STZ administration, there were no significant differences in the
average body weights of all the 15 groups of experimental animals. By the end of the
first week after DM was experimentally induced, the weights of groups D, E, F, and G
were significantly reduced as shown in Table 3.4. This weight loss continued for four
weeks in diabetic control animals (135.50 g to 86.25 g). Administration of
A. paniculata ethanol leaf extract (250 mg/kg b.w. and 500 mg/kg b.w.) and
glibenclamide significantly increased the body weights in diabetic rats as compared to
diabetic control rats. The effect of 500 mg/kg b.w. of extract on the body weight of
diabetic rats was better than glibenclamide.
Table 3.4 : Effect of treatment of Andrographis paniculata leaf extract on body
weight of streptozotocin induced diabetic rats (n=6)
Groups Average body weight (g)
Day 1 Day 7 Day 14 Day 21 Day 28
Group A
Normal control rats
(0.9% NaCl)
142.50 ± 5.0d
146.25 ± 2.50b
152.50 ± 5.0c
160.0 ± 4.08c
166.25±4.78c
Group D
STZ induced diabetic
control
135.50 ± 5.77bc
115.0 ± 5.77a
105.0 ± 5.77a
95.0 ± 4.08a
86.25 ± 4.78a
Group E
STZ+ glibenclamide
(0.5 mg/kg b.w.)
132.50 ± 5.0bc
120.0 ± 8.16a
125.0 ± 5.77b
123.75 ± 4.78b
126.25 ± 2.50b
Group F
STZ+extract
(250 mg/kg b.w.)
120.0 ± 0.00a
117.0 ± 2.44a
118.75 ± 2.50b
122.50 ± 2.88b
123.75 ± 4.78b
Group G
STZ+extract
(500 mg/kg b.w.)
125.0 ± 5.77ab
118.75 ± 2.50a
121.25 ± 2.50b
125.0 ± 4.08b
125.50 ± 5.25b
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
91
Change in the body weight of diabetic rats after administration of ethanol leaf
extract of T. cordifolia is summarised in Table 3.5. There was a decrease in the body
weight of diabetic rats as compared to normal rats. Administration of the extract
(250 mg/kg b.w. and 500 mg/kg b.w.) did not significantly increase the body weights
of groups F1 and G1 rats as compared to glibenclamide treated rats. As compared to
the steady decrease in the body weights of diabetic control rats, the effect of
500 mg/kg b.w. of the extract on body weight was significant.
Table 3.5 : Effect of treatment of Tinospora cordifolia leaf extract on body
weight of streptozotocin induced diabetic rats (n=6)
Groups Average body weight (g)
Day 1 Day 7 Day 14 Day 21 Day 28
Group A
Normal control rats
(0.9% NaCl)
142.50 ± 5.0b
146.25 ± 2.50b
152.50 ± 5.0c
160.0 ± 4.08c
166.25±4.78d
Group D
STZ induced diabetic
control
135.50 ± 5.77ab
115.0 ± 5.77a
105.0 ± 5.77a
95.0 ± 4.08a
86.25 ± 4.78a
Group E
STZ+ glibenclamide
(0.5 mg/kg b.w.)
132.5 ± 5.0ab
120.0 ± 8.16a
125.0 ± 5.77b
123.75 ± 4.78b
126.25 ± 2.50c
Group F1
STZ+extract
(250 mg/kg b.w.)
125.0 ± 5.77a
117.50 ± 15.0a
112.50 ± 12.58ab
110.0 ± 9.12b
103.75 ± 9.46ab
Group G1
STZ+extract
(500 mg/kg b.w.)
125.0 ± 5.77a
117.50 ± 12.50a
115.0 ± 12.90ab
112.50 ± 9.57b
110.0 ± 14.10bc
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
The effect of administration of T. foenum-graecum ethanol leaf extract on
body weights of diabetic rats is summarised in Table 3.6. The body weight of animals
in groups F2 and G2 increased on treatment with the extract of T. foenum-graecum leaf
over a period of 28 days which can be compared to that of group E animals. At the
end of the experimental period there was a significant difference in the weights of
groups F2 and G2 as compared to group D. Animals in group A showed a steady
increase in their body weights during the experimental period.
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
92
Table 3.6 : Effect of treatment of Trigonella foenum-graecum leaf extract on body
weight of streptozotocin induced diabetic rats (n=6)
Groups Average body weight (g)
Day 1 Day 7 Day 14 Day 21 Day 28
Group A
Normal control rats
(0.9% NaCl)
142.50 ± 5.0b
146.25 ± 2.50b
152.50 ± 5.0c
160.0 ± 4.08c
166.25±4.78c
Group D
STZ induced
diabetic control
135.50 ± 5.77ab
115.0 ± 5.77a
105.0 ± 5.77a
95.0 ± 4.08a
86.25 ± 4.78a
Group E
STZ+ glibenclamide
(0.5 mg/kg b.w.)
132.50 ± 5.0ab
120.0 ± 8.16a
125.0 ± 5.77b
123.75 ± 4.78b
126.25 ± 2.50b
Group F2
STZ+extract
(250 mg/kg b.w.)
127.50 ± 5.0a
117.50 ± 2.88a
116.25 ± 4.78b
120.0 ± 4.08b
122.5 ± 2.88b
Group G2
STZ+extract
(500 mg/kg b.w.)
133.75 ± 4.78ab
121.25 ± 2.50a
123.75 ± 2.50b
124.50 ± 4.20b
125.0 ± 4.08b
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
Streptozotocin induced diabetes is characterized by a severe loss in body
weight (Al-Shamaony et al., 1994; Kalaiarasi and Pugalendi, 2009). The decrease in
the body weight of diabetic rats in our study was due to the loss or degradation of
structural proteins (since structural proteins are known to contribute to the body
weight) to provide amino acids for gluconeogenesis during insulin deficiency
resulting in muscle wasting and weight loss (Swanston-Flatt et al., 1990; Rajkumar
et al., 1991). Due to insulin deficiency, protein content is decreased in muscular tissue
by proteolysis (Babu et al., 2007). Murray et al. (2003) have shown that protein
synthesis is decreased in all tissues due to decreased production of ATP and absolute
or relative deficiency of insulin. In the present study, diabetic control rats showed
marked reduction in their body weights when compared to normal rats. The weight
loss was reverted by administration of A. paniculata, T. cordifolia and T. foenum-
graecum extracts to the diabetic rats for a period of 28 days. The ability of the extracts
to protect body weight loss in diabetic rats seems to be the result of their ability to
reduce hyperglycemia (Whitton and Hems, 1975). Zhang and Tan (2000a) in their
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
93
study on antihyperglycemic activity of ethanol extract of aerial parts of A. paniculata
have shown a significant increase in the body weight of STZ diabetic rats receiving
the extract. Prince and Menon (2000) have shown the ability of T. cordifolia root
extract in preventing loss of body weight in alloxan induced diabetic rats. The results
of the present study substantiate the earlier work done on different parts of these
plants.
Effect of leaf extracts on food and water intake
The mean food and water intake of rats receiving A. paniculata ethanol leaf
extract are represented in Tables 3.7 and 3.8. The untreated diabetic rats had severe
polyphagia and polydipsia by the end of the experimental period with respective
increase in food and fluid intake. The mean food and water intake were significantly
lower in the extract treated group (groups F and G) than the glibenclamide treated
group and the diabetic control group. Extract of 500 mg/kg b.w. had more pronounced
effect on food and water intake than 250 mg/mg b.w. of extract. The normal control
animals showed a gradual increase in their food and water intake which can be
considered normal.
Table 3.7 : Effect of treatment of Andrographis paniculata leaf extract on food
intake in streptozotocin induced diabetic rats (n=6)
Groups Treatment Food intake (g/rat/week)
Day 7 Day 14 Day 21 Day 28
A Normal control rats
(0.9% NaCl) 57.75 ± 1.25
b 63.25 ± 1.70
c 65.50 ± 1.29
c 65.75 ± 0.95
d
D STZ induced diabetic
control 56.0 ± 1.41
ab 59.0 ± 0.81
b 59.50 ± 1.29
b 59.75 ± 2.21
c
E STZ+ glibenclamide
(0.5 mg/kg b.w.) 54.0 ± 1.41
a 49.50 ± 2.38
a 44.50 ± 1.29
a 44.0 ± 1.15
b
F
STZ+extract
(250 mg/kg b.w.) 54.50 ± 1.29
a 49.0 ± 0.81
a 42.50 ± 2.08
a 42.0 ± 2.16
ab
G
STZ+extract
(500 mg/kg b.w.) 53.25 ± 0.95
a 47.75 ± 1.70
a 42.0 ± 1.41
a 39.75 ± 1.70
a
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
94
Table 3.8 : Effect of treatment of Andrographis paniculata leaf extract on water
intake in streptozotocin induced diabetic rats (n=6)
Groups Treatment Water intake (ml/rat/week)
Day 7 Day 14 Day 21 Day 28
A Normal control rats
(0.9% NaCl) 93.50 ± 2.38
a 97.50 ± 1.29
a 99.25 ± 1.25
b 101.75 ± 1.50
b
D STZ induced diabetic
control 104.50 ± 1.29
c 111.0 ± 3.16
b 116.75 ± 2.21
c 122.25 ± 2.21
c
E STZ+ glibenclamide
(0.5 mg/kg b.w.) 99.50 ± 1.73
b 94.75 ± 2.75
a 91.0 ± 0.81
a 90.50 ± 1.29
a
F
STZ+extract
(250 mg/kg b.w.) 96.25 ± 1.25
ab 93.75 ± 1.70
a 88.50 ± 1.29
a 87.50 ± 1.29
a
G
STZ+extract
(500 mg/kg b.w.) 96.0 ± 2.58
ab 94.75 ± 2.87
a 89.25 ± 4.19
a 87.25 ± 0.95
a
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
The changes in the food and water intake of rats treated with T. cordifolia
ethanol leaf extract are represented in Tables 3.9 and 3.10. At the end of the 28th
day
period, there was a decrease in both food and water intake in the extract treated
groups (groups F1 and G1) when compared to the diabetic control group. But, in the
glibenclamide treated group, the reduction of food and water intake was much lower
than the extract treated groups.
Table 3.9 : Effect of treatment of Tinospora cordifolia leaf extract on food intake
in streptozotocin induced diabetic rats (n=6)
Groups Treatment Food intake (g/rat/week)
Day 7 Day 14 Day 21 Day 28
A Normal control rats
(0.9% NaCl) 57.75 ± 1.25
b 63.25 ± 1.70
d 65.50 ± 1.29
d 65.75 ± 0.95
d
D STZ induced diabetic
control 56.0 ± 1.41
ab 59.0 ± 0.81
c 59.50 ± 1.29
c 59.75 ± 2.21
c
E STZ+ glibenclamide
(0.5 mg/kg b.w.) 54.0 ± 1.41
a 49.50 ± 2.38
a 44.50 ± 1.29
a 44.0 ± 1.15
a
F1
STZ+extract
(250 mg/kg b.w.) 54.75 ± 1.25
ab 53.75 ± 0.95
b 52.25 ± 1.89
b 49.50 ± 1.29
b
G1
STZ+extract
(500 mg/kg b.w.) 54.75 ± 1.70
ab 51.75 ± 0.95
ab 49.50 ± 1.29
b 47.25 ± 1.70
ab
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
95
Table 3.10 : Effect of treatment of Tinospora cordifolia leaf extract on water
intake in streptozotocin induced diabetic rats (n=6)
Groups Treatment Water intake (ml/rat/week)
Day 7 Day 14 Day 21 Day 28
A Normal control rats
(0.9% NaCl) 93.50 ± 2.38
a 97.50 ± 1.29
a 99.25 ± 1.25
c 101.75 ± 1.50
c
D STZ induced diabetic
control 104.50 ± 1.29
c 111.0 ± 3.16
b 116.75 ± 2.21
d 122.25 ± 2.21
d
E STZ+ glibenclamide
(0.5 mg/kg b.w.) 99.50 ± 1.73
b 94.75 ± 2.75
a 91.0 ± 0.81
a 90.50 ± 1.29
a
F1
STZ+extract
(250 mg/kg b.w.) 100.25 ± 2.75
b 99.50 ± 1.73
a 97.75 ± 1.50
bc 97.0 ± 1.82
b
G1
STZ+extract
(500 mg/kg b.w.) 99.75 ± 1.70
b 97.25 ± 1.70
a 95.0 ± 0.81
b 92.75 ± 1.70
a
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
The food and water intake of experimental animals treated with T. foenum-
graecum ethanol leaf extract are shown in Tables 3.11 and 3.12. Treatment with the
extract lowered the food and water intake of the groups F2 and G2. The result was
more evident with respect to rats treated with 500 mg/kg b.w. of extract than
250 mg/kg b.w. which was comparable to group E animals.
Table 3.11 : Effect of treatment of Trigonella foenum-graecum leaf extract on
food intake in streptozotocin induced diabetic rats (n=6)
Groups Treatment Food intake (g/rat/week)
Day 7 Day 14 Day 21 Day 28
A Normal control rats
(0.9% NaCl) 57.75 ± 1.25
b 63.25 ± 1.70
c 65.50 ± 1.29
d 65.75 ± 0.95
d
D STZ induced diabetic
control 56.0 ± 1.41
ab 59.0 ± 0.81
b 59.50 ± 1.29
c 59.75 ± 2.21
c
E STZ+ glibenclamide
(0.5 mg/kg b.w.) 54.0 ± 1.41
a 49.50 ± 2.38
a 44.50 ± 1.29
ab 44.0 ± 1.15
b
F2
STZ+extract
(250 mg/kg b.w.) 54.25 ± 0.95
a 51.25 ± 1.25
a 46.0 ± 1.41
b 41.50 ± 1.29
ab
G2
STZ+extract
(500 mg/kg b.w.) 54.25 ± 1.50
a 48.0 ± 2.16
a 42.50 ± 2.08
a 39.50 ± 1.29
a
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
96
Table 3.12 : Effect of treatment of Trigonella foenum-graecum leaf extract on water
intake in streptozotocin induced diabetic rats (n=6)
Groups Treatment Water intake (ml/rat/week)
Day 7 Day 14 Day 21 Day 28
A Normal control rats
(0.9% NaCl) 93.50 ± 2.38
a 97.50 ± 1.29
b 99.25 ± 1.25
b 101.75 ± 1.50
c
D STZ induced diabetic
control 104.50 ± 1.29
c 111.0 ± 3.16
c 116.75 ± 2.21
c 122.25 ± 2.21
d
E STZ+ glibenclamide
(0.5 mg/kg b.w.) 99.50 ± 1.73
b 94.75 ± 2.75
ab 91.0 ± 0.81
a 90.50 ± 1.29
b
F2
STZ+extract
(250 mg/kg b.w.) 96.75 ± 1.25
ab 93.75 ± 0.95
ab 89.25 ± 0.95
a 88.5 0± 1.29
ab
G2
STZ+extract
(500 mg/kg b.w.) 96.5 ± 2.08
ab 91.0 ± 1.82
a 87.75 ± 2.06
a 85.0 ± 3.36
a
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
The increase in food and water intake in diabetic rats is a classical symptom of
DM which was observed in all diabetic rats. The increase which was seen in the initial
period was decreased after the administration of the plant extracts for a period of
28 days. The metabolic disorders were corrected as shown by the reduction in
polyphagia, polyuria and polydipsia in diabetic rats treated with plant extracts. This
could be the result of improved glycemic control produced by ethanol extracts of
A. paniculata, T. cordifolia and T. foenum-graecum which was better seen with the
higher doses. The results obtained in our study supports the earlier study by Zhang and
Tan (2000a) on crude extract of A. paniculata aerial parts at a dose of 400 mg/kg b.w.
Effect on kidney parameters
Serum and urinary creatinine levels and serum urea levels of normal and
diabetic rats treated with A. paniculata ethanol leaf extract is shown in Table 3.13.
Diabetic control rats exhibited higher serum creatinine, urinary creatinine and serum
urea levels compared to those of normal rats. The creatinine and urea levels were
significantly decreased by glibenclamide and the extract due to 28 days of treatment. In
groups F and G animals there was a reduction in serum creatinine (74.1% and 71.9%),
urinary creatinine (63.4% and 60.6%) and serum urea (50.5% and 49.3%) levels as
compared to diabetic untreated animals. In the normal control rats fed with the extract
(groups B and C) the creatinine and urea levels were similar to the normal control rats.
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
97
Table 3.13 : Effect of treatment of Andrographis paniculata leaf extract on serum
creatinine, urinary creatinine and serum urea levels in normal and
streptozotocin induced diabetic rats (n=6)
Groups Treatment
Serum
creatinine
(mg/dl)
Urinary
creatinine
(mg/day)
Serum urea
(mg/dl)
A Normal control rats
(0.9% NaCl) 0.47 ± 0.04
a 18.72 ± 1.83
a 23.74 ± 0.73
a
B
Normal rats with extract
(250 mg/kg b.w.) 0.44 ± 0.04
a 18.42 ± 0.75
a 23.79 ± 0.26
a
C
Normal rats with extract
(500 mg/kg b.w.) 0.43 ± 0.08
a 18.25 ± 0.34
a 23.72 ± 0.43
a
D STZ induced
diabetic control 1.82 ± 0.17
b 51.52 ± 3.76
c 61.94 ± 1.54
d
E STZ+ glibenclamide
(0.5 mg/kg b.w.) 0.60 ± 0.07
a 23.61 ± 2.38
b 29.73 ± 0.59
b
F
STZ+extract
(250 mg/kg b.w.) 0.51 ± 0.01
a 20.25 ± 0.91
ab 31.40 ± 0.62
c
G
STZ+extract
(500 mg/kg b.w.) 0.47 ± 0.04
a 18.85 ± 0.71
a 30.63 ± 0.46
bc
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
Table 3.14 represents the creatinine and urea levels in normal and diabetic rats
treated with T. cordifolia ethanol leaf extract. There was a reduction in the creatinine
and urea levels in the extract fed diabetic rats. Extract of 500 mg/kg b.w. showed
greater reduction in the serum creatinine (65.3%), urinary creatinine (47.9%) and
serum urea (47.0%) levels. In the diabetic control rats there was a significant increase
in the creatinine and urea levels. In the normal rats fed with the extract (groups B1 and
C1) the creatinine and urea levels were normal.
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
98
Table 3.14 : Effect of treatment of Tinospora cordifolia leaf extract on serum
creatinine, urinary creatinine and serum urea levels in normal and
streptozotocin induced diabetic rats (n=6)
Groups Treatment
Serum
creatinine
(mg/dl)
Urinary
creatinine
(mg/day)
Serum urea
(mg/dl)
A Normal control rats
(0.9% NaCl) 0.47 ± 0.04
ab 18.72 ± 1.83
a 23.74 ± 0.73
a
B1
Normal rats with extract
(250 mg/kg b.w.) 0.48 ± 0.02
ab 18.45 ±1.04
a 23.62 ± 0.43
a
C1
Normal rats with extract
(500 mg/kg b.w.) 0.43 ± 0.04
a 18.45 ± 1.86
a 23.65 ± 0.34
a
D STZ induced
diabetic control 1.82 ± 0.17
d 51.52 ± 3.76
d 61.94 ± 1.54
e
E STZ+ glibenclamide
(0.5 mg/kg b.w.) 0.60 ± 0.07
abc 23.61 ± 2.38
b 29.73 ± 0.59
b
F1
STZ+extract
(250 mg/kg b.w.) 0.68 ± 0.07
c 28.45 ± 1.92
c 35.53 ± 0.56
d
G1
STZ+extract
(500 mg/kg b.w.) 0.63 ± 0.03
bc 26.80 ± 2.64
bc 32.78 ± 0.68
c
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
The effect of T. foenum-graecum ethanol leaf extract on serum and urinary
creatinine and serum urea levels is shown in Table 3.15. The extract at a concentration
of 500 mg/kg b.w. showed a greater decrease in serum creatinine (73.6%), urinary
creatinine (62.7%) and serum urea (50.6%) levels when compared to diabetic
untreated rats. There were no changes in the creatinine and urea levels in normal
extract treated controls (groups B2 and C2) as compared to the normal controls.
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
99
Table 3.15 : Effect of treatment of Trigonella foenum-graecum leaf extract on
serum creatinine, urinary creatinine and serum urea levels in
normal and streptozotocin induced diabetic rats (n=6)
Groups Treatment
Serum
creatinine
(mg/dl)
Urinary
creatinine
(mg/day)
Serum urea
(mg/dl)
A Normal control rats
(0.9% NaCl) 0.47 ± 0.04
ab 18.72 ± 1.83
a 23.74 ± 0.73
a
B2
Normal rats with extract
(250 mg/kg b.w.) 0.41 ± 0.08
a 18.72 ± 0.62
a 23.62 ± 0.79
a
C2
Normal rats with extract
(500 mg/kg b.w.) 0.36 ± 0.05
a 18.32 ± 0.36
a 23.42 ± 0.74
a
D STZ induced
diabetic control 1.82 ± 0.17
c 51.52 ± 3.76
c 61.94 ± 1.54
d
E STZ+ glibenclamide
(0.5 mg/kg b.w.) 0.60 ± 0.07
b 23.61 ± 2.38
b 29.73 ± 0.59
b
F2
STZ+extract
(250 mg/kg b.w.) 0.50 ± 0.03
ab 22.22 ± 2.30
ab 32.63 ± 0.29
c
G2
STZ+extract
(500 mg/kg b.w.) 0.48 ± 0.01
ab 19.17 ± 0.84
a 30.55 ± 0.44
b
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
Diabetic nephropathy is the leading cause of DM related morbidity and
mortality. The pathogenesis of diabetic nephropathy is related to chronic
hyperglycemia and hemodynamic alterations in renal microcirculation and structural
changes in glomerulus as evident by the significant elevation in creatinine and urea
levels (Cryer, 2001). Measurement of creatinine and urea levels reflects the function
of kidneys (Smith et al., 2006). Most of the reports on toxic effects of herbal
medicines and dietary supplements are associated with hepatoxicity. But, reports of
other toxic effects including kidney, nervous system, blood, cardiovascular,
dermatologic effects, mutagenecity and carcinogenicity have also been published in
medical literature (Niggermann and Gruber, 2003; Pak et al., 2004). Streptozotocin
induced diabetes is associated with generation of free radicals and oxidant tissue
damage, increasing the risk of renal complications (Elena et al., 2001). The ROS
produced affect the renal functions by promoting varieties of vasoactive mediators
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
100
such as thromboxane, which in turn causes renal vasoconstriction and alter the
glomerular filtration rate (Craven et al., 1992). Hence measurement of these two
parameters enables the study of toxic effects of the drug on the kidney.
The increased creatinine and urea levels in the diabetic rats indicates renal
damage due to abnormal glucose regulation, including elevated glucose and
glycosylated protein tissue levels, hemodynamic changes within the kidney tissue and
increased oxidative stress (Veeramani et al., 2008). Treatment of ethanol leaf extracts
of A. paniculata, T. cordifolia and T. foenum-graecum caused a reduction in the
creatinine and urea levels thereby enhancing the renal function that is generally
impaired in diabetic rats. The results of the present study are in agreement with other
previous studies on the herbal formulation D-400 (Dubey et al., 1994), mesocarp
extract of Balanites aegyptiaca (Mansour and Newairy, 2000), fruit extract of
Terminalia catappa (Nagappa et al., 2003), chloroform root extract of A. paniculata
(Rao, 2006), ginger and clove oils (Atef et al., 2007) and Annona squamosa extract
(Kaleem et al., 2008) which showed lowering of creatinine and urea levels on
supplementation with various plant extracts.
The normal levels of creatinine and urea in the normal healthy rats fed with
the extracts of A. paniculata, T. cordifolia and T. foenum-graecum at a concentration
of 250 mg/kg b.w. and 500 mg/kg b.w. for a period of 28 days revealed the non-toxic
nature of the ethanol extract and also showed the normal kidney function. No
mortality was observed in the extract treated rats and behaviour of the treated rats also
appeared normal. Rajalakshmi et al. (2009) in their study on stem extract of
T. cordifolia have shown the non-toxic nature of the methanol extract. Similar results
were observed in the ethanol leaf extract of T. cordifolia.
Effect on liver antioxidant enzymes
As shown in Table 3.16, STZ induced diabetic control rats showed a
significant decrease in SOD, catalase and GSH-Px levels as compared to the normal
rats. Treatment with A. paniculata ethanol leaf extract for 28 days produced a
significant increase in the activities of the antioxidant enzymes comparable to that of
glibenclamide treated group. Treatment with 500 mg/kg b.w. of extract produced
more pronounced effect with an increase in the activities of SOD (10.29 U/mg
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
101
protein), catalase (8.57 U/min/mg protein) and GSH-Px (5.31 U/mg protein). The
activities of the enzymes in the non diabetic rats treated with extracts (groups B and
C) were similar to the normal control rats.
Table 3.16 : Effect of ethanol extract of Andrographis paniculata leaves on liver
antioxidant enzymes in normal and streptozotocin induced diabetic
rats (n=6)
Groups Treatment
Superoxide
dismutase
(units/mg
protein)
Catalase
(units/min/mg
protein)
Glutathione
peroxidase
(units/mg protein)
A Normal control rats
(0.9% NaCl) 10.50 ± 0.54
b 10.70 ± 1.13
c 5.73 ± 0.28
c
B
Normal rats with extract
(250 mg/kg b.w.) 10.00 ± 0.49
b 10.60 ± 0.66
c 5.70 ± 0.14
c
C
Normal rats with extract
(500 mg/kg b.w.) 10.32 ± 0.44
b 10.97 ± 0.81
c 5.82 ± 0.16
c
D STZ induced
diabetic control 3.23 ± 0.30
a 6.37 ± 0.48
a 3.45 ± 0.36
a
E STZ+ glibenclamide
(0.5 mg/kg b.w.) 10.02 ± 0.33
b 8.82 ± 0.63
b 5.26 ± 0.27
bc
F
STZ+extract
(250 mg/kg b.w.) 9.78 ± 0.43
b 8.05 ± 0.20
b 4.82 ± 0.21
b
G
STZ+extract
(500 mg/kg b.w.) 10.29 ± 0.40
b 8.57 ± 0.29
b 5.31 ± 0.26
bc
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
The activities of the liver antioxidant enzymes in normal and diabetic rats
treated with T. cordifolia ethanol leaf extract is summarised in Table 3.17. Treatment
with the extract increased the enzyme activities but, the increase was lower when
compared to the glibenclamide treated rats. The effect of 500 mg/kg b.w. of extract
was more than 250 mg/kg b.w. of extract. There was an increase in the activity of
SOD in the non diabetic rats treated with 250 mg/kg b.w and 500 mg/kg b.w. of the
extract. Marginal increase in the activities of catalase and GSH-Px were found with
group C1 rats.
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
102
Table 3.17 : Effect of ethanol extract of Tinospora cordifolia leaves on liver
antioxidant enzymes in normal and streptozotocin induced diabetic
rats (n=6)
Groups Treatment
Superoxide
dismutase
(units/mg
protein)
Catalase
(units/min/mg
protein)
Glutathione
peroxidase
(units/mg
protein)
A Normal control rats
(0.9% NaCl) 10.50 ± 0.54
cd 10.70 ± 1.13
d 5.73 ± 0.28
cd
B1
Normal rats with extract
(250 mg/kg b.w.) 10.89 ± 0.14
cd 10.57 ± 0.48
d 5.75 ± 0.26
cd
C1
Normal rats with extract
(500 mg/kg b.w.) 11.41 ± 0.82
d 11.10 ± 0.31
d 5.93 ± 0.19
d
D STZ induced
diabetic control 3.23 ± 0.30
a 6.37 ± 0.48
a 3.45 ± 0.36
a
E STZ+ glibenclamide
(0.5 mg/kg b.w.) 10.02 ± 0.33
c 8.82 ± 0.63
c 5.26 ± 0.27
bc
F1
STZ+extract
(250 mg/kg b.w.) 7.88 ± 0.47
b 7.10 ± 0.54
ab 4.84 ± 0.26
b
G1
STZ+extract
(500 mg/kg b.w.) 8.63 ± 0.53
b 7.95 ± 0.26
bc 4.98 ± 0.10
b
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
Table 3.18 shows the level of antioxidant enzyme activities in liver of normal
and diabetic rats treated with T. foenum-graecum ethanol leaf extract. Administration
of the leaf extract for 28 days produced a marked increase in the enzyme activities.
There was a significant increase in the activity of SOD (11.50 U/mg protein), catalase
(8.32 U/min/mg protein) and GSH-Px (5.48 U/mg protein) in diabetic rats treated with
500 mg/kg b.w. of extract which was greater than the glibenclamide treated rats.
There was an increase in the enzyme activities in group B2 and C2 animals.
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
103
Table 3.18 : Effect of ethanol extract of Trigonella foenum-graecum leaves on
liver antioxidant enzymes in normal and streptozotocin induced
diabetic rats (n=6)
Groups Treatment
Superoxide
dismutase
(units/mg
protein)
Catalase
(units/min/mg
protein)
Glutathione
peroxidase
(units/mg protein)
A Normal control rats
(0.9% NaCl) 10.50 ± 0.54
bc 10.70 ± 1.13
d 5.73 ± 0.28
cd
B2
Normal rats with extract
(250 mg/kg b.w.) 12.39 ± 0.75
d 11.12 ± 0.40
d 6.08 ± 0.18
de
C2
Normal rats with extract
(500 mg/kg b.w.) 14.15 ± 0.26
e 11.35 ± 0.42
d 6.30 ± 0.24
e
D STZ induced
diabetic control 3.23 ± 0.30
a 6.37 ± 0.48
a 3.45 ± 0.36
a
E STZ+ glibenclamide
(0.5 mg/kg b.w.) 10.02 ± 0.33
b 8.82 ± 0.63
c 5.26 ± 0.27
bc
F2
STZ+extract
(250 mg/kg b.w.) 10.08 ± 0.43
b 7.52 ± 0.61
ab 4.93 ± 0.16
b
G2
STZ+extract
(500 mg/kg b.w.) 11.50 ± 1.26
cd 8.32 ± 0.40
bc 5.48 ± 0.17
c
Values are expressed as mean ± S.E.M.
Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)
There is an increasing evidence in both experimental and clinical studies
which suggests that diabetes is associated with oxidative stress, leading to an
increased production of ROS, including superoxide radical, hydrogen peroxide and
hydroxyl radical (Young et al., 1995; Baynes and Thorpe, 1999; Arulselvan and
Subramanian, 2007; Gokce and Haznedaroglu, 2008; Bagri et al., 2009). Recently,
much attention has been focussed on the role of oxidative stress and it has been
suggested that oxidative stress may constitute the key and common events in the
pathogenesis of different diabetic complications (Maritim et al., 2003; Sepici-Dincel
et al., 2007). Several lines of evidences indicate that free radicals may play an
essential role in the mechanism of β cell damage and diabetogenic effect of STZ
(Ohkuwa et al., 1995). Streptozotocin induced hyperglycemia induces free radical
generation which thereby leads to DNA damage, protein degradation, lipid
peroxidation and finally culminating into damage to various organs of the body like
liver, kidney, brain and eyes (Feldman, 1988; Saxena et al., 1993; Garg and Bansal,
2000; Yazdanparast et al., 2007).
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
104
Diabetics and experimental animal models exhibit high oxidative stress due to
persistent and chronic hyperglycemia, which thereby depletes the activity of
antioxidative defence system and thus promote de novo free radical generation
(Nazirogilu and Butterworth, 2005; Kamalakannan and Prince, 2006). Free radical
production caused by hyperglycemia may occur via at least three different routes: non
enzymatic glycation (Cariello et al., 1992), auto-oxidation of glucose (Jiang et al.,
1990) and intracellular activation of the polyol pathway (Cariello, 2000). An
imbalance of oxidant/antioxidant defence systems result in alterations in the activity
of antioxidant enzymes such as SOD, catalase and GSH-Px (Maritim et al., 2003).
Antioxidant enzymes have been shown to play an important role in maintaining
physiological levels of oxygen and hydrogen peroxide by hastening the dismutation of
oxygen radicals and eliminating organic peroxides and hydroperoxides generated
from inadvertent exposure to STZ (Pari and Latha, 2004).
Enzymatic antioxidants such as SOD and catalase are considered primary
enzymes since they are involved in the direct elimination of ROS (Arulselvan and
Subramanian, 2007). Superoxide dismutase is the first line of defence against free
radical attacks. Its function is to catalyse the conversion of superoxide radicals to
hydrogen peroxide and hence diminishes the toxic effects due to this radical or other
free radicals derived from secondary reaction (Manonmani et al., 2005). The
hydrogen peroxide produced by SOD is excreted as water, based on the activity of
GSH-Px and catalase, thereby protecting the body from oxygen toxicity (Punitha
et al., 2005). Hence in the present study activities of SOD, catalase and GSH-Px in
non diabetic and diabetic rats treated with extracts were evaluated.
In the present study, the activity of SOD, catalase and GSH-Px were decreased
in liver homogenate of diabetic control rats compared to normal rats, indicating
dysfunction in antioxidant defensive system which could be due to free radical
induced inactivation or glycation of the enzyme in diabetic state (Al-Azzawie and
Alhamdani, 2006). Schettler et al. (1994) suggested that the reduced antioxidant
production is due to increased oxygen metabolites causing a decrease in the activity of
the antioxidant defence system. Various studies in the past reported conflicting results
regarding the status of antioxidant enzymes in DM. Majority of the authors reported
the decreased enzymatic antioxidant activities in diabetic rats (Pari and Latha, 2004;
Pavana et al., 2007; Sarkhail et al., 2007; Kapoor et al., 2009). A few authors have
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
105
shown an increased activity of catalase in diabetic erythrocytes (Terekhina et al.,
1998) or tissues (Cekic et al., 1999). Hazem et al. (2007) reported elevated levels of
enzymatic antioxidants SOD and reduced catalase in diabetic rats.
Treatment with A. paniculata, T. cordifolia and T. foenum-graecum ethanol
leaf extracts for a period of 28 days reversed the activities of these enzymatic
antioxidants, which could be due to decreased oxidative stress. Zhang and Tan
(2000b) reported reduced activities of hepatic SOD, catalase and GSH-Px in untreated
diabetic rats. Oral administration of ethanol extract of aerial parts of A. paniculata for
a period of 14 days resulted in a significant elevation of activities of SOD and
catalase, but the extract had no significant effect on GSH-Px activity (Zhang and Tan,
2000b). But, the present study with A. paniculata ethanol leaf extract has shown the
increased activities of all the three antioxidant enzymes and shows agreement with the
earlier study of Trivedi and Rawal (2001). Alcoholic root extract of T. cordifolia
administered orally for six weeks normalized the antioxidant status of SOD, catalase
and GSH-Px (Prince and Menon, 2001; Prince et al., 2004a; Prince et al., 2004b). The
results from the present study on T. cordifolia ethanol leaf extract support the earlier
studies. Trigonella foenum-graecum seeds have been shown to normalise the
antioxidant functions of liver enzymes in diabetic rats (Ravikumar and Anuradha,
1999; Anuradha and Ravikumar, 2001; Genet et al., 2002). Similar results were
observed in diabetic rats treated with T. foenum-graecum leaf extract in the present
study. Various other plant extracts, Punica granatum (Bagri et al., 2009), Rosmarinus
officinalis (Bakirel et al., 2008) and Phlomis anisodonta (Sarkhail et al., 2007) have
shown significant increase in the activities of SOD, catalase and GSH-Px on
administration to diabetic animals. Normal antioxidant enzyme levels in non diabetic
control rats treated with A. paniculata and T. cordifolia extracts indicates the normal
functioning of the liver and also the non-toxic nature of the extracts. The
improvement in the enzyme activities in non diabetic control rats treated with
T. foenum-graecum extracts is indicative of better liver function. The result of our study
is in agreement with the earlier study on fenugreek seeds (Muralidhara et al., 1999).
Theoretically, hypoglycemic plants act through a variety of mechanisms such
as improving insulin sensitivity, augmenting glucose dependent insulin secretion and
stimulating the regeneration of islets of langerhans in pancreas of STZ induced
diabetic rats (Sezik et al., 2005). Moreover, the role of antioxidant compounds in both
protection and therapy of diabetics have been considered in various scientific
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
106
researches. Treatment of STZ injected diabetic animals with N-acetly-L-Cysteine, a
well known antioxidant, prevented hyperglycemia through reduced oxidative stress
and restoring β cell function (Takatori et al., 2004). In this regard, it is anticipated that
the ethanol leaf extracts of A. paniculata, T. cordifolia and T. foenum-graecum acts by
decreasing the oxidative damage to pancreatic tissue. However, by this speculation,
we do not exclude the possibilities of other mechanisms by which the ethanol leaf
extracts exerts their effects.
Histopathological studies
Figures 3.1 and 3.2 are the photomicrographs of the pancreas of a normal rat
showing normal architecture and normal islets of langerhans. Figures 3.3 and 3.4 are the
photomicrographs of the pancreas of diabetic untreated rat. There were lymphocytic
infiltrations. Atrophy and destruction of β cells were marked. Islet cells were small and
shrunken. Figures 3.5 and 3.6 are photomicrographs of the pancreas of diabetic rat
treated with glibenclamide. There was an increase in the number of islet cells.
Hematoxylin and eosin sections of the pancreas of diabetic rats treated with
ethanol leaf extract of A. paniculata are shown in Figures 3.7 to 3.10. In animals
treated with 250 mg/kg b.w. and 500 mg/kg b.w. of extract there were regenerative
changes in tissue architecture of islet cells of pancreas. In animals treated with 500
mg/kg b.w. of extract, there was a marked increase in the number and size of islet
cells especially in the β cell region compared to animals treated with 250 mg/kg b.w.
of extract. Figures 3.11 to 3.14 are the photomicrographs of the pancreas of the
diabetic rats treated with 250 mg/kg b.w. and 500 mg/kg b.w. of T. cordifolia ethanol
leaf extract. In animals treated with 250 mg/kg b.w. of extract, there was a slight
improvement in the architecture of the islet cells as compared to diabetic control rats.
But in animals treated with 500 mg/kg b.w. of extract, there was a further
improvement in the architecture of islet cells with many cells showing normal shape
and size. Figures 3.15 to 3.18 are the photomicrographs of the pancreas of diabetic
rats treated with 250 mg/kg b.w. and 500 mg/kg b.w. of T. foenum-graecum ethanol
leaf extract. In diabetic rats treated with 250 mg/kg b.w. of the extract, the restoration
of the normal cellular population and size of islets were noted especially in the central
β cell region. In animals treated with 500 mg/kg b.w. of the extract, there was not
only an increase in the cellular population and size of the islets but also an increase in
the number of islets. The regeneration noted in the β cell regions was comparable to
that noted with glibenclamide.
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
107
Fig. 3.1: Normal rat Fig. 3.2: Normal rat
pancreas (10×) pancreas (40×)
Fig. 3.3: Diabetic untreated Fig. 3.4: Diabetic untreated
rat pancreas (10×) rat pancreas (40×)
Fig. 3.5: Glibenclamide treated Fig. 3.6: Glibenclamide treated
rat pancreas (10×) rat pancreas (40×)
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
108
Fig. 3.7: A. paniculata treated Fig. 3.8: A. paniculata treated
(250 mg/kg b.w.) diabetic (250 mg/kg b.w.) diabetic
rat pancreas (10×) rat pancreas (40×)
Fig. 3.9: A. paniculata treated Fig. 3.10: A. paniculata treated
(500 mg/kg b.w.) diabetic rat (500 mg/kg b.w.) diabetic
pancreas (10×) rat pancreas (40×)
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
109
Fig. 3.11: T. cordifolia treated Fig. 3.12: T. cordifolia treated
(250 mg/kg b.w.) diabetic (250 mg/kg b.w.) diabetic
rat pancreas (10×) rat pancreas (40×)
Fig. 3.13: T. cordifolia treated Fig. 3.14: T. cordifolia treated
(500 mg/kg b.w.) diabetic (500 mg/kg b.w.) diabetic
rat pancreas (10×) rat pancreas (40×)
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
110
Fig 3.15: T. foenum-graecum treated Fig 3.16: T. foenum-graecum treated
(250 mg/kg b.w.) diabetic (250 mg/kg b.w.) diabetic
rat pancreas (10×) rat pancreas (40×)
Fig. 3.17: T. foenum-graecum treated Fig. 3.18: T. foenum-graecum treated
(500 mg/kg b.w.) diabetic (500 mg/kg b.w.) diabetic
rat pancreas (10×) rat pancreas (40×)
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
111
Pancreas is a compound tubular, alveolar, partly exocrine and partly endocrine
gland. The exocrine part of the pancreas is in the form of serous acini, secreting the
secretions into intralobular duct. The endocrine part of the pancreas is in the form of
numerous rounded collections of cells known as islet of langerhans, embedded within
the exocrine part. Each islet is separated by the surrounding alveoli by a thin layer of
reticular tissue. The average islet in rats is 150 µm in diameter and contains about 45 ng
of insulin. There are four major endocrine cell types in mammalian islets; the insulin
producing β cells, the glucagon producing α cells, the somatostatin producing δ cells and
pancreatic polypeptide producing pp cells. The β cells are polyhedral, being truncated
pyramids and are usually well granulated with secretory granules 250-300 nm in
diameter. It has been estimated that each rat β cell contains about 10,000 granules
(Bonner-Weir and Smith, 1994).
Streptozotocin (N-[methylnitrocarbamoyl]-D-glucosamine) is well known for
its selective pancreatic islet β cell cytotoxicity and in many animal species STZ
induces diabetes that resembles human hyperglycemic non-ketotic DM (Weir et al.,
1981; Papaccio et al., 2000). Further, rats treated with STZ displays many of the
features seen in human subjects with uncontrolled DM and are invaluable when
studying the mechanism by which hyperglycemia may contribute to complications
such as nephropathy, retinopathy and neuropathy (Obrosova et al., 2005). So, in the
present investigation, the histological changes in the tissues of pancreas of diabetic
rats and effect of the extracts of A. paniculata, T. cordifolia and T. foenum-graecum
on the islet cells of pancreas were studied.
Microscopic examination shows abundant patches of β cells in the pancreas of
normal rats which are absent in diabetic pancreas (Anil et al., 1996). The decrease in
the cellularity, small and shrunken islets and destruction of β cells within islets of
langerhans observed in diabetic rats in the present study reflects the cytotoxicity of
STZ (Mitra et al., 1996; Szudelski, 2001; Xiu et al., 2001; Selvan et al., 2008). The
β cells in some islets of diabetic pancreas were found to be fusiform. The change in
shape of cells can be attributed to the partial damage by STZ. Aybar et al. (2001) have
reported that the use of lower dose of STZ produces an incomplete destruction of
pancreatic β cells eventhough rats become permanently diabetic. Streptozotocin
destroys β cells selectively and a single adequate dose produces lasting hyperglycemia
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
112
and insulin deficiency (Szaleczky et al., 1999). It has been reported that STZ enters
β cells via a glucose transporter (GLUT2) and causes alkylation of DNA. Other
studies indicated that cytotoxic effects of STZ are dependent upon DNA alkylation by
site specific action with DNA bases (Benneth and Pegg, 1981) and by free radical
generation during STZ metabolism (Bolzan and Bianchi, 2002).
Andrographis paniculata ethanol extract significantly increased the number
and size of islet cells especially in the β cell region. A recent study has shown that
andrographolide-lipoic acid conjugate (andrographolide analogue) had both
hypoglycemic and β cell protective effects (Zhang et al., 2009). The protective effect
of A. paniculata ethanol leaf extract may be due to the andrographolide or other
diterpenoids, flavonoids or polyphenols present in the plant (Matsuda et al., 1994;
Cheung et al., 2001; Pholphana et al., 2004; Rao et al., 2004).
A study by Rajalakshmi et al. (2009) on T. cordifolia stem have shown that
the alterations in the islet cells caused due to STZ was corrected to normal by
methanol extract treatment at a dose of 250 mg/kg b.w. for a period of 100 days. The
result obtained after treatment with T. cordifolia leaf extract to diabetic rats in the
present study is consistent with the earlier results and has shown the reversal of tissue
architecture of number of islet cells in diabetic treated rats but not to the extent of
becoming normal. It may be due to the shorter experimental period of 28 days as
compared to 100 days. A study by Puranik et al. (2010) on T. cordifolia stem extract
did not show any change in the altered architecture of the islet cells of pancreas even
after the treatment and the results of our study are contrary to this study. The
antihyperglycemic activity of T. cordifolia leaf extract may be through the increased
insulin secretion by pancreatic β cells or due to increased entry of glucose into the
peripheral tissues and organs (Prince and Menon, 2000). The antidiabetic activity of
T. cordifolia leaves may be due to the presence of diterpene lactones, phenols and
aliphatic compounds (Singh et al., 2003).
There was regeneration of islet cells especially in the β cell region in the
pancreatic tissues of rats treated with T. foenum-graecum ethanol leaf extract.
Nagappa et al. (2003) in their study had shown the regeneration of β cell due to the
presence of β carotene in Terminalia catappa fruits. The activity of T. foenum-
Chapter – 3: In vivo antidiabetic activity studies Results and Discussion
113
graecum ethanol leaf extract in the present study might be due to the presence of
β carotene (Srinivasan, 2006) or other phytochemicals present in its leaves.
Photomicrographical data in our studies reinforce the healing of pancreas, by
T. foenum-graecum leaf extract as a plausible mechanism of their antidiabetic activity.
Various other studies have reported the regeneration islet cells of pancreas.
Shanmugasundaram et al. (1990) have reported the activity of Gymnema sylvestre leaf
extract in regenerating the damaged endocrine tissue and increasing β cell numbers
partially. Sharma et al. (2003) have reported that oral administration of Eugenia
jambolana seed extract reversed the abnormalities in the islet of langerhans of alloxan
induced diabetic rabbits.
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